ALsubs

PBL 2

2008-01-26T19:37:00.001-08:00

Problem Statement
There are outbreaks of viral, fungal and protozoa diseases among platoons of army soldiers in Indonesia. Soldiers reported sick after 2 weeks of jungle warfare training. It is of concern to the ministry that there are also sporadic reports of avian flu in the nearby villages. In view of these outbreaks, you have been tasked to conduct a pre-mission briefing with blogs and poster to educate future batches of soldiers.

Environment in the tropical jungle:
1. High exposure to different kinds of virus, fungal and protozoa.
2. Dark and moist environment which allows growth of microorganisms.
3. High amounts of decay (leaf, dead plants and animals) which microorganisms can thrive in.
4. Indonesia is in tropical area. The environment is always warm and humid in its jungle.
5. Short supply of food and clean water.
6. poor hygiene conditions due to continuous training of the soldiers.
7. Source of food may come from jungle or nearby village. The food may be cook improperly to cause infection.Disease can be acquired from water, soil, animals, plants, and human to human interaction.

Possible fugal infection in tropical jungle in Indonesia

2008-01-26T19:10:00.000-08:00

1. Superficial mycoses
Tinea versicolor or pityriasis versicolor

Description :
Tinea versicolor or pityriasis versicolor is a common skin infection caused by Malassezia furfur. This yeast is normally found on the human skin and only becomes troublesome under certain circumstances, such as a warm and humid environment. In tropical climate, the condition is more common than in temperate zones and as many as 40% of some apparently healthy populations may be affected.

Microscopic: tinea versicolor, "spaghetti and meatballs" appearance
Taken from http://missinglink.ucsf.edu/lm/DermatologyGlossary/img/Dermatology%20Glossary/Glossary%20Clinical%20Images/KOHtineaversicolor.jpg

Transmission:
The development of disease is thought to follow a shift in the balance between the host and resident yeast flora. There are many factors contributing to this change. Infection is more prone to be caused by environmental risk factors and individual host susceptibility such as genetic susceptibility. Most of the transmission is through direct contact with the yeast.

Signs and Symptoms:
· Generally oval or irregularly-shaped spots of 1/4 to 1 inch in diameter, often merging together to form a larger patch
· Occasional fine scaling of the skin producing a very superficial ash-like scale
· Pale, dark tan, or pink in color, with a reddish undertone that can darken when the patient is overheated, such as in a hot shower or during/after exercise
· Sharp border
· commonly affect the back, underarm, upper arm, chest, lower legs, and neck. Occasionally it can also be present on the face.
· In people with dark skin tones, pigmentary changes such as hypopigmentation (loss of color) are common, while in those with lighter skin color, hyperpigmentation (increase in skin color) are more common. These discolorations have led to the term "sun fungus".

Treatment:
· Topical antifungal medications such as selenium sulfide, ketoconazole, clotrimazole, miconazole or terbinafine is applied to dry skin and washed off after 10 minutes, repeated daily for 2 weeks.
· Oral antifungal prescription only medications include ketoconazole or fluconazole in a single dose, or ketoconazole daily for 7 days, or itraconazole daily for 3-7 days. The single-dose regimens can be made more effective by having the patient exercise 1-2 hours after the dose, to induce sweating. The sweat is allowed to evaporate, and showering is delayed for a day, leaving a film of the medication on the skin.

Prevention:Infection may be reduced by intermittent application of topical agents (such as tea tree oil) or adding a small amount of anti-dandruff shampoo to water used for bathing.

2. Cutaneous mycoses
Dermatophytosis

Description:
Dermatophytosis is caused by fungi in the genera Microsporum, Trichophyton and Epidermophyton. Belonging to pathogenic members of the keratinophilic (keratin digesting) soil fungi. Microsporum and Trichophyton are human and animal pathogens. Epidermophyton is a human pathogen.Dermatophytes grow best in warm and humid environments and are, therefore, more common in tropical and subtropical regions. The geographic distribution varies with the organism. M. canis, M. nanum, T. mentagrophytes, T. verrucosum and T. equinum occur worldwide. T. simii (found in monkeys) occurs only in Asia.

Transmission :
Infection occurs by contact with arthrospores (asexual spores formed in the hyphae of the parasitic stage) or conidia (sexual or asexual spores formed in the “free living” environmental stage). Infection usually begins in a growing hair or the stratum corneum of the skin. Dermatophytes do not generally invade resting hairs, since the essential nutrients they need for growth are absent or limited. Hyphae spread in the hairs and keratinized skin, eventually developing infectious arthrospores. Transmission between hosts usually occurs by direct contact with a symptomatic or asymptomatic host, or direct or airborne contact with its hairs or skin scales. Geophilic dermatophytes, such as M. nanum and M. gypseum, are usually acquired directly from the soil rather than from another host.

Signs and Symptoms:

Treatment:
Tinea capitis, tinea barbae and tinea faciei are generally treated with systemic antifungals. Topical lotions or shampoos are sometimes used to decrease shedding of fungi and spores. Tinea corporis can usually be treated with nonprescription antifungals. Prescription drugs may be required if the fungus infects the hairs and recrudescence occurs. Tinea manuum is usually treated with topical drugs and emollients.

Prevention:
Control of the disease in animals can prevent some cases of dermatophytosis in humans. Infected animals should be treated and the premises and fomites should be disinfected. Gloves and protective clothing should be used during contact with infected animals. Such contact should be avoided as much as possible. Similar measures can prevent infections with anthropophilic dermatophytes.

3. Subcutaneous mycoses
Chromoblastomycosis

Description:
The fungi that cause subcutaneous mycoses normally reside in soil or vegetation. They enter the skin or subcutaneous tissue by traumatic inoculation with contaminated material. In general, the lesions become granulomatous and expand slowly from the area of implantation. Extension via the lymphatic draining the lesion is usually slow. These mycoses are usually confined to the subcutaneous tissues, but in rare cases they become systemic and produce life-threatening disease. Chromoblastomycosis is mainly caused by phialophora verrucosa, fonsecaea pedrosoi, Rhinocladiella aquaspersa, Fonsecaea compacta, and Cladophilophora carrionii.

Cladosporium (Cladophialophora) carrionii, magnified 475X. The C. carrionii fungus is a common cause of chromoblastomycosis infections, and is particularly prevalent in arid and semi-arid areas, most often in tropical and subtropical zones
Taken from http://pathmicro.med.sc.edu/mycology/cladophialophora.jpg

Transimission:
The organisms responsible for Chromoblastomycosis are found in the soil, endemic in the tropicas. They are introduced into tissues by trauma from eg :thorns or splinters. Transmission from person to person does not occur.

Signs and symptoms:
· a small red papule (skin elevation)
· Painless lesion
· Satellite lesion form if patient scratches
· Formation of cauliflower-like warty swellings which spread over the years
· Foul discharge may occur in secondary infection
· Nodular pattern becomes tumorous, large, rough and ulcerating
· Several complications may occur

Treatment:
Sugical excision with wide margins is the therapy of choice for small lesions. Chemotherapy with flucytosine or itraconazole may be efficacious for larger lesions. Local applied heat is also beneficial. Relapse is common.

Prevention:
There is no known preventative measure aside from avoiding the traumatic inoculation of fungi.

4. Endemic mycoses
Histoplasmosis

Description:
Histoplasmosis, also known as Darling's disease, is caused by the dimorphic fungus Histoplasma capsulatum. This sexually reproducing fungal species is naturally found in the soil (often associated with avian and bat guano) and is endemic in tropical areas of the world. The fungus exists in a hyphal form in the soil and exists as small conidia in the human host. In C.albicans both the yeast and hyphal forms are important in virulence while in H.capsulatum (and other systemic mycoses) only the conididal form is associated with disease. Inhalation of the infectious propagules by normal healthy individuals usually results in a self limiting sub-clinical infection.

Asexual spores (conidia). Tuberculate macroconidia of Histoplasma capsulatum.
Taken fromhttp://aapredbook.aappublications.org/week/061_02.jpg

Transmission:
Histoplasmosis can be transmissed through breathing in airborne spores. The initial infection often occurs without causing symptoms, and most persons usually will not develop subsequent disease, unless the exposure was heavy.
Long-term smokers and those with preexisting lung disease may be at higher risk for developing the disease.
People with severely damaged immune systems are vulnerable to a very serious disease known as progressive, disseminated histoplasmosis. Nationwide, about 5 percent of people with AIDS will develop histoplasmosis. In geographic areas where the fungus is common, people with AIDS are at higher risk for disseminated histoplasmosis.

Signs and symptoms:
· symptoms start within 3 to 17 days after exposure; the average is 12-14 days. Most affected individuals have clinically-silent manifestations and show no apparent ill effects.
· The acute phase of histoplasmosis is characterized by non-specific respiratory symptoms, often cough or flu-like. Chest X-ray findings are normal in 40-70% of cases.
· Chronic histoplasmosis cases can resemble tuberculosis; disseminated histoplasmosis affects multiple organ systems and is fatal unless treated.
· Severe infections can cause hepatosplenomegaly, lymphadenopathy, and adrenal enlargement.
· Lesions have a tendency to calcify as they heal.
· Ocular histoplasmosis damages the retina of the eyes. Scar tissue is left on the retina which can experience leakage, resulting in a loss of vision.

Treatment:
Antifungal medications are used to treat severe cases of acute histoplasmosis and all cases of chronic and disseminated disease. Typical treatment of severe disease first involves treatment with amphotericin B, followed by oral itraconazole. In many milder cases, simply itraconazole is sufficient. Asymptomatic disease is typically not treated. Past infection results in partial protection against ill effects if reinfected.

Prevention:
Avoid areas where disease is prominent.

References:

http://rds.yahoo.com/_ylt=A0oGkm.8ZplH.GsA13BXNyoA;_ylu=X3oDMTExZnBsOTUwBHNlYwNzcgRwb3MDNQRjb2xvA3NrMQR2dGlkAwRsA1dTMQ--/SIG=121lfb013/EXP=1201322044/**http://en.wikipedia.org/wiki/Tinea_versicolor

http://books.google.com.sg/books?id=VZfAfeyo4aoC&pg=PA261&dq=pityriasis+versicolor+transmission&sig=M9a38jxgbHc3e1A3wbi98mFkkYM

http://www.cfsph.iastate.edu/Factsheets/pdfs/dermatophytosis.pdf

http://en.wikipedia.org/wiki/Chromoblastomycosis

http://rds.yahoo.com/_ylt=A0oGklJAiJlHqTgAv0xXNyoA;_ylu=X3oDMTExNmthcDFsBHNlYwNzcgRwb3MDNARjb2xvA3NrMQR2dGlkAwRsA1dTMQ--/SIG=11vemseqp/EXP=1201330624/**http://en.wikipedia.org/wiki/Histoplasmosis

http://rds.yahoo.com/_ylt=A0oGkyPmj5lHmRMAJvZXNyoA/SIG=17ujo8kmk/EXP=1201332582/**http%3a//66.218.69.11/search/cache%3fei=UTF-8%26p=Aspergillosis%2bprevention%26fr=yfp-t-501%26fp_ip=SG%26u=www.mayoclinic.com/health/aspergillosis/DS00950/DSECTION%253D9%26w=aspergillosis%2bprevention%26d=DuVBJnDuQHvH%26icp=1%26.intl=us

Done be: Liu Qian and Dorene

MMIC PBL 2

2008-01-26T01:43:00.000-08:00

Viral Infections

In the jungle environment, viral transmission can occur through soil, contaminated food or water, breathing in of viruses in the form of aerosals and by insect vectors such as mosquitoes e.g. dengue. The rate or speed of transmission of virus infections depends on factors that include population density, the number of susceptible individuals, the quality of health care and the weather. The following are some examples of Virus that can be found in the jungle envirnment.

Arboviruses

Arboviruses are transmitted through arthropod vectors. Arthropods refer to the
insects, arachnids, crustaceans and others. The arthropods transmit the virus upon biting the host, allowing the virus to enter the bloodstream causing viraemia.

Avian influenza A

Description:

Influenza, commonly known as flu, is an infectious disease caused by RNA viruses of the family Orthomyxoviridae. Avian influenza is an infection caused by birds which are infected with flu viruses. These influenza viruses occur naturally among birds. Wild birds worldwide carry the viruses in their intestines, but usually do not get sick from them. Infected birds shed influenza virus in their saliva, nasal secretions, and feces. Susceptible birds become infected when they have contact with contaminated secretions or excretions from these infected birds. Influenza A viruses have also infected many different animals including ducks, chickens, pigs, whales, horses, and seals. However, certain subtypes of influenza A virus are specific to certain species, except for birds, which are hosts to all known subtypes of influenza.

Transmission:

Direct from birds or from avian virus-contaminated environments to people or through an intermediate host, such as a pig. The influenza viruses are transmitted from wild aquatic birds to domestic poultry giving rise to human influenza pandemics.

Signs and Symptoms:

Conjunctivitis, influenza-like illness symptoms, severe respiratory illness, nausea, vomiting and neurologic changes.
Treatment: Antiviral medication of oseltamivir is administered.
Prevention: Persons exposed to avian influenza A-infected or potentially infected poultry are recommended to follow good infection control practices including careful attention to hand hygiene and to use personal protective equipment. In addition, they should be vaccinated against seasonal influenza and should take influenza antiviral agents for prophylaxis. Exposed persons should be carefully monitored for symptoms that develop during and in the week after exposure to infected poultry or to potentially avian influenza-contaminated environments. Also, humans should avoid consumption of poultry from the nearby villages as they face a high risk of influenza infection.

Influenza Virus- Taken from http://www.fsu.edu/

Chikungunya Virus

Description:

Chikungunya is an alphavirus in the Togaviridae family of viruses. It is a viral disease that is almost self limiting and rarely fatal. The symptoms are similar to those of dengue. However, unlike dengue, there is no hemorrhagic or shock syndrome form.

Transmission:

Spread through bites from Aedes aegypti and Culex mosquitoes.

Signs and Symptoms:

Fever which can reach 39°C, a petechial or maculopapular rash usually involving the limbs and trunk, and arthritis affecting multiple joints. The fever typically lasts for two days and then comes down abruptly. However, other symptoms, namely joint pain, intense headache, insomnia, vomiting, epitaxis and an extreme degree of prostration last for a variable period; usually for about 5 to 7 days.

Treatment:

Chloroquine is gaining ground as a possible treatment for the symptoms associated with chikungunya and as an antiviral agent to combat the Chikungunya virus.

Prevention :

Protection against any contact with the disease-carrying mosquitoes. These include using insect repellents with substances like DEET (N, N’-Diethyl-3-methylbenzamide), icaridin and PMD (p-menthane-3,8-diol). Mosquito nets can be used to protect those who may rest during the day. The effectiveness of such nets can be improved by treating them with permethrin (pyrethroid insecticide). Wearing bite-proof long sleeves and trousers also offers protection. In addition, garments can be treated with pyrethoids, a class of insecticides that often has repellent properties. Securing screens on windows and doors will help to keep mosquitoes out of the house. Mosquito coils can be used too. Mosquito control is also especially important by draining water from coolers, tanks, barrels, drums and buckets and emptying coolers when not in use etc.

Chikungunya Virus- Taken from http://www.flickr.com/

West Nile Virus

Description:

Arbovirus that spreads to people from the bite of the mosquito infected with the virus. The mosquitoes are active under warm conditions and temperate climates like Asia’s. Thus, the West Nile virus infection generally occurs under these conditions .

Transmission:

Mosquitoes get infected with West Nile virus by feeding on infected birds. Once infected with the virus, a mosquito will transmit the virus to other animals or birds when they take another blood meal.

Signs and Symptoms:

Most people infected with West Nile virus do not often become ill. If they do, they have mild illness with fever, headache, eye pain, muscle aches, joint pain, a rash on the trunk, swollen lymph nodes, nausea and vomiting. Symptoms of severe illness include extreme muscle weakness, inflammation of the brain (encephalitis), paralysis, and coma. Symptoms usually occur 3 to 15 days after an infected mosquito bites a person.

Treatment:

There is no specific treatment for West Nile virus infection. A physician may provide treatment to relieve the symptoms of the illness. In severe cases hospitalization may be required.

Prevention:

West Nile control is achieved through mosquito control, by elimination of mosquito breeding sites, larviciding active breeding areas and encouraging personal use of mosquito repellents containing DEET.

West Nile Virus- Taken from http://en.wikipedia.org/wiki/West_Nile_virus

Flavivirus

It belongs to a family of viruses transmitted by mosquitos and ticks that cause some important diseases including dengue, yellow fever, tick-borne encephalitis virus, and Japanese encephalitis (JE) virus. It causes rare viral infections and it primarily occurs in tropical areas of the world.

Japanese encephalitis virus

Description:

Japanese encephalitis is a mosquito-borne viral disease. In subtropical and tropical regions, risk of infection is associated with the rainy season. In tropical areas, sporadic cases may occur at any time of the year. Disease is endemic and epidemic in Asia such Indonedia,Japan,Hong Kong, Malaysia. The Japanese encephalitis virus (JEV) infects the lumen of the endoplasmic reticulum and rapidly accumulates substantial amounts of viral proteins for the JEV.

Transmission:

The virus is transmitted by various mosquitoes of the genus Culex. It infects pigs, various wild birds and humans. Mosquitoes become infective after feeding on viraemic pigs or birds.

Signs and Symptoms:

Most infections are asymptomatic. Symptomatic ones begin clinically as a flu-like illness with headache, fever, and often gastrointestinal symptoms. Confusion and disturbances in behaviour also may occur. It may progress to encephalitis, and in one third of cases, the illness may be fatal. Another one third of cases survive with serious neurologic after effects such as paralysis or other forms of brain damage.

Treatment:

There is no specific treatment for Japanese encephalitis. Antibiotics are not effective against viruses, and no effective anti-viral drugs have been discovered.

Prevention:

Japanese Encephalitis vaccination and wear mosquito repellents containing DEET as an active ingredient. Main protection rests in taking all possible care against being bitten. The mosquitoes, which transmit Japanese B Encephalitis tend to bite mainly in the evening time though day biting in shady areas may also occur.

Dengue virus

Description:

The dengue virus causes dengue and dengue hemorrhagic fever. The dengue virus is composed of single-stranded RNA, and it has four serotypes, known as DEN-1, 2, 3, and 4.

Transmission:

The transmission cycle of dengue virus by the mosquito Aedes aegypti begins with a dengue-infected person. The person will have viremia that lasts for about five days. During the viremic period, an uninfected female Aedes aegypti mosquito bites the person and ingests blood that contains dengue virus. The mosquito then bites a susceptible person and transmits the virus to him or her, as well as to every other susceptible person the mosquito bites for the rest of its lifetime. The virus is inoculated into humans with the mosquito saliva. It localizes and replicates in various target organs, for example, local lymph nodes and the liver. The virus is then released from these tissues and spreads through the blood to infect white blood cells and other lymphatic tissues.

Signs and Symptoms:

Manifestation starts with a sudden onset of fever, with severe headache, muscle and joint pains (myalgias and arthralgias) and rashes. The dengue rash is characteristically bright red petechia and usually appears first on the lower limbs and the chest; in some patients, it spreads to cover most of the body. There may also be gastritis with some combination of associated abdominal pain, nausea, vomitting or diarrhoea.

Treatment:

The mainstay of treatment is supportive therapy. Increased oral fluid intake is recommended to prevent dehydration. Supplementation with intravenous fluids may be necessary to prevent dehydration and significant concentration of the blood if the patient is unable to maintain oral intake. A platelet transfusion is indicated in rare cases if the platelet level drops significantly (below 20,000) or if there are significant bleeding.

Prevention:

Mosquito control is the primary prevention of dengue. This can be done through the elimination or reduction the mosquito vector for dengue. Public spraying for mosquitoes is the most important aspect of this vector. Application of larvicides such as Abate® to standing water is more effective in the long term control of mosquitoes.

Dengue Virus. Taken from http://www.topblog.ws/

Tick-borne encephalitis virus(TBEV)

Description:

TBE is an important infectious disease of in many parts of Europe, the former Soviet Union, and Asia, corresponding to the distribution of the ixodid tick reservoir. It is a human viral infectious disease involving the central nervous system. The virus can infect the brain (encephalitis), the membrane that surrounds the brain and spinal cord (meningitis) or both (meningoencephalitis).

Transmission:

It is transmitted by the bite of infected deer ticks or (rarely) through the non-pasteurized milk of infected cows.

Signs and Sympoms:

The incubation period of TBE is usually between 7 and 14 days and is asymptomatic. Fever, malaise, anorexia, muscle aches, headache, nausea, and/or vomiting follows after that.

Treatment:

Hospitalization and supportive care based on syndrome severity. Anti-inflammatory drugs, such as corticosteroids, may be considered under specific circumstances for symptomatic relief. Intubation and ventilatory support may be necessary. There are four main catgeories of treatment for TBE. Phosphrenyl, both a therapeutic and prophylactic agent for TBE, interferon treatment (like interferon for Hepatitis C), antibiotic treatment for possible tickborne coinfections and phytotherapy.

Prevention:

TBEV infection can be prevented by using insect repellents and protective clothing to prevent tick bites. A vaccine is available in some disease endemic areas.

Ixodes dammini - Taken from
http://rds.yahoo.com/_ylt=A0S020mibJtHpIYAJl.jzbkF/SIG=125jigaog/EXP=1201454626/**http://www.cdc.gov/ncidod/EID/vol3no2/telford.htm

Nipah virus

Description:

Nipah virus is a newly recognized zoonotic virus. Nipah is closely related to another newly recognized zoonotic virus, called Hendra virus. Both Nipah and Hendra are members of the virus family Paramyxoviridae. Certain species of fruit bats are the natural hosts of both Nipah and Hendra viruses

Transmission:

From animal to animal. Animal to human transmission is uncertain, but appears to require close contact with contaminated tissue or body fluids from infected animals. Nipah antibodies have been detected in pigs, other domestic and wild animals. Despite frequent contact between fruit bats and humans there is no serological evidence of human infection among bat carers.

Signs and Symptoms:

"Influenza-like" symptoms, high fever and muscle pains (myalgia). The disease may progress to inflammation of the brain (encephalitis) with drowsiness, disorientation, convulsions and coma.

Treatment:

No drug therapies have yet been proven to be effective in treating Nipah infection. Treatment relies on providing intensive supportive care. However, there is some evidence that early treatment with the antiviral drug, ribavirin, can reduce both the duration of feverish illness and the severity of disease.

Prevention:

Humans should avoid animals that are known to be infected and use appropriate personal protective equipment devices when it is necessary to come into contact with potentially infected animals.

Nipah virus. Taken from http://www.antropozoonosi.it/

References:

Arthropod Cell Culture Systems. (2006). Mosquito Cell Lines. Retrieved January 20, 2008 from http://books.google.com/books?id=ACTMENR2U90C&pg=PA31&lpg=PA31&dq=virus+in+forest&source=web&ots=eE4RtzIhwP&sig=jRYypyuK5gQM4Vx6MJH7GE0864c

eMedicine from WebMD. (2008). Viral Hemorrhagic Fevers. Retrieved January 22, 2008 from http://www.emedicine.com/ped/topic2406.htm

Tropical Infectious Diseases. (2006). Arboviral diseases. Retrieved January 23, 2008 from http://books.google.com/books?id=YKJlKP16_CwC&pg=PA279&lpg=PA279&dq=virus+in+forest&source=web&ots=rD1H19Gm1W&sig=bpcLcD_cxUOy2_9XVW5L2LFr-kg#PPA127,M1

Wikipedia, the free Encyclopedia. (2008). Tick-borne encephalitis viruses. Retrieved January 23, 2008 from http://en.wikipedia.org/wiki/Tick-borne_meningoencephalitis

Wikipedia, the free Encyclopedia. (2008). West Nile Virus. Retrieved January 23, 2008 from http://en.wikipedia.org/wiki/West_Nile_virus

Done by Cassandrea Teng, Sasikala S. and Vinodhini Jayaram

Protozoal Diseases
These diseases are commonly found spread in the jungles and forests through sources such as water (from the river, lakes), food (from infected animals) and mosquitoes. Most of the diseases listed below are found in Indonesia forests whereas others are found worldwide.

Giardiasis
• Diarrheal illness caused by the protozoa Giardia lamblia.
• Description: Giardiasis is an infection of the small intestine caused by Giardia lamblia, a flagellate protozoan. This disease is prevalent among people in the jungles and forests.
• Transmission: Infection usually occurs through ingestion of G. cysts in water (unfiltered drinking-water or recreational water, i.e. water in lakes, rivers, ponds, or streams that can be contaminated with feces from humans or animals) contaminated by the faeces of infected humans or animals.
• Clinical signs & symptoms: Anorexia, nausea, chronic watery diarrhoea, abdominal cramps, bloating, frequent loose greasy stools, fatigue and weight loss.
• Prevention: Avoid drinking unfiltered or recreational water. Boil water for longer periods, at least twenty minutes to destroy any heat-resistant cysts.
• Treatment: Metronidazole or quinacrine hydrochloride.

Giardia lamblia

Taken from:
http://www.pathobio.sdu.edu.cn/sdjsc/webteaching/Course/webteach/Protozoan/Giardia-20lamblia/GiardiaTroph(1).jpg

Amebiasis
• Intestinal disease caused by the protozoa (amoeba) Entamoeba histolytica.
• Description: In amebiasis, the commensal amoeba produces proteolytic enzymes that enable penetration of intestinal mucosa and invasion of other parts of the body. This produces flask-shaped ulcers “ameboma” in the liver, lungs & brain.
• Transmission: Fecal-oral transmission occurs through ingestion of mature cyst through contaminated water (in this case, from lakes in the jungle).
• Clinical signs & symptoms: Fulminating dysentery, diarrhea, weight loss, fatigue, abdominal pain, and amebomas.
• Prevention: Avoid drinking untreated water and observe good hygiene practices such as hand-washing. Vegetables should be cooked before consumption as some villages use human feces as fertilizers for their crops.
• Treatment: Metronidazole for the treatment of intestinal amebiasis or hepatic abscess. Asymptomatic patients are treated with a lumenal amoebicide i.e. paromomycin.

E. histolytica

Taken from: http://www.sfda.gov.sa/NR/rdonlyres/E99EC3AD-9B45-4BF3-80BF-251F41E1D1BF/439/Ehistolytica05.jpg

Cryptosporidiosis
• Diarrheal disease caused by the protozoa, Cryptosporidium parvum.
• Description: This disease is caused by microscopic parasites of Cryptosporidium. The oocysts excyst in the small intestine of an infected person or animal, where the trophozoites attach to the gut wall. The parasite is protected by an outer shell that allows it to survive outside the body for long periods of time and makes it very resistant to chlorine-based disinfectants.
• Life cycle of Cryptosporidium parvum:

Taken from: http://www.dpd.cdc.gov/dpdx/images/ParasiteImages/AF/Cryptosporidiosis/Cryptosporidium_LifeCycle.gif
Oocysts release sporozoites, which release trophozoites. Several stages ensue, with the formation of schizonts and merozoites. Finally, micrgametes and macrogametes form. They unite to produce a zygote, which differentiates into oocysts.
• Transmission: Fecal-oral transmission of oocysts. Once an animal or person is infected, the parasite lives in the intestine and passes in the stool. Cryptosporidium is found in soil, water, or surfaces that have been contaminated with the infected human or animal feces e.g. recreational water contaminated with Cryptosporidium parvum.
• Clinical signs & symptoms: Stomach cramps or pain, dehydration, nausea, vomiting, fever and weight loss.
• Prevention: Avoid drinking unfiltered or recreational water. Purification of water supply such as filtration can help to remove any cysts in the water. These cysts are resistant to chlorination.
• Treatment: Paromomycin is effective in reducing diarrhea.

Malaria
• Blood-borne disease caused by the protozoa, Plasmodium falciparum and Plasmodium vivax.
• Description: Plasmodium falciparum is dangerous because it digests the red blood cell's hemoglobin and also, it changes the adhesive properties of the cell it inhabits. This change in turn causes the cell to stick to the walls of blood vessels. It becomes especially dangerous when the infected blood cells stick to the capillaries in the brain, obstructing blood flow, a condition called cerebral malaria. P. vivax and P. falciparum are found predominantly in Southeast Asia.
• The Life Cycle of Malaria Parasite:

Taken from: http://www.lbl.gov/MicroWorlds/xfiles/malariawhatis.html

The life cycle of the malaria parasite begins when an infected mosquito transmits malaria sporozoites to a new host. The sporozoites travel to the liver, invade hepatocytes and multiply thousands of times over the following two weeks before rupturing out of the liver into the blood stream. During the 1st 48 hours after infecting a erythrocyte, the parasite undergoes several phases of development.

First phase: Ring stage in which the parasite begins to metabolize hemoglobin.
Second phase: Trophozoite stage during which the parasite metabolizes most of the hemoglobin, gets larger, and prepares to reproduce more parasites.
Last stage: The parasite divides asexually to form a multinucleated schizont. The erythrocyte bursts open and the parasites are dispersed to infect more red blood cells.

• Transmission: Plasmodium falciparum and Plasmodium vivax are transmitted by the bites of about 60 species of mosquitoes in the genus Anopheles.

Taken from: http://www.lbl.gov/MicroWorlds/xfiles/malariawhatis.html

• Clinical signs & symptoms: Moderate to severe shaking chills, profuse sweating as body temperature falls, high fever, general feeling of unease and discomfort (malaise), headache, nausea, vomiting and diarrhea.
• Prevention: Bednets, insecticides, and antimalarial drugs are effective. Apply mosquito repellent.
• Treatment: Chloroquine, sulfadoxine-pyrimethamine (Fansidar®), mefloquine (Lariam®), atovaquone-proguanil (Malarone®) and quinine.

Cyclosporaisis
• Diarrheal disease caused by the protozoa, Cyclospora cayetanensis.
• Description: Cyclosporaisis is endemic in tropical and sub-tropical regions, especially when the disease is in its best season for spreading. These warmer temperatures are needed to get oocysts to sporulate rapidly. The only hosts C. cayetanensis uses are humans. The protozoan lives out its lifecycle intracellularly within the host’s epithelial cells and gastrointestinal tract.
• Transmission: Occurs through the oral-fecal route, and begins when a person ingests oocysts in fecally contaminated food or water.
• Clinical signs & symptoms: Prolonged watery diarrhea, abdominal cramping, weight loss, anorexia, myalgia, and occasionally vomiting and/or fever.
• Prevention: Avoid drinking water from river and lakes without boiling and add water sterilization tablets to the river water before drinking.
• Treatment: Seven-day course of oral trimethoprim-sulfamethoxazole.

Cyclospora cayetanensis

Taken from: http://en.wikipedia.org/wiki/Cyclospora_cayetanensis

Toxoplasmosis
• Disease caused by the protozoa, Toxoplasma gondii.
• Description: The parasite infects most warm-blooded animals, including humans, but the primary host is the felid (cat) family. The cycle in the cat begins with ingestion of cysts in raw meat, e.g. mice. There is a possibility that T. gondii in the cat’s feces get passed on to intermediate hosts such as pig and lambs grazed in soil contaminated with infected cat feces. Human infection occurs from consuming under-cooked meat from these animals.
• Transmission: By the ingestion of raw or partly cooked meat, especially pork, lamb, transmitted by contaminated cat feces, Drinking water contaminated with Toxoplasma gondii.
• Clinical signs & Symptoms: Asymptomatic.
• Prevention: Ensure that all food are cooked thoroughly and drink treated water.
• Treatment: Combination of pyrimethamine and sulfadiazine, trisulfapyrimidines, spiramycin, clindamycin, trimethoprim sulfamethoxazole.

Toxoplasma gondii

Taken from:
http://www.que.at/images/aktuelles/toxoplasmose/toxoplasma_gondii.jpg

Balantidiasis
• Disease caused by the protozoa, Balantidium coli.
• Description: Balantidium coli is a species of ciliate protozoan. Cysts are the infective stage, responsible for transmission of balantidiasis. The host acquires cysts through ingestion of contaminated food or water. After ingestion, excystation occurs in the small intestine, and the trophozoites colonize the large intestine.
• Transmission: Occurs through fecal-oral, person-to-person and water transmission. Hosts include pigs, wild boars, rats, primates (including humans), horses. Pigs are the most important reservoir hosts, though they show few symptoms. There is a possibility that the soldiers come into contact with the feces of an infected wild boar in the jungle or drinking of contaminated water.
• Clinical signs & Symptoms: Persistent diarrhea, occasionally dysentery, abdominal pain, and weight loss
• Prevention: Drink treated water and reduce contact with feces.
• Treatment: Tetracycline with iodoquinol and metronidazole as alternatives.

Balantidium coli shown in wet mount

Taken from: http://www.flickr.com/photos/euthman/268022978/

References:

Levinson, W. (2006). Revise of Medical Microbiology and Immunology. USA: The McGraw-Hill Companies.

International Travel and Health. (2005). CHAPTER 5: Infectious Diseases of Potential Risk for Travellers. Retrieved January 19, 2008, from http://whqlibdoc.who.int/publications/2005/9241580364_chap5.pdf

Centre for Disease Control. (2004). Giardiasis. Retrieved January 19, 2008, from http://www.cdc.gov/ncidod/dpd/parasites/giardiasis/factsht_giardia.htm

Centre for Disease Control. (2007). Cryptosporidium Infections. Retrieved January 25, 2008, from http://www.cdc.gov/ncidod/dpd/parasites/cryptosporidiosis/factsht_cryptosporidiosis.htm

MayoClinic. (2006). Malaria. Retrieved January 25, 2008, from http://www.mayoclinic.com/health/malaria/DS00475/DSECTION=2

MicroWorlds. (2001). What is Malaria? Retrieved January 24, 2008, from http://www.lbl.gov/MicroWorlds/xfiles/malariawhatis.html

K-State. (2007). Cyclospora cayetanensis. Retrieved January 25, 2008, from http://www.k-state.edu/parasitology/cyclospora/cyclospora.html

Wikipedia. (2008). Toxoplasmosis. Retrieved January 25, 2008, from http://en.wikipedia.org/wiki/Toxoplasmosis

Carlo Denegri Foundation. (2008). Balantidium coli. Retrieved January 24, 2008, from http://www.cdfound.to.it/HTML/bal1.htm

MedicineNet. (2004). Definition of Balantidium. Retrieved January 25, 2008, from http://www.medterms.com/script/main/art.asp?articlekey=31339

Sodeman, W. J. (2002). Intestinal Protozoa: Amebas. Retrieved January 25, 2008, from http://gsbs.utmb.edu/microbook/ch079.htm

Medical Microbiology Lecture Notes

Done by: Sally and Shu Hui TG02

2007-12-09T09:46:00.001-08:00

Case	Suspected Diagnosis	Suspected Microorganisms	Lab Investigations
Khong Fay Seah	UTI	Escherichia coli Staphylococcus saprophyticus Proteus mirabilis Klebsielle pneumoniae Enterobacter Serratia Pseudomonas aeruginosa Morganella morganii Enterococcus faecalis Chlamydiae	Gram staining to classify the bacteria into gram-positive and gram-negative groups. Urine culture on blood agar, MacConkey, EMB. Antibiotic Susceptibility Test using ampicillin, cephalosporin, aminoglycosides, trimethoprim-sulfamethoxazole, norfloxacin, novobiocin, cefotaxime etc. Biochemical Tests like TSI, Oxidase, Indole, Catalyse, Coaulase, Methyl Red an Urease etc.
Kwan Siew Yan	Enterocolitis (most likely the bacterial type)	Shigella species Enteroinvasive E.Coli (EIEC) Enterohermorrhagic E.coli (EHEC) Salmonella species eg: S. enteritidis, S. typhimurium Campylobacter jejuni Vibro parahaemolyticus Vibro cholerae Clostridium difficile C. perfringens Yersinia species eg: Y. Enterocolitica Bacillus cereus Entamoeba histolytica	Stool culture to isolate and identify any parasites or bacteria Microscopy with gram staining Antibody Susceptibility Test Biochemical Test like oxidase, catalase
Maisy Hong	UTI	Escherichia (E.) coli Staphylococcus saprophyticus Klebsiella Enterococci bacteria Proteus mirabilis ureaplasma urealyticum Mycoplasma hominis Chlamydia Mycoplasma Corynebacterium Pseudomonas aeruginosa	Urine culture Antibiotic Susceptibility Test Staining and Microscopy
Tong Wei Hong	Bronchitis	Respiratory adenovirus Rhinovirus Histoplasma capsulatum Cryptococcus neoformans Pneumocystis jiroveci Mycoplasma pneumoniae Chlamydia pneumoniae Streptococcus pneumoniae Staphylococcus aureus K.pneumoniae P.aeruginosa Haemophilus influenzae Moraxella catarrhalis	Culture using chocolate, blood, Mannitol Salt agar etc. Gram staining EIA Antibiotic Susceptibility Test using penicillin, vancomycin, doxycycline etc. Other Biochemical Test like catalase, oxidase etc.
Wong Fei Hong	Wound Infection	Staphylococcus aureus Enterococci Escherichia coli Pseudomonas aeruginosa Proteus mirabilis Klebsiella pneumoniae Proteus mirabilis Enteric Gram negative bacilli Beta-hemolytic streptococci Streptococcus milleri Streptococcus pyogenes Gram-negative aerobes Methicillin resistant Staphylococcus aureus	Culture of wound using blood agar, BAP, chocolate agar, Xylose Lysine Sodium Deoxycholate (XLD) and MacConkey agar etc. Gram staining and microscopy Biochemical Test like indole, methyl red, cytochrome oxidase, phenylalanine deaminase etc. Others like fungal culture if necessary
Ong Fei Fei	UTI & probably vaginal infection	Causes Vaginal infection: Yeast eg. Candida vulvovaginitis Gardnerella vaginalis, Mobiluncus species, Mycoplasma hominis Trichomonas eg. Trichomonas vaginalis Neisseria Gonorrhoea Chlamydia trachomatis Causes UTI: Most Frequent Enterococci Streptococcus agalactae(Group B Streptococcus) Enterobacteriaceae Pseudomonas Streptococcus pyogens(Group A Streptococccus) Streptococcus aureus S.saprophyticus Candida species. Less frequent Gardenerella vaginalitis Ureaplasma urealyticum Mycoplasma hominis Mobiluncus Leptospira Mycobacterium species Chlamydia trachomatis(males) Other Associated with multisystem disease Salmonella(with gastroenteritis) Schistosoma haematobium Crytococcus neoformans Trichosporon beigelii Trichomonas vaginalis Aspergillus Penicillium Adenovirus Herpes simplex virus	Wet Mount Whiff's Test Chlamydia Rapid Test Gram staining Antibiotic Susceptibility Test using Carbenicillin, Cinoxacin, Lomefloxacin, norfloxacin, or Loracarbef etc.

2007-12-09T08:59:00.001-08:00

Learning Issues for this PBL:

Definition of the possible diagnosis stated for each case
Possible causes
Microorganisms that cause the disease
Different ways of laboratory diagnosis of the microorganisms (i.e. by urine culture and antibiotic sensitivity tests)
Treatment

Case 1 (Shu Hui)

Patient: Khong Fay Seah (27,F)

Key Points:
Possible diagnosis: Urinary Tract Infection

Picture taken from:

http://images.google.com.sg/imgres?imgurl=http://www.supplementnews.org/images/urinary-tract-infection.jpg&imgrefurl=http://www.supplementnews.org/urinary-tract-infection/index.htm&h=396&w=271&sz=84&hl=en&start=1&tbnid=vqS-42dNv8LZyM:&tbnh=124&tbnw=85&prev=/images%3Fq%3Durinary%2Btract%2Binfection%26gbv%3D2%26svnum%3D10%26hl%3Den

UTI is an infection of any part of the urinary tract, consisting the kidneys, ureters, bladder, and urethra. If only the bladder is infected, the condition is known as cystitis. Infection of the kidneys and urethra are known as pyelonephritis and urethritis respectively. An infection occurs when microorganisms, usually from the intestinal tract, find their way into the urethra and multiply there.
- Patient is female
UTI occurs most commonly in women due to a shorter urethra and close proximity of the urethra to the anus. In addition, the vagina may be colonised by the fecal flora.
Complaints of fever, chills and dysuria

Fever and chills are the symptoms of pyelonephritis. The most prominent characterisitic of cystitis is dysuria, i.e. pain on urination.
Urine specimen

Lab requests a urine specimen for culture and antibiotic sensitivity testing in order to confirm diagnosis.

List of possible microoragnisms that can cause UTI

Table 1: Classification of the different bacteria.

Chlamydiae is another microorganism that may cause UTI. However, Chlamydiae cannot be classified as they are obligate intracellular bacteria that cannot be gram-stained.

Investigation required:

1. Microscopy of Gram-stained specimens

Significance:

Screening procedure that helps to determine the presence or absence of bacteria in the urine specimen. Gram-staining also helps to classify bacteria into two major groups-Gram-positive and gram-negative.

A drop of urine is placed on the glass slide and heat-fixed. Gram-staining is done and the slide is viewed under oil immersion. Gram-positive bacteria are seen as violet and gram-negative bacteria appear red or pink. The size, shape, arrangement and no. of types of bacteria in the urine are observed too.
2. Urine Culture

Significance: To obtain a pure culture of the microorganism for identification.

Urine is cultured on:
Differential media

- Blood agar
- supports the growth of many bacteria and detects hemolysis.
Selective and Differential media

- MacConkey agar or eosin-methylene blue (EMB)
- Selects against gram-positive bacteria and differentiates between lactose fermenters and non-fermenters.
- Lactose fermenters form coloured colonies while non-fermenters form colourless colonies.

The plates are incubated at 37°C for 24 hours under aerobic conditions as all the possible microorganisms suggested are either facultative anaerobes or obligate aerobes(pls refer to table 1).

3. Antibiotic Susceptibility Test

Significance:
The pure cultures of bacteria obtained are tested against antibiotics for their sensitivity or resistance to different anti-microbial drugs. This disk diffusion test is used to determine the antibiotics to be used to treat an infection.

A standardised suspension of bacteria is spread across the surface of the Muller-Hinton agar and paper discs containing antibiotics are placed on the surface. After incubation, the diameter of the zone of growth inhibition is measured and the size of the zone of inhibition is directly proportional to the sensitivity of the organism to the antibiotic.

4. Others: Biochemical tests

Significance:

These tests can be used to identify bacteria as different bacteria have different enzymes that can catalyse various chemical reactions.

Table 2 below shows a summary of the laboratory tests expected results (gram-staining in table 1, urine culture, biochemical tests and antibiotic sensitivity tests) for the different microorganisms. By using this table, the bacteria that caused UTI in patient 1 can be identified.

Table 2: Expected results for different laboratory tests.

Chlamydiae cannot be gram-stained. Instead, it can be seen with Giemsa stain. The method of detection of Chlamydiae is by polymerase chain reaction(PCR) test using the urine specimen.

Case 2 (Dorene)

Patient: Kwan Siew Yan (29,F)

Key Points:
- Possible diagnosis: Enterocolitis
- Complaint of diarrhoea
Enterocolitis is the inflammation of both small and large intestine. Enteritis refers to inflamation of the small intestine while colitis refers to inflamation of the large intestine. Symptoms of Enterocolitis include fever, abdominal swelling, nausea, vomitting, Diarrhea, rectal bleeding and sluggishness. In this patient case, she was only reported to have diarrhea.

There are many different type of Enterocolitis : Necrotizing enterocolitis, pseudomembranous enterocolitis and other bacterial enterocolitis (caused by Shigella, Salmonella, and EPEC.)

Necrotizing enterocolitis happens primarily in premature infants, where the small and large intestine under necrosis. The earlier the baby in prematurely born, the later the stages of Enterocolitis. There is no known organism that causes this type of Enterocolitis . As the baby has weak immune system, normal flora that resides in body after ingestion could be the causative agent. However, since in this case, the age of the patient is 29, the Necrotizing Enterocolitis is not the correct diagnosis.

Necrotic Colon (Dead Tissue) (arrow 1)
Source: http://medimages.healthopedia.com/large/necrotizing-enterocolitis.jpg

Pseudomembranous enterocolitis is a combination of both acute inflammation and necrosis of both small and large intestines. It starts with affecting the 1st layer of the intestine, mucosa layer, and slowly enxtend into submucosa and in severe cases, deeper into othe layers. Pseudomembranous enterocolitis is recognized by severe diarrhea that last for a several days, causing dehydration and toxicity in the body on the 1st week (1-7 days). There is also no known cause of the diease but usually occcurs in patient treated with broad-spectrum antibiotics ( eg: clindamycin (Cleocin) and broad-spectrum penicillins and cephalosporins )and in patient after undergoing abdominal surgery.

Bacterial enterocolitis is inflammation of small and large intestine by bacterial infection. Some of the bacteria infects include Campylobacter jejuni in America, or Shigella, Salmonella, and EPEC. They are infected with these bacterial through fecal-oral route, where they accidentally ingested contaminated water or foods such as dairy products containing such bacteria.

List of possible microorganisms that caused enterocolitis:

1. Shigella species
Gram-negative, non-motile, non-spore forming rod-shaped bacteria closely related to Escherichia coli and Salmonella
Due to ingestion of egg and tuna salads, lettuce, milk
Causes fever, abdominal cramping, diarrhea, occassional vomitting

2. Enteroinvasive E.Coli (EIEC)
gram-negative rod-shaped bacterium
due to ingestion of contaminated food and water
Causes fever, watery diarrhea, abdominal cramping, fever
3. Enterohermorrhagic E.coli (EHEC) Eg: E.coli serotype 0157:H7
- Gram-negative rod-shaped bacterium.
- Enterohemorrhagic strain of the bacterium Escherichia coli.
- Due to ingestion of undercooked ground beef, cider causes water diarrhea progressing to bloody diarrhea, abdominal cramping.
- No feveror vomitting seen.
4. Salmonella species eg: S. enteritidis, S. typhimurium
Rod-shaped Gram-negative enterobacteria.
Due to ingestion of eggs, dairy producs, fowl and beef.
Causes diarrhea, fever, vomiting, and abdominal cramps.
5. Campylobacter jejuni
Curved, rod-shaped, Gram-negative microaerophilic, oxidase positive, catalase positive.
Bacteria commonly found in animal feces .
Due to ingestion of contaminated water and unpasteurised milk.
Causes enteritis, which is characterised by abdominal pain, diarrhea, fever, and malaise.

6. Vibro parahaemolyticus
- Curved rod-shaped, Gram-negative oxidase positive, facultatively aerobic bacteria and does not form spores.
- This species is motile, with a single, polar flagellum.
- Due to ingestion of shellfish.
- Causes pain, vomitting, fever, watery diarrhea.
7. Vibro cholerae
Gram negative bacterium with a curved-rod shape.
Due to ingestion of contaminated water.
Causes diarrhea.

8. Clostridium difficile
Gram positive, anaerobic bacillus.
Due to overuse of antibiotics that decreases normal flora.
Causes inflammation of intestine, diarrhea.

9. C. perfringens
Gram-positive, rod-shaped, anaerobic, spore-forming bacterium.
Due to poorly prepared meat and poultry
Causes tissue necrosis, bacteremia, emphysematous cholecystitis, and gas gangrene.

10. Yersinia species eg: Y. Enterocolitica
Gram-negative coccobacillus-shaped bacterium.
Due to ingestion of milk and pork.
Causes fever, severe abdominal pain, diarrhea.

11. Bacillus cereus
Endemic, soil-dwelling, Gram-positive, rod shaped, beta hemolytic bacteria .
Due to ingestion of boiled and fried rice, meats and vegetable.
Causes nausea, vomitting, abdominal cramping, watery diarrhea.

12. Entamoeba histolytica
- Anaerobic parasitic protozoan, part of the genus Entamoeba.
- Due to ingestion of cyst form of E. histolytica in food .
- Causes fulminating dysentery, diarrhea, weight loss, fatigue, abdominal pain, and adenomas.
- Amoeba can actually 'bore' into the intestinal wall, causing lesions and intestinal symptoms
- Microscopy:
- detection of cysts/ova in stool sample
- Gram staining to determine gram positive or gram negative bacteria
- Culture:
- Stool culture in both aerobic and anaerobic environment to isolate and identify presence of parasites and bacteria.
- Antibiotics Susceptibility Test:
- It is used to identify antibiotic treatment. Antibiotics used are:
  
  Penicillin
  Gentamycin
  Ampicillin
  Erythromycin
  Cloxacillin
  Chloramphenicol
  Cefuroxime
  Piperacillin Ceftazidime
  Ciprofloxacin
  Bactrim
  Imipenam
- Other tests:
- Oxidase test and catalase test ; to determining the type of bacteria.
- Occult blood test: determine if bleeding occurs in the intestine.
The first step towards indentification of the bacteria is to culture the bacteria in agar plates (eg: MH agar and Blood agar) in both aerobic condition and anaerobic condition. The suspected bacteria cultured will be use for gram staining and other diagnostic tests.

Gram staining for differentiating between gram positive and gram negative bacteria. A purple colored stain shows that the bacteria is gram positive while a pink stain shows that the bacteria is gram negative.

From microscopic examination, there is a need to examine the morphology of the bacteria using the stained slide. The bacteria can be in the form of bacillus or coci. There is also a possibility of detecting yeast/ ova in the specimen itself.

If needed, other tests, like catalase test, oxidase test will be used to further identify the bacteria.

Case 3 (Sasi)

Patient: Maisy Hong (67,F)

Key Points:
- Fever
- Chills – feeling cold after an exposure to a cold environment; shivering, accompanied by paleness and feeling cold.
Bladder distension
- Bladder distension: Abnormal enlargement of the bladder. It results from an inability to excrete urine, which results in its accumulation.
  
  Distention can be caused by a mechanical or anatomic obstruction, neuromuscular disorder or the use of certain drugs. It is relatively common in all ages and both sexes and is most common in older men with prostate disorders that cause urine retention.
On indwelling catheter
- Indwelling catheter: Any catheter which is inserted into the bladder and allowed to remain in the bladder is called an indwelling catheter. Catheters allow drainage or injection of fluids or access by surgical instruments.
  If the patient has severe distention, an indwelling urinary catheter is inserted to help relieve discomfort and prevent bladder rupture. If more than 700 ml is emptied from the bladder, compressed blood vessels dilate and may make the patient feel faint. Typically, the indwelling urinary catheter is clamped for 30 to 60 minutes to permit vessel compensation.
  
  There are two kinds of indwelling catheters: urethral and supra pubic. A urethral catheter is inserted into the bladder through the urethra. A supra pubic catheter is inserted into the bladder through a hole in the abdomen, a few inches below the tummy button.
  
  As patient is unable to excrete urine, a catheter is inserted into the bladder.
  
  Additional fact about catheterization :
The bladder wall is coated with various mannosylated proteins, such as Tamm-Horsfall proteins (THP), which interfere with the binding of bacteria to the uroepithelium. As binding is an important factor in establishing pathogenicity for these organisms, its disruption results in reduced capacity for invasion of the tissues. Moreover, the unbound bacteria are more easily removed when voiding. The use of urinary catheters (or other physical trauma) may physically disturb this protective lining, thereby allowing bacteria to invade the exposed epithelium.
Possible diagnosis: UTI (clinical symptoms are fever and chills)

In this case the patient might have acquired Urinary Tract Infection due to the insertion of the indwelling catheter. Catheters for men are longer than catheters for women. Because of the location and shorter length of the catheter, women with a urethral catheter are generally more susceptible to urinary tract infections (UTIs) as bacteria from the anus can be passed easily up the urethra with the insertion of the catheter. Also, girls have shorter urethras than guys, and the opening lies closer to the rectum and vagina where bacteria are likely to be.
Bacteria on a catheter can also cause UTI (bladder infection). So, it is important that catheters be clean during insertion and removed/ replaced at a regular basis.

Hence, the presence of a catheter within the urinary tract increases the likelihood of urinary tract infection. It may also increase the difficulty of treating the infection.
If a urinary catheter is left in place for long periods of time, in the form of an indwelling catheter, bacteria will inevitably grow in it. A harmful infection may occur if the number of bacteria becomes large or if specific pathologic bacteria grow in the urinary tract.
Although a variety of bacteria can cause UTIs, most (80 to 90 percent) are due to Eschericia coli, a bacterium that is common in the gastrointestinal tract and is routinely found in stool. Other bacteria that may cause UTIs include species of: Proteus, Klebsiella, Enterococcus and Staphylococcus.
E. coli are normally found in the digestive tract and on the skin around the rectal and vaginal areas. When the bacteria enter the urethra, they can make their way up into the bladder and cause an infection.
Picture taken from http://www.calder.med.miami.edu/

List of possible microorganisms:
The bacterial strains that cause UTIs are the following:
- Escherichia (E.) coli is responsible for between 75% and 90% of uncomplicated cystitis cases in younger women and in more than half the cases in older women (over 50). In most cases of UTI, E. coli, which originates as a harmless microorganism in the intestines, spreads to the vaginal passage, where it invades and colonizes the urinary tract. Some bacteria may be able to invade into deeper tissue in the bladder, where they survive to reinfect the patient after resolution of the previous infection.
- Staphylococcus saprophyticus accounts for 5% to 15% of UTIs, mostly in younger women. Interestingly, infections caused by this bacterium have a seasonal variation, with a higher incidence in the summer and fall than in the winter and spring.
- Klebsiella, Enterococci bacteria, and Proteus mirabilis account for most of remaining bacterial agents that cause UTIs. They are generally found in UTIs in older women.
- Rare bacterial causes of UTIs include ureaplasma urealyticum and Mycoplasma hominis, which are generally harmless organisms.
- Chlamydia and Mycoplasma infections tend to remain limited to the urethra and reproductive system. Unlike E. coli, Chlamydia and Mycoplasma may be sexually transmitted, and infections require treatment of both partners.
- Also, other suspected bacteria include
  - Corynebacterium
  - Pseudomonas aeruginosa
Laboratory Investigation:
- (A"clean catch" urine sample by washing the genital area and a "midstream" sample of urine in a sterile container had been requested).
- (This method of collecting urine helps prevent bacteria around the genital area from getting into the sample and confusing the test results).
  
  Urinalysis - In the urinalysis test, the urine is examined for white and red blood cells and bacteria.
  
  A urinalysis involves a physical and chemical examination of urine. In addition, the urine is spun in a centrifuge to allow sediments containing blood cells, bacteria, and other particles to collect. This sediment is then examined under a microscope.
  
  A urinalysis, then, offers a number of valuable clues for an accurate diagnosis:
  - Simply observing the urine for color and cloudiness can be important.
  - Acidity is measured.
  - White blood cells (leukocytes) are counted. A high count in the urine is referred to as pyuria. (A leukocyte count over 10 per microliter is considered to indicate pyuria.) This is very accurate in identifying the disease when it's present, but it also tests positive in many people who do not have a UTI. Pyuria is usually sufficient for a diagnosis of UTI in nonhospitalized patients if other standard symptoms (or just fever in small children) are also present.
    (Treatment can be started without the need for further tests if the following )
    
    Urinalysis results are present in patients with symptoms and signs of UTIs:
  - A high white cell count.
  - Cloudy urine.
1. Urine culture:

Urine culture uses a urine specimen that is placed on an agar plate, then incubated in the laboratory for 24 to 48 hours.
- It is then examined for the presence of bacterial growth. Urinary tract infection is nearly always caused by a single species of bacteria, notably E. coli.
- Cultures have limitations, however. If a mix of different species is found, the test is considered contaminated and is redone.
- In addition, even if E. coli is identified, variants of this bacteria. Certain types may indicate a higher risk for a second infection, while others may even be protective against recurring infections. Furthermore, some organisms, such as Chlamydia, which is a sexually transmitted organism, may not be detected.
- Even if bacteria are present in the culture, a diagnosis of UTI depends on symptoms and gender:
- The presence in a culture of at least 100,000 bacteria milliliter of any single type of bacterium per milliliter of urine usually provides conclusive evidence of infection in women with symptoms.
- A count of 100,000 bacterial per milliliter in a woman without symptoms indicates asymptomatic bacteriuria. The decision to treat depends on the woman's risk factors for complications.
- In young women with symptoms of cystitis, a diagnosis of infection can reasonably be made with counts as low as 1000 bacteria per milliliter.
- Men are considered to have an infection with a count of only 1,000.
- Sample will have to be spread out onto culture plates to grow the bacteria, to see if there are any in the sample that might be the cause of the infection - this will generally take at least 24 hours. If there are any suspect bacteria there, they will probably need to be identified further and also checked out to ensure that they are not resistant to the effect of antibiotics - at least another 24 hours.
2. Antibiotic susceptibility:
Bacteria are grown in a culture and tested against different antibiotics to see which drug best destroys the bacteria.
- For microbes like Chlamydia and Mycoplasma, special bacterial cultures done. A doctor suspects one of these infections when a person has symptoms of a UTI and pus in the urine, but a standard culture fails to grow any bacteria.
3. Staining and Microscopy:
If physicians suspect that bacteria other than E. coli may be present, a Gram stain is used to help predict the species. This is a staining procedure used to make bacteria visible through a microscope. Many bacteria are categorized by the terms gram-positive and gram-negative.
- Bacteria that turn pink from staining are called gram-negative
- Those that turn blue are called gram-positive.
Escherichia coli is gram negative and the most common cause of UTIs. If physicians suspect that bacteria other than E. coli are causing a UTI, a Gram stain is useful for identifying other species.
- In this process, purple dyes are poured over bacteria that have been spread out thinly on a microscope slide and the cell walls of the bacteria (made out of peptidoglycan) take up the colour.
- If a solvent is then applied to the slide, bacteria which have only got a cell wall still keep their purple colour, but bacteria which have got an extra cell membrane (made out of phospholipid) outside their cell wall quickly lose the purple stain and become colourless; in order to be able to see these bacteria under the microscope a second red stain is then used.
  - Bacteria that manage to keep the original purple dye have only got a cell wall - they are called Gram positive.
  - Bacteria that lose the original purple dye and can therefore take up the second red dye have got both a cell wall and a cell membrane - they are called Gram negative.
  - In this case, for example, Escherichia coli is Gram negative
  - Gram stain not possible for Chlamydia and Mycoplasma as they do not have conventional cell walls at all and specialised techniques are often required to diagnose infections caused by these bacteria.
Case 4 (Cassendrea)

Patient: Tong Wei Hong (68,M)
Key Points:
- Complaints of fever, chills, excessive phlegm & breathing difficulties
- Possible Diagnosis: Bronchitis
Bronchitis is inflammation of the large airways that branch off the trachea (bronchi), usually caused by infection but sometimes caused by irritation from a gas or particle. It generally begins with the symptoms of a common cold which includes runny nose, sore throat, fatigue, chilliness, and back and muscle aches. A slight fever may be present. The onset of cough signals the beginning of acute bronchitis. With viral bronchitis, small amounts of white mucus are often coughed up. When the coughed-up mucus changes from white to green or yellow, the condition may have been complicated by a bacterial infection. Airway hyper reactivity, which is a short-term narrowing of the airways with impairment or limitation of the amount of air flowing into the lungs, is common with acute bronchitis.

The symptoms presented by the patient fit the profile of a person with acute bronchitis. Bronchitis is an inflammation of the main air passages to the lungs.

In bronchitis, areas of the bronchial wall become inflamed and swollen, and mucus increases. As a result, the air passageway is narrowed. Bacteria and viruses are usually present
Source:

List of possible microorganisms that caused bronchitis and their respective laboratory investigation:
- Infectious agents causing acute bronchitis:
  Viruses such as respiratory adenovirus, rhinovirus, influenza A/B
  Fungal infections like Histoplasma capsulatum, Cryptococcus neoformans, Pneumocystis jiroveci, and Coccidioides immitis.
- Bacterial infections such as Mycoplasma pneumoniae and Chlamydia pneumoniae ,Streptococcus pneumoniae, Staphylococcus aureus, K.pneumoniae, P.aeruginosa Haemophilus influenzae, and Moraxella catarrhalis. Yellow or green sputum suggests a bacterial infection.
  - Mycoplasma pneumoniae:
    
    - lack peptidoglycan wall
    - resistant to the effects of penicillins and other beta-lactam antibiotics, which act by disrupting the bacterial cell wall
    - Spread through respiratory droplet transmission
    - Attachment sites include the upper and lower respiratory tract, causing pharyngitis, bronchitis and pneumonia
    - Susceptible to second generation macrolide antibiotics, doxycycline and second generation quinolones
    - M. pneumoniae is grown on Eaton's agar.
  - Chlamydia pneumoniae:
  - common cause of pneumonia
  
  - Symptoms of infection include cough, fever, and difficulties breathing
  - Examination of sputum or the secretions of the respiratory tract may reveal signs of the bacteria
  - most common antibiotic active against Chlamydia pneumoniae is doxycycline
  - Streptococcus pneumonia
    
    - Gram positive, cocci, seen in pairs or chain.
    - When cultured on blood agar plates with added optochin antibiotic disk, they show alpha- hemolytic colonies and a clear zone of inhibition around the disk
    - catalase negative
    - resistant to cephalosporins, macrolides (such as erythromycin), tetracycline, clindamycin and the quinolones
    - susceptible to vancomycin
Source: http://en.wikipedia.org/wiki/Image:Streptococcus_pneumoniae.jpg

The bacterium Streptococcus pneumoniae, a common cause of pneumonia, photographed through an electron microscope
- Haemophilus influenza
- non-motile Gram-negative
coccobacillus
- generally aerobic, but can grow as a facultative anaerobe
- Gram-stained to show Grain - ve, coccobacilli, with no specific arrangement
- growth on blood agar would show the satellitism phenomenon, which is the growth of H. influenzae around a streak of S. aureus line of growth.
- On Chocolate agar, it shows small, convex, smooth, pale, grey or transparent colonies.
- catalase and oxidase positive.
- Staphylococcus aureus
- Gram-positive
coccus, which appears as grape-like clusters when viewed through a microscope
- large, round, golden-yellow colonies, often with β-hemolysis, when grown on blood agar plates.
- facultative anaerobe
- catalase positive and coagulase-positive
- culture in Mannitol Salt Agarà produces yellow-colored colonies
- resistance to penicillin such as methicillin, oxacillin, cloxacillin, dicloxacillin and flucloxacillin
- Moraxella catarrhalis
- gram-negative, aerobic, oxidase-positive
diplococcus
- colonise and cause respiratory tract-associated infection
- produce beta-lactamases and are resistant to penicillin
- Susceptible to fluoroquinolones and most second and third generation cephalosporins.
Virusesàserology tests such as enzyme immunoassay (EIA)
- EIA is another term for ELISA, Enzyme-Linked ImmunoSorbent Assay. It is a biochemical technique used to detect the presence of an antibody or an antigen in a sample. An unknown amount of antigen is affixed to a surface and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme. When a particular substance is added, this enzyme acts on it and converts it to a detectable signal such as fluorescence. This fluorescence will be measured and will indicate amount of anitgen present.
- Sputum Culture: cultures are done on the sputum to detect and identify bacteria or fungi that are causing an infection in the lungs or bronchi
  - A sample of sputum is cultured on a medium that promotes the growth of bacteria or fungi.
  - If no bacteria or fungi grow, the culture is negative.
  - If organisms that can cause infection (pathogenic
    organisms) grow, the culture is positive.
  - The type of bacterium or fungus will be identified with a microscope or by chemical tests.
  - If bacteria or fungi that can cause infection grow in the culture, sensitivity testing may be done to the type of bacteria or fungi present.
Microscopy: fungal stain and gram stain
- The sputum is stained and viewed under the microscope
- The distinctive morphology of the various microorganisms will aid in the identification of the pathogen causing the infection
Antibiotic susceptibility testing
- antibiotic disk diffusion test which uses antibiotic-impregnated wafers to test whether particular bacteria are susceptible to specific antibiotics
- Bacteria are grown on agar plates in the presence of thin wafers containing relevant antibiotics
- If the bacteria are susceptible to the antibiotics, an area of clearing surrounds the wafer where bacteria are not capable of growing (zone of inhibition)
- This can be used for the identification of various microorganisms as different organisms are susceptible to different antibiotics
- By using a combination of antibiotic, identification can be done
Case 5 (Vinodhini)

Patient: Wong Fei Hong (37,M)

Key Points:
Wound Infection

Complaints of fever, swelling around operation wound

A wound is the result of physical disruption of the skin, one of the major obstacles to the establishment of infections bybacterial pathogens in internal tissues. When bacteria breachthis barrier, infection can result (1, 7). The most commonunderlying event for all wounds is trauma. Trauma may be accidentalor intentionally induced. The latter category includes hospital-acquiredwounds, which can be grouped according to how they are acquired,such as surgically and by use of intravenous medical devices

Signs of wound infection consists of redness, warmth, and tenderness in the area of the wound, pus—a foul-smelling, yellowish-white fluid coming from the wound and fever. It is obvious that the patient is having wound infection after his operation.

List of possible microorganisms:

Wound infections may be due to a variety of organisms.Some examples of microorganisms that cause wound infection are:

Staphylococcus aureus

Enterococci

Escherichia coli

Pseudomonas aeruginosa

Proteus mirabilis

Klebsiella pneumoniae

Proteus mirabilis

Enteric Gram negative bacilli.

beta-hemolytic streptococci

Streptococcus

milleri

Streptococcus pyogenes

Gram-negative aerobes

Methicillin resistant Staphylococcus aureus

Laboratory Investigation:

MICROSCOPY

Gram Stain: A Gram stain is done by staining the slide with purple and red stains, then examining it under a microscope.

Note: If many white blood cells and bacteria are seen, it is an early confirmation of infection. The color of stain retained by the bacteria (purple or red), their shape (such as round or rectangular), and their size provide valuable clues as to their identity, and help the physician predict which antibiotics might work best even before the entire test is completed. Bacteria that stain purple are called gram-positive; those that stain red are called gram-negative.

CULTURE:

Note: Bacteria can be grouped into two categories: aerobes and anaerobes. Aerobes are bacteria that need oxygen to live; anaerobes live only where there is no oxygen.

Deep wounds, closed-off from oxygen, are an ideal environment for an anaerobic infection to develop. Foul-smelling odor, gas, or gangrene at the infection site are signs of an infection caused by an anaerobic bacteria. Routine cultures typically only look for aerobic bacteria. If the physician tells the laboratory to include a culture for anaerobes, a portion of the wound sample will be put on culture plates, or in a tube of culture broth, and incubated in a special chamber without oxygen.

Bacteria present in the wound sample will multiply and appear as visible colonies on the plates, or as cloudiness in the tube of broth. They are identified by the appearance of their colonies, the results of biochemical tests, and information from Gram staining part of the bacterial colony.

The most common types of microorganism that causes surgical wound infection are Staphylococcus aureus/MRSA, Streptococcus pyogenes, Enterococci and Pseudomonas aeruginosa.
- Processing of Specimens: Media
Blood agar plate (BAP) -- Staphylococcus aureus, Streptococcus pyogenes
- S. aureus is a Gram-positive coccus, which appears as grape-like clusters when viewed through a microscope and has largened, round, golden-yellow colonies, often with β-hemolysis, when grown on blood agar plates.
- Streptococcus pyogenes is a Gram-positive coccus that grows in long chains depending on the culture method.^[1]
  S. pyogenes displays group A antigen on its cell wall and beta-hemolysis when cultured on blood agar plate. S. pyogenes typically produces large zones of beta-hemolysis, the complete disruption of erythrocytes and the release of hemoglobin, and it is therefore called Group A (beta-hemolytic) Streptococcus
Contains mammalian blood (usually sheep), typically at a concentration of 5–10%. BAP are an enriched, differential media used to isolate fastidious organisms and detect hemolytic activity. β-hemolytic activity will show complete lysis of red blood cells surrounding colony, while α-hemolysis will only partially lyse hemoglobin and will appear green. γ-hemolysis (or non-hemolytic) is the term referring to a lack of hemolytic activity.

http://www.answers.com/topic/agarplate-redbloodcells-jpg

Blood agar plates are often used to diagnose infection. On the left is a positive Staphylococcus infection; on the right a positive Streptococcus culture.
- Morphological studies:
STAINING CHARACTERISTICS:

Prepare a Gram stain of these organisms.
Study the morphological and staining characteristics.

Procedure for Gram staining:

Step	Stain	Purpose	Procedure	Colour of Cells
1.	Crystal violet	As Primary Stain. It colours the cytoplasm violet regardless of cell type	Flood the smear with crystal violet solution for 1 minute Then rinse with water	Violet
2.	Iodine	As a mordant. Iodine combines with crystal violet to form an insoluble complex inside the cell. Complex resists decolourization	Flood the smear above with iodine solution for 30 seconds. Then rinse with water	Violet
3.	95% Ethanol	As a decolourising agent. The violet dye complex is retained by Gram positive cells, but is readily removed from Gram negative cells.	Flood the smear with 95% ethanol for a few seconds e.g, until the violet colour disappears. Then rinse with water.	Gram positive appear violet, while gram negatives appear colourless
4.	Safranin or Dilute carbol fuchsin	As a counter stain. Stain bacterial cells light pink or red. Gram negatives that have been decolourized will absorb safranin, while Gram positives will not.	Flood the smear above with safranin solution for 1 min. Then rinse with water and blot dry.	Gram positives remain violet while Gram negatives appear pink/ red

CULTURE:

Streak the nasal swab for the isolation of Staphylococcus aureus, Streptococcus pyogenes from the normal flora on each of BAP and Chocolate agar plates.
Incubate in CO2 incubator at 37oC for 24 hours.
Study and record the colony characteristics

MacConkey agar (MAC) - Klebsiella pneumonia, Escherichia coli

A selective and differential media used to differentiate between Gram negative bacteria while inhibiting the growth of Gram positive bacteria. The addition of bile salts and crystal violet to the agar inhibits the growth of most Gram positive bacteria, making MacConkey agar selective. Lactose and neutral red are added to differentiate the lactose fermenters, which form pink colonies, from lactose nonfermenters that form clear colonies.
Enterococci- Gram-positive cocci

Enterococci are facultative anaerobic organisms, i.e. they prefer the use of oxygen, but they can survive in the absence of oxygen. They typically exhibit gamma-hemolysis on sheep's blood agar.
Pseudomonas aeruginosa is a Gram-negative, aerobic, rod-shaped bacterium

Pseudomonas aeruginosa bacterial culture on a Xylose Lysine Sodium Deoxycholate (XLD) agar plate.
The xylose lysine agars were developed for the differentiation of pathogens from non-pathogens and to support the growth of the more fastidious enteric organisms. The basal XL Agar is nutritionally designed to permit the development of all species.
Xylose Lysine Deoxycholate (XLD) contains deoxycholate as inhibitor of Gram-positive organisms and allows the growth and differentiation of enteric pathogens.
The Phenol red indicator changes from red to yellow under acid conditions.
A 0.25 percent concentration of sodium deoxycholate in XLD provides nearly complete inhibition of Gram-positive microorganisms.
Proteus mirabilis is a Gram-negative, facultatively anaerobic bacterium

The micro-organism tests:
Indole negative and Nitrogen Reductase positive (no gas bubble produced)
Methyl Red positive and Vogues-Proskauer negative
Catalase positive and Cytochrome Oxidase negative
Phenylalanine Deaminase positive

ANTIBIOTIC SUSCEPTIBILITY:

A sensitivity test, also called an antibiotic susceptibility test, is also done. The bacteria are tested against different antibiotics to determine which will treat the infection by killing the bacteria.

OTHERS:

If the physician thinks the wound may be infected with a mold or yeast, a fungal culture is also done. The wound sample is spread on special culture plates that are treated to encourage the growth of mold and yeast. Different biochemical tests and stains are used to identify molds and yeast.

Case 6 (Liu Qian)

Patient: Ong Fei Fei (37,F)

Key Points:

Complaints of fever, pain during urination and virginal discharge
Possible diagnosis: UTI

Investigation of vaginal discharge in sexually active adult women should involve the collection of both endocervical and high vaginal swabs. Low vaginal swabs are to be avoided as they are likely to be contaminated with perineal/ faecal flora. High vaginal swabs (HVS) should be placed in transport medium to prevent drying and to allow the survival of anaerobes.

Urinary Tract Infection:

Urinary tract infection (UTI) is one of the most common sources of infection. It is important as it can cause troublesome and recurrent symptoms and may point to unsuspected anomalies of the urinary tract. Outcome of infection is of concern as it is associated with future complications including poor renal growth, recurrent adult pyelonephritis, impaired glomerular function, early hypertension and end-stage renal disease. The aim of management should be prompt diagnosis, rapid treatment and the detection of any underlying cause that might predispose to further infection or lead to long-term renal damage.

Urinary tract is made up of two sections: the lower urinary tract and the upper urinary tract. Lower urinary tract contains the bladder and urethra. Upper urinary tract contains two kidneys and the tube that connects them, called the ureters. An infection occurs when bacteria stick to the walls of the urethra, multiplying and moving up the urethra to the bladder. The urethra is the tube that carries urine from the bladder to outside the body. If a lower urinary tract infection is not treated, the infection may spread up through the ureters, and into the kidneys.

Different types of UTI:

Urethritis is an infection or inflammation of the urethra. This can be due to other things besides the organisms usually involved in UTI's. In particular, many sexually transmitted diseases (STD's) appear initially as urethritis.
Cystitis is an infection of the bladder and is the most common form of UTI. Cystitis can often occur at the same time as urethritis. It can be aggravated if the bladder does not empty completely when you urinate.
Ureteritis is infection of a ureter. This can occur if the bacteria entered the urinary tract from above or if the ureter-to-bladder valves don't work properly and allow urine to "reflux" from the bladder into the ureters.
Pyelonephritis is an infection of the kidney. This can happen with infection from above, or if reflux into the ureters is so bad that infected urine refluxes all the way to the kidney. Kidney infections can cause kidney damage or even failure if left untreated for an extended period of time.

Vaginal discharge:

The female genital tract has a complex microbial flora. Bacteria commonly present in large numbers include anaerobic streptococci, diphtheroids, coagulase negative staphylococci, and haemolytic streptococci. Other common commensals which can act as pathogens include Candida spp, Staphylococcus aureus, f haemolytic streptococci including Str agalactiae, and Actinomyces spp. In bacterial vaginosis the concentration of Gardnerella vaginalis, increases.

White: Thick, white discharge is common at the beginning and end of your cycle. Normal white discharge is not accompanied by itching. If itching is present, thick white discharge can indicate a yeast
infection.

Clear and stretchy: This is "fertile" mucous and means you are ovulating.

Clear and watery: This occurs at different times of your cycle and can be particularly heavy after exercising.

Yellow or Green: May indicate an infection, especially if thick or clumpy like cottage cheese or has a foul odor.

Brown: May happen right after periods, and is just "cleaning out" your vagina. Old blood looks brown.

Spotting Blood/Brown Discharge: This may occur when you are ovulating/mid-cycle. Sometimes early in pregnancy you may have spotting or a brownish discharge at the time your period would normally come. If you have spotting at the time of your normal period rather than your usual amount of flow, and you have had sex without using birth control, you should check a pregnancy test.

Possible microoragnisms that can cause Vaginal discharge:

Yeast, also called Candida, a type of fungi that is part of the normal flora of human skin but can also cause infections.(eg:Candida vulvovaginitis)
Bacteria found in the female genital tract that is the cause of bacterial vaginosis.(eg: Gardnerella vaginalis, Mobiluncus species, Mycoplasma hominis)
Trichomonas, a type of protozoa, an organism made up of one cell (eg: Trichomonas vaginalis)
Neisseria Gonorrhoea
Chlamydia trachomatis

Possible microoragnisms that can cause UTI:

Most Frequent

Enterococci
Streptococcus agalactae(Group B Streptococcus)
Enterobacteriaceae
Pseudomonas
Streptococcus pyogens(Group A Streptococccus)
Streptococcus aureus
S.saprophyticus
Candida species.

Less frequent

Gardenerella vaginalitis
Ureaplasma urealyticum
Mycoplasma hominis
Mobiluncus
Leptospira
Mycobacterium species
Chlamydia trachomatis(males)

Other Associated with multisystem disease

Salmonella(with gastroenteritis)
Schistosoma haematobium
Crytococcus neoformans
Trichosporon beigelii
Trichomonas vaginalis
Aspergillus
Penicillium
Adenovirus
Herpes simplex virus

Diagnosis Plan:

Fever and pain during urination are the symptoms of Urinary Tract Infection. Virginal discharge is normally present. But a change in color or amount of discharge, accompanied by other symptoms, may indicate that you have an infection. So I suspect that the bacteria go from vaginal to the urethra and cause Urinary infection.

Vaginal swabs were cultured both anaerobically and aerobically on the surface of freshly prepared brain heart infusion agar plate supplemented with vitamin K (0.5 mg/l) and Haemin (5mg/l), blood agar and chocolate agar plates. Additional Bacteroides Bile Esculin agar, Neomycin Vancomycin Chocolate agar plates were inoculated for anaerobic culture. Agar plates were examined after 48 hrs, 96 hrs and 7days and isolates were identified.

To confirm Vaginitis, vaginial fluid pH, wet mount preparation, ad KOH microsopy should be done. The interpretation can follow Figure 1.

Possible microoragnisms that can cause Vaginal discharge:	Laboratory investigation	Commend
Yeast, also called Candida, a type of fungi that is part of the normal flora of human skin but can also cause infections.(eg:Candida vulvovaginitis)	Examine the KOH preparation under the microscope ("Wet Mount"). Multiple strands of thread-like hyphae confirm the presence of yeast. Whiff" test : Positive	http://www.fpnotebook.com> GynVaginitisYeast.jpg
Bacteria found in the female genital tract that is the cause of bacterial vaginosis.(eg: Gardnerella vaginalis, Mobiluncus species, Mycoplasma hominis)	Test the pH. If >5.0, this suggests Gardnerella. Mix one drop of KOH with some of the discharge on a microscope slide. The release of a bad-smelling odor confirms Gardnerella. Vaginal epithelial cells studded with coccoid bacteria are "clue cells" signifying Gardnerella. Whiff" test : Negative
Trichomonas, a type of protozoa, an organism made up of one cell (eg: Trichomonas vaginalis)	Mix one drop of saline with some discharge ("Wet Mount"). Under the microscope, large (bigger than WBCs), moving micro-organisms with four flagella are trichomonads. Whiff" test : can be positive Additional test: DNA probe tests and Culture	www.k-state.edu/.../625tutorials/Protozoa01.html
Neisseria Gonorrhoea	N. Gonorrhoea should begin with a Gram stain. If intracellular gram-negative diplococci are present, N. Gonorrhoea infection is presumed and treatment should be initiated. Gonococcal cultures should confirm the diagnosis. It was inoculated onto modified Thayer-Martin medium.Cultures were examined following overnight incubation, and if they were negative on initial evaluation, they were examined again following an additional 24 and 48 h of incubation. Typical colonies containing gram-negative diplococci and giving a positive oxidase reaction were presumptively identified as N. gonorrhoeae. The identities of presumptive N. gonorrhoeae colonies were then con- firmed with fluorescein-conjugated monoclonal antibodies.
Chlamydia trachomatis	Chlamydia Rapid Test(commercial kit)

Figure1: Possible microoragnisms that can cause Vaginal discharge and and Laboratory investigation respectively.

If vaginitis is not confirmed, urine sample must be asked for further investigation.

Urine culture is the most straightforward and simplest method to diagnosis urinary tract infection. Urine was cultures in both Blood agar and Macconkey agar. It was "dogma" that a finding of 10⁵colony-forming units per ml (CFU/ml) or more was "positive". In addition, urine microscopy must be done to quantify the WBC.

Gram stain should be performed for the positive cultures to determine the Gram nature and morphology of the Bacteria. (eg: Gram positive Bacilli)

To identify the type of bacteria, colonies are isolated from the plate and enzyme tests are done.

After all, the antiicobial susceptibility test should be done to guide the treatment.

Antimicobials usually tested for UTI

Enterobacteriaceae
- Carbenicillin
- Cinoxacin
- Lomefloxacin,norfloxacin, or ofloxacin
- Loracarbef
- Nitrofurantoin
- Sulfisoxazole
- Trimethoprim
Pseudomonas aeruginosa and other non-enterobacteraceae
- Carbenicillin
- Ceftizoxine
- Tetracycline
- Lomefloxacin, norfloxacin, or ofloxacin
- Sulfisoxazole
Staphylococci
- Lomefloxacin, norfloxacin, or ofloxacin
- Nitrofurantoin
- Sulfisoxazole
- Trimethoprim
Enterococci
- Ciprofloxacin
- Norfloxacin
- Nitrofurantoin
- Tetracycline
Streptococci
- Norfloxacin
- Nitrofurantoin

Haematology and LMQA

2007-11-25T19:43:00.000-08:00

Name: Dorene

Subject: Haematology
The tests done in the lab are FBC, ESR, blood grouping, coagulation test, haemoglobin electrophoresis. I would like to share with you all haemoglobin electrophoresis test and bloog grouping.

In Haemoglobin electrophoresis, it tests for beta-thalassemia, found most commonly in heteroygous state as beta-thalassemia minor or beta-thalassemia trait. In homozygous state, which is known as beta-thalassemia major, is a lethal disease, as there is no efficiemt treatment for the disease.

Adult blood contains promary haemoglobin A (HbA) and a small percentage of hemoglobin A2, (HbA2) and trace amount of fetal hemoglobin (HbF). For patients with thalassemia, there will be elevated percentage of HbA2 and HbF and this test will determine the quantity of them. The machine used for the test, VARIANT, is a fully automated HPLC (High-performance liquid chromatography) system that can separate and determine area percentages for hemoblogin A2, and F.

Method:

First, pipette 5 microlite of whole blood from patient into separate a 1.5ml vial. Add 1.0ml of hemolysis reagent to each sample vial and mix well to enhance hemolysis effect Finally load them into the machine. For the machine, first press "power" button and let it run on it's own as warm-up. next, press test menu> 1 (1 refers to beta-thalassemia short program). press enter and the test will start running.

Principle of the test:

the machine uses dual wavelength filter photometer at 415 and 690nm to monitor hemoglobin concentration. 415 nm detects the absorbance changed when reagents/buffers are added while 690nm corrects the effects caused by mixing buffers with different ionic strength.

when the test starts, the machine will extract the specimen every ~6 minutes into the analysis stream. elution buffer of different ionic strength will be release each time and pass through the analysis cartridge. the ionic strength of the buffer will be sLlowly increase which cause a higher concentration of hemoglobin to be eluted from the analysis stream. higher ionic strength of the mixture will force more strongly retained hemoglobin to be eluted. Changes in aborbance cause by elution of hemoglobin (difference in absorbance from the start of experiment to the end of experiment) will be detected by the photometer when different ionic strength were added and this absorbance will be converted to absolute values for interpretation.

Interpretation of concentration:

heterozygous beta-thalassemia - HbA2 - 4-9%, HbF - 1-5%
homozygous beta-thalassemia - HbA2 - normal or increased, HbF - 80-100%

Blood grouping

Source:http://www.visualsunlimited.com/browse/vu308/vu308235.html

In the lab, it uses microtiter plate and tile for blood grouping. first one drop of commerical anti A, B, AB, D are added into consecutive well (G F E D) respective. one drop of A B and O are then added into well (C B A). The lab number of the specimen are then written on the plate. next, the spin down the specimen to separate serum from the red cells. One drop of serum are then added each into well C B A, containing A, B, O blood cells. next dilute the rbc in saline to have 3-5% concentration. and one drop of it will be added each into well G F E D containing anti A,B,AB,D. mix the blood using a vibrator and centrifudge it at 900rpm for 1.5min to concentrate the agglutins.

Results : agglutination = positive. no agglutination = negative.

Quality Hierarchy Model in LMQA

In LMQA, it teaches us about quality control, quality assurance, quality system, quality management and total quality mangement. In our lab, i noticed there is also such system and would like to share about it more with you all.

1. Quality Control - eg:Quality controls are performed in all machines by technicans and documented down everyday.

2.Quality Assurance - eg: Quality of all reagents, plates, equipment are checked and approved upon arrival before using by supervisior and technicans. Having external QC to ensure precision and accuracy.

3.Quality System -eg: work on areas to improve efficiency and quality of the system.

4.Quality Management - eg: having meeting every week for management level to work on the problems encountered and determine ways of troubleshooting. Work on areas to improve efficiency and quality of the system.

5. Total quality management - eg:development of lab automation in the laboratory(it's half automated currently), upgrade of the device and standards of the laboratory.

reply questions

2007-11-21T16:26:00.000-08:00

sorry for the late reply caz busy with the MP.

1.What's the clinical significance of the PYR test and what does PYR stand for? June
The clinical significant of PYR test is to help microbiologist to detect the pyroglutamate aminopeptidase found in group A streptococci (Streptococcus pyogenes) and group D enterococci. PYR stands for pyrrolidonyarylamidase

2. What do I expect to see after the developing soultion is added onto the test area if the specimen is negative for PYRase activity? Eugene Wong
A negative test is indicated by no color change.

3. "PYR test kit uses Test Cards impregnated with L-pyroglutamic acid 7-amino-4-methyl-coumarin (7AMC) and dimethylamino-cinnamaldehyde for the detection of PYRase activity."how does L-pyroglutamic acid 7-amino-4-methyl-coumarin (7AMC) and dimethylamino-cinnamaldehyde aid in the detection of PYRase activity?"The enzymatic hydrolysis of this substrate by enterococci, Group A streptococci and Citrobacter spp. produces a purple colour followed by the addition of the Developing Solution."enzymatic hydrolysis of what? L-pyroglutamic acid 7-amino-4-methyl-coumarin (7AMC) and dimethylamino-cinnamaldehyde to give purple colour? Cass

Some Enzyme in Group A streptococci and Citrobacter spp can hydrolysis the L-pyroglutamic acid 7-amino-4-methyl-coumarin (7AMC) and dimethylamino-cinnamaldehyde, they react together to give a purple color.

4. can u explain what is PYRase activity? Juexiu
PYRase activity means the ability of the bacteria to product PYRase.

Liu Qian
0503935I
TG02

Microbiology

2007-10-31T18:54:00.000-07:00

Hi everybody! Time pssed so fast, SIP is going to end soon!
This week, I am going to share something in the microbiology lab.
Microbiology lab is a place where safety issues are particular important. So I want to talk about the four-biosafety levels.

Biosafety level 1
Biosafety Level 1 is suitable for work involving well-characterized agents not known to consistently cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment. Work is generally conducted on open bench tops using standard microbiological practices. Laboratory personnel have specific training in the procedures conducted in the laboratory.(Eg: normal routine bench in the microbiology lab)

Biosafety level 2
Biosafety Level 2 is similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It differs from BSL-1 in that laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists; access to the laboratory is limited when work is being conducted.
Certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets.(Eg: the bench working on the respiratory specimen)

Biosafetty level 3
Biosafety Level 3 is applicable to the place where work is done with exotic agents which may cause serious or potentially lethal disease through the inhalation route. Laboratory personnel have specific training in handling pathogenic and potentially lethal agents, and are supervised by competent scientists who are experienced in working with these agents. All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. The laboratory has special engineering and design features.

Biosafety level 4
Biosafety Level 4 is required for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease. The laboratory director strictly controls the access to the laboratory. The facility is either in a separate building or in a controlled area within a building, which is completely isolated from all other areas of the building.

I want to share one more biochemistry test kit in the micro-lab also. It is PYR rapid colorimetric test.

PYR
PYR is a rapid colorimetric test for the determination of PYRase activity in Streptococci and Citrobacter spp. PYRase activity distinguishes Group A streptococci and enterococci from other streptococcal groups including Group D streptococci.
PYR test kit uses Test Cards impregnated with L-pyroglutamic acid 7-amino-4-methyl-coumarin (7AMC) and dimethylamino-cinnamaldehyde for the detection of PYRase activity. The enzymatic hydrolysis of this substrate by enterococci, Group A streptococci and Citrobacter spp. produces a purple colour followed by the addition of the Developing Solution.

Specimen preparation
When identifying enterococci and Group A streptococci, fresh primary or secondary cultures grown overnight on non selective media such as blood agar give best results. Colonies tested must be Gram-positive cocci and catalase-negative. In case of insufficient growth, a subculture should be performed.When identifying Citrobacter spp. from Salmonella spp. and Enterbacteriaceae, colonies from non-selective media such as (XLD Medium, MLCB Agar, Desoxycholate Citrate Agar, Salmonella Shigella Agar, Brilliant Green Agar or Hektoen Enteric Agar) may be tested. Colonies should be Gram-negative, oxidase-negative and urease-negative.

Test procedure
1. Apply one suspect colony (0.5mm or larger) onto the test area (enough to make a visible smear).

2. Moisten test area with 1 drop of Buffer.

3. Incubate the inoculated Test Card at room temperature (15-30°C) for 5 minutes.

4. Dispense I drop of Developing solution onto the test area. Development of a vivid purple colour on and around the colonies within 20 seconds confirms PYRase activity.

www.labcenter.com.br/novidades.htm

ok that's all~

Enjoy reading:)

Liu Qian

0503935I

2007-10-25T09:55:00.000-07:00

Name :Dorene Chen

Department:Microbiology

Hello people,sorry for late blog posting, for this week I will talk about one special type of testing I had during my attachment :Seminal Analysis.

Clinical Significance:

Semen analysis is used to determine male fertility status or to determine the success of a vasectomy.

Semen is composed of spermatoza suspended in seminal fluid, which provides nutrition and volume to transfer the spermatoza to endocervical mucus.

Male in fertility can be affected by a number of causes eg:
1) Decrease in the number of viable sperm
2) Sperm with abnormal morphology
3)Abnormalities of seminal fluid

In determining successfulness of vasectomy, sperm count should decrease slowly from a time span of 6 weeks to 3 months for even longer.

Specimen must be fresh, perferably lesser than 2 hours as viability decreases as time increases. Sample is collected through masturbationm after a minimum of 3-7 days of sexual abstinence and kept into a clean, wide mouthed plastic container and kept at body temperature best.

Method:

Vital Stain (for viability)
1) mix one drop of semen with 2 drops of 1% Eosin Y
2) add 3 drops of 10% Nigrosin solution after 30 seconds and mix
3)place a drop of mixture into a glass slide to make a smear.
4) Viavle spermatoza will be unstained and non viable spermatopza will be stained.( spermatoza shape with purple background seen)
5) Percentage of viabililty can be accessed.

Motility test
1) fill KOVA slide chamber with 20 microlite of neat semen (undiluted, pure specimen)
2) examine under microscope for motility as well as percentage of active, sluggish, non-progressive and non-motile sperms

Sperm count, leucocytes and morphology of sperm

1) specimen is diluted with sodium bicarbonate-formalin solution in 1:20 ratio ( 50 microlite of semen with 950 microlite of solution)
2) fill KOVA slide chamber with 20 microlite of diluted semen suspension
3) examine under microscope for number of spermatoza and leucocytes.
4) access the percentage of normal spermatoza, and abnormal morphology eg: double headed, no head, 2 tails, no tail, wrong head- tail ratio (head too big) etc etc.

Lastly apearance, viscosity, liquefaction time (time taken for semen to be liquid-like), anstinence, time collected, volume of specimen, time received and pH (must be alkaline) should be reported in to seminal analysis as well.

Counting Spermatoza: chose only 1 large grid.

Source : http://www.clt.astate.edu/wwilliam/cls_1531_seminal_fluid_lab_1_.htm

undiluted Specimen
Source :http://www2.uni-jena.de/hautkl/elbandro.htm

2007-10-16T23:02:00.000-07:00

STUDENT: Cassandrea TG02
LABORATORY: Immunochemistry

The ARCHITECT

o Uses chemiluminescent microparticle immunoassay for the qualitative detection of the antigen or antibody in the serum and plasma of the paitent.

o Two-step immunoassay:
 Sample and antibody/antigen coated with paramagnetic microparticles are combined
 After washing, acridinium-labeled antibody/antigen is added.
 After another round of washing, solutions are added to the reaction mixture, resulting in the chemiluminescent reaction measured by relative light units (RLUs)
 The RLUs is compared to the cutoff signal determined by calibration which will determine if the results are reactive or non-reactive

o Two versions of ARCHITECT in the immunochemistry
ARCHITECT 2000 and ARCHITECT I 2000 SR (newer version)

o Types of test run in ARCHITEST 2000 tests involving hormones like beta-hCG, FSH, LH, Estradiol , prolactin and GH

o Types of test run in ARCHITECT I 2000 SR tests involving viruses like HIV, Syphilis, Hepatitis B and Rubella and thyroid tests which consist of free T4, TSH and calcitonin.

Clinical significance of measuring:

 HBeAg
o First detectable in the early phase of hepatitis B after appearance of hepatitis B surface antigen (HBsAg) early detection of disease
o Both antigen rise rapidly during viral replication in acute infection
o However, patients may not have HBeAg during infection but are still positive for hepatitis B if the antibodies (anti-HBe) to HBeAg are present

 Anti-Hbe
o Used to monitor the convalescence and recovery from hepatitis B infected individuals
o Presence of anti-Hbe and loss of HbeAg after acute infection is a good indication of recovery
o Samples with less than 10.0mIU/mL considered non-reactive
o Samples with greater than or equal to 10.0mIU/mL are considered reactive.

 HbsAg
o Aid in the diagnosis of suspected HBV infection and monitor the status of infected individuals
o Diagnosis of acute or chronic hepatitis HbsAg reactivity should be correlated with patient history and other hepatitis B serological markers
o Negative results need not be tested further but reactive results are retested

 -hCG
o hCG is a sialoglycoprotein initially secreted by the trophoblastic cells of placenta shortly after implantation of fertilized ovum into uterine wall
o Rapid rise in hCG levels after conception makes it an excellent marker for conformation and monitoring of pregnancy
o The hCG increases to peak concentration then decrease an plateau
o HCG levels can be useful in the prediction of spontaneous abortions, aiding in the detection of ectopic pregnancy and multiple gestation

 Ferritin
 High molecular weight iron–containing protein that functions as iron storage compound
 25% of iron in the body is present in various storage forms; 2/3 of the iron stores exist in the form of Ferritin
 Ferritin provides a more sensitive, specific and reliable measurement for determining iron deficiency at an early stage

2007-10-07T17:56:00.000-07:00

Hey Guys

Sorry for the late BLogginG. Today i am going to talk about how Amniotic fluid is being set-up.

Amniotic fluid set up

Purpose: The most common reasons for prenatal diagnosis of chromosome disorders of the fetus are advanced maternal age, family history of a chromosome abnormality, abnormal maternal serum screen or fetal defect identified by ultrasonography.

Reason for using amniotic fluid: Amniotic fluid contains cells derived from the amnion, from the gastrointestinal tract and from the skin of the fetus. Some of these cells are viable and are capable of undergoing cell division in vitro. Through amniocentesis, a sample is collected for the purpose of culturing the cells to obtain chromosomes that reflect the fetal karyotype. The in situ culture technique is used where single AF cells are plated onto a sterile glass coverslip in a petri dish where they grow in situ to form discrete colonies.

Steps involved:

1) Usually AF is collected in a tube or sometimes it comes in the syringe.

2) Spin down the tubes containing AF at 1200 rpm for 10 mins

3) Once you have spun down the sample, check the appearance of supernatant and the pellet size

4) After spinning down the tubes, discard the supernatant and dislodge the pellet

5) In one of the tubes, add 1 ml of alpha-AM media and mix it well and in the other tubes add 1 ml of Alpha-bio media. (These are the two media used in my lab for all samples: AF, Chorionic villi etc)

6) There is a need to do 4 cultures. Thus four petri dishes are labeled wit the patients’ name, lab number. (labeled as A,B,C and D)
Note: Each dish has a coverslip attached to the surface of the dish

7) For the dishes A and B, aliquot 0.5 ml of the cell suspension (media used alpha- AM) into each dish

8) As for dishes C and D, 0.5 ml of cell suspension (media used alpha-bio) is added to each dish.

9) Make sure the cell suspension is confined to the coverslips. This is to allow the cell attachment and growth of cells on the surface of the coverslip. (Harvest cells that grow on the coverslipknown as in situ)

10) After which, place the dishes into the respective 37oCCO2 incubator for 4-5days. Culture A and c into incubator A and Culture B and D in incubator B (for backup)

11) And also make sure that you have placed in dishes in the right tray (so that the next day who ever is going to flood the dishes with media knows which dish to flood. )

Diagram to summarize:

Vinodhini
TGO2

Answers to your questions (Sasi's)

2007-10-02T03:37:00.000-07:00

Answer to Chaur Lee’s query:

The factors that influence ESR results can be listed under two headings. First, those physical changes in blood plasma and cells, which to some degree the test is measuring and secondly those technical variables that must be avoided when performing the test.

Physical Changes in Blood
The ESR comes about via the interaction between red cells and plasma, therefore any physical variation in either component of blood will affect the result.

The rate of fall is dependent on: - Shape and size (density and surface area) of the red blood cell.Large cells fall faster than small cells. The presence of fewer cells allows for a faster fall rate that increased cell numbers. Cell shape variations , for e.g. abnormal cell morphologies such as sickling (HbS), may slow the rate of sedimentation. Increased albumin slows the fall of cells. An increase in other plasma proteins may increase the rate of fall. Changes in the red cells due to anaemia also usually increases the rate.

- Viscosity of the plasma. This is affected by a number of factors such as concentration of albumin or gamma globulins. Plasma proteins that affect the ESR include the immunoglobulins (IgM) and a number of the acute phase proteins including fibrinogen, C-reactive protein, alpha-1-anti-trypsin, haptoglobin, etc. These acute phase proteins increase with tissue damage (e.g. lupus or rheumatoid arthritis), pregnancy, chronic inflammation and chronic infection and hence increase the ESR rate.

Technical Variables
A number of external factors will affect the result.
Correct mixing of the blood sample before setting up the test is one of the most important factors influencing the ESR result.
Also, the rate of fall is affected by the tube diameter, angle of the tube sides to vertical, temperature, amount of vibrations present, and length of the tube. If a 3o tilt from vertical is present, it is possible that an error factor of 30% may be introduced in the test. A narrow lumen in the ESR tube will slow down the sedimentation rate giving lower results.

Other factors
Females tend to have higher ESR, and menstruation and pregnancy can cause temporary elevations.
Drugs such as dextran, methyldopa (Aldomet), oral contraceptives, penicillamine procainamide, theophylline, and vitamin A can increase ESR, while aspirin, cortisone, and quinine may decrease it.
Age also affects ESR. ESR increases with age in general.

Answer to Charmaint’s query:
Generally, ESR is a marker for inflammation.
It has been also a marker for temporal arteritis and rheumatoid disease as mentioned in my blog. Its also a marker for Lupus.
Apart from that, ESR can also be used as a prognosis marker of ischemic stroke and heart failure.
Many other diseases also increase the ESR: Infection, kidney disease, anemia, diseases involving white blood cells, cancer, and autoimmune and inflammatory diseases.

Answer to Kent’s query:
My lab’s using the Westergren method.
The Westergren method employs a 200 mm, 2.5 mm diameter tube vertically aligned column. The column is filled with blood anticoagulated with EDTA. The distance that the column of blood falls in one hour is recorded and reported in mm/ at the end of 1st hour.
The Wintrobe method employs a shorter tube (100mm) and a different anticoagulant (ammonium oxide and potassium oxalate) is used to mix with the blood.
It has one advantage over the Westergren method as it is able to correct for anaemia while testing patients with marked anemia provides no useful ESR data when using the Westergren method.
Though the Wintrobe method is sensitive for mild elevations, it has a higher false positive rate.
The Westergren method is also more sensitive for changes at elevated levels and may be more useful where the ESR is being used to evaluate the response to therapy for instance, in diseases such as temporal arteritis. Hence, the Wintrobe procedure is not used in most laboratories, the Westergren method being preferred.

Answer to Alex’s query:
Yes Alex, ESR is not diagnostic for any particular disease. It only aids in the diagnosis of the two diseases mentioned in my blod.
The two diseases that Ive mentioned, Temporal arteritis and Polymyalia Rheumatica are basically inflammatory diseases. For instance, ‘Temporal arteritis’ means means "inflammation of the temporal arteries." Since ESR measures inflammation, it is used to aid in the diagnostic aspect of these diseases, to confirm if there is inflammation in the first place. (The ESR rate in these diseases will be always be higher than normal.). When an inflammatory process is present, the high proportion of fibronogen in the blood causes red blood cells to stick to each other. The red cells form stacks called 'rouleaux' which settle faster.
Hence, ESR acts as a marker for these inflammatory diseases.
As for your query on the reference ranges:
The size of the red blood cell affects the ESR reading. The size changes according to the age. For instance, red blood cells are larger in neonates, though smaller in other children. Hence, the reference range is calculated accordingly.

Answer to Elaine’s query:
In our lab, we run control once in the morning daily using the high, normal and low controls which are commercially prepared. We also run another Internal Qc using internally pooled blood (from real patients) three times a day to make sure the precision of the FBC readings amongst the three machines stay constant throughout the day.
And Elaine, guess there was a misinterpretation of what I’ve mentioned about ABO grouping and ESR. Sorry! for not being clear. What I meant was, in our lab, once the EDTA tubes arrive at the haematology department, FBC is run first. Then, ESR is done. Only after these two tests are complete, the respective EDTA tubes are directed to ABO bloodgrouping. ESR is given the next priority after FBC as only then, there is a gurantee that the volume of the sample would be sufficient for ESR. If the respective EDTA tubes happen to go through all the other tests before ESR is run, by the time the tubes reach the ESR section, the volume of blood may not be sufficient for an effective ESR to be run. Hence, I did not mean that ABO bloodgrouping has to be done together with ESR hehe. Hope I’ve cleared your doubts on that.

Haematology

2007-09-29T06:21:00.000-07:00

Hi everybody. This week's my turn again to share what I've learnt with you'll.

Occult Blood Test(Blood in the stools)

Clinical Interpretation
Fecal occult blood test is performed as part of routine physical examination during examination of the rectum and it is a screening tool for colorectal cancer. Blood in the stools could indicate gastrointestinal diseases such as inflammatory bowel disease, gastric ulcers, etc.

Materials needed
-Test card(contains guaiac)
-Developing solution(contains hydrogen peroxide and denatured ethyl alsohol)
-Wooden applicator sticks

Test Principle
The test is based on the oxidation of guaiac by hydrogen peroxide to a blue compound. The heme portion of hemoglobin that is present in the stool will catalyse the oxidation of alpha-guaiaconic acid to form a blue colouration.

A small fecal sample is collected and a thin smear is applied on the test card using wooden applicatior sticks. The cover flap is closed and we have to wait for 2-3 mins for the stool to be absorbed into the test paper. The back of the test card is opened and a drop of the developer is added to the the guaiac paper directly over the smear and both positive and negative controls. Results are read after 1 minute and a blue colouration is indicative of occult blood. A brown colouration indicates negative results.

Patients should avoid red meat and NSAIDs as these substances can cause false positive results. Occult blood test must be done with screening endoscopy to detect colorectal cancer as a negative result for OB does not guarantee that a patient does not have colorectal cancer.

Enjoy reading!

Cheong Shu Hui
TG02

Haematology-ESR ( Sasi's)

2007-09-24T19:10:00.000-07:00

Hi! Sasi here! Its my turn to blog! So, here I go....

Recently I have been attached to the Haematology department.

In this blog, I would like to highligh one test I frequently was assigned to do besides ABO blood grouping and FBC (which has been previously elaborated by Lizzie). The test would be ESR.

ESR stands for Erythrocyte Sedimentation rate. The rate at which the red blood cells falls is measured and reported in mm/h. It is an easy, cheap, non specific test that has been used for years to help diagnose conditions associated with acute and chronice inflammation including infections, cancers and auto immune diseases. According to a senior at work, I learnt that ESR is said to be non specific because increases in the rate do not tell exactly where the inflammation is in the body or what is causing it. For this reason, ESR is typically used in conjunction with other tests like Full Blood Count, Anitinuclear antibody etc to aid in the diagnosing aspect. ESR is particularly useful in diagnosing two specific inflammatory diseases, Temporal arteritis and Polymyalia Rheumatica. A high ESR is one of the main test results use to support the diagnosis. ESR is also used to monitor disease activivity and response to therapy in both of these diseases.

In our haematology lab, once the EDTA bloody tubes arrive,they are run in the FBC analyzers first. After FBC is run, ESR is run. ESR is given the very next priority as it has to be ensured that the blood volume is sufficient for the test. (Hence, that is why ABO is done last). After preparing the worklist for the ESR test, I had to alliguot the blood into the small tubes provided by the ESR commercial kit, mix it well with the anticoagulant in the tubes and make sure the sample is homogenous. Long pipettes are provided in the kit. These pipettes are thrusted into the tubes and I have to make sure the blood level reaches the brim of the pipette. Once the tubes are fit into the automated readers, it would take approximately 40 mins for the readings to be out.

When an inflammatory process is present, the high proportion of fibronogen in the blood causes red blood cells to stick to each other. The red cells form stacks called 'rouleaux' which settle faster. The height of the remaining plasma is read.

Samples with results more than 50 have to be rerun.The reference range applies as follows:

(ESR 95% limits) (Adults)
Age (years) 20 55 90
Men 12 14 19
Women 18 21 23

Children
Newborn: 0 to 2 mm/hr.
Neonatal to puberty: 3 to 13 mm/hr.
Newborn: 0-5 mm/hr.
Neonatal to puberty: 0-15 mm/hr.

A very high ESR may occur due to a marked increase in globulins, that can be due to a severe infection. The doctor would then order for other follow-up tests like cultures depending on the patient's symptoms.

A low ESR is seen with polycythemia which is the condition where a patient makes too many red blood cells or with extreme leokocytosis where the patient has too many white blood cells or with protein abnormalities. Some changes in red cell shape such as sickle cells in sickle cell anemia aso lowers the ESR.

That marks the end of my blog. Feel free to drop ya questions if any :) Take care!

Sasi
Tgo2
0503804g

Answers to ur questions!!!

2007-09-22T21:16:00.000-07:00

Dear all, thanks for reading my post and I can see that most of you are very curious about why the patient has to lie down on the left side and not the right side after consuming the tablet. Well, the answer is actually shown in the second picture which I have posted. If you have noticed, you will realise that the human stomach has a larger surface area on the left side or you can say that the stomach is tilted to the left. Therefore, when the patient lie on the left side after consumption of the tablet, it allows the tablet to be in contact with a larger surface area of the stomach, thus more H. pylori (if present in the patient) can act on the tablet effectively and results will be more accurate too.
The reason why the patient has to be seated after lying down is to allow the labelled carbon dioxide (produced as a result of the presence of H. pylori) to move up to the lungs for breathing out into the bag. (answered gail, shu hui and vino)

Is there anything in the bag and how the breath bag is sent to the lab? Will results be inaccurate if the breath bag is exposed to the surrounding air?
The breath bag is a sterile bag which contain no anticoagulant. There is no special procedure that must be followed during the sending of bag to the lab. Once patient breathes out into the bag, it must be capped immediately. Of course results will be inaccurate if the patient does not capped the bags properly, therefore the bags will be sealed in another big plastic bag when sent to the lab. As this test is usually conducted in the hospital, the breath bag will be sent immediately up to the lab for processing. The processing and sample handling part is less complicated. However, the med techs do act fast when they are processing the samples. For example, when they remove cap on the breath bag, they will quickly fit the mouth of the breath bag to the machine. In another words, they will try to lessen the exposure of the breath inside the bag to the surrounding air .The tedious part will have to be sample collection. (answered ci liang, martin and liu qian)

Why patient cannot chew the tablet?
Because chewing may reduce the actual amount of the tablet that goes into the stomach and results can be inaccurate. Patient's result may be negative when actually very little tablet is present in the stomach, more of it is remained on the patient's teeth. (answered Kangting)

Why not do a culture for the detection of the microorganism?
This is a good question. Yes, biopsy check during endoscopy with a rapid urease test, histological examination and microbial culture are much better and relaible choice for the detection of the microorganism. In fact, histological examination is the gold standard for the detection of H. pylori. But seriously thinking, all the above three methods are evasive. Let's say if a patient is under treatment and doctor want to see the effectiveness of the treatment, do you think the patient will like it if he has to do endoscopy every now and then. It will be uncomfortable to the patient. Therefore, with UBT, patient's treatment progress can be updated and he does not have to undergo any evasive procedure.
This is also the advantage of UBT. UBT is the gold standard for the determination of treatment process. (answered jia hao)

Additional info:
Disadvantage of UBT is that it is subjected to non-conformance by patient as there are many instructions that must be followed before taking the test.
They are:

Fast for 8-9 hrs before test because food can interfere with results
Stop taking Proton Pump Inhibitors (PPI) eg. omeprazole, lansoprazole one week before the test because they are acid-reducing medication which can cause the stomach condition to become less acidic. This is no good for the thriving of H. pylori. Helicobacter spp. are the only known microorganisms that can thrive in the highly acidic environment of the stomach.
Stop taking antibiotics eg amoxicillin, tetracycline 4 weeks before test. This test measures active H. pylori infection. If antibiotics are suppressing the amount of H. pylori present in the stomach, results will not be accurate too.

That's all.
Sally

Biochemistry/ Immunology

2007-09-15T22:18:00.000-07:00

Hello to all of you. For this week, I shall talk about a very special test called the Urea Breath Test (UBT). Firstly, we have to know that this is not a commonly requested test. It is unique in the sense that detection of an illness can be done using the patient's breath sample instead of common samples like blood or urine.

Purpose of UBT

To identify infections by Helicobacter pylori, a spiral bacterium implicated in gastritis, gastric ulcer, and peptic ulcer disease.

Helicobacter pylori is a helical shaped, gram-negative bacterium. It infects various area of stomach and duodenum. Its helical shape is thought to have evolved to penetrate and favour its motility in the mucus gel layer.

Helicobacter pylori

Principle of UBT

Patient will swallow urea labelled with an uncommon isotope, either radioactive carbon-14 or non-radioactive carbon-13. For the two different forms of urea, different instrumentation is required; carbon-14 is normally measured by scintillation, whereas carbon-13 by isotope ratio mass spectrometry (IRMS). For carbon-13, a baseline sample is taken before taking the urea tablet and used to compare with the post urea sample. In this case, my lab uses the carbon-13 method and so 2 samples are collected from each patient.

UBIT-IR300 is an infrared spectral analyzer that measures the change in the carbon isotope (13CO2/12CO2) in carbon dioxide in breath air gas.

During the resting period after taking of the tablet, the bacterium (if present in the stomach) will metabolize the labelled urea and produce labelled carbon dioxide that can be detected in the breath. Detection of isotope-labelled CO2 in exhaled breath indicates that urea in the breath is split. This also means that urease (enzyme used by H. pylori to metabolize urea) is present in the stomach, hence the bacteria are present. Refer to top.

Procedure of test

Patient has to breathe into the first sample bag (before taking the UBIT tablet). This bag will be labelled as 'Baseline'.

Immediately (within 5 secs), swallow one UBIT tablet on an empty stomach with 100 ml of water. Do not chew, crush or dissolve the tablet.

After taking the UBIT tablet, lie down on your left side for 5 min.

Remain seated for a further 15 min.

20 min after taking the UBIT tablet, collect breath again using the 2nd sample bag which will be labelled as 'Sample'. The two sample bags will be analysed uisng the machine UBIT-IR300.

Interpretation of results

This is also the reason why a 'pre' and 'post' breath are collected from the patient so that the difference between the pre and post urea measurements can be compared to a cut-off value. Results below the value are assumed to be negative, those above is positive.

That's all.

Sally

0503315D

Hematology

2007-09-09T06:47:00.000-07:00

It’s my turn to blog again. This week I was attached to hematology. Our lab use ruby to do the full blood count and retic count and D10for beta-thalassemia. There is a test we never learned in the school before is D- dimmer. I want to share with you here.

D-DIMER
D-dimer is a blood test to diagnose thrombosis. A negative result can rules out thrombosis, a positive result can indicate thrombosis but can not rule out other potential etiologies. It is to exclude thromboembolic disease where the probability is low.

Principle
D-dimers are unique in that they are the breakdown products of a fibrin mesh that has been stabilized by Factor XIII. This is the final step in the generation of a thrombus. D-dimer assays rely on monoclonal antibodies to bind to this specific protein fragment.
Types of assays
l ELISA
l Latex turbidimetric assay (automated immunoassay)
l Enhanced microlatex
l Latex-enhanced photometric

Reference range

Values exceeding 250, 300 or 500 ng/ml (different for various kits) are considered positive.

Next I want to share some information about beta-thalassemia.
There are two forms of beta thalassemia. They are thalassemia minor and thalassemia major.
Thalassemia minor: The individual with thalassemia minor has only one copy of the beta thalassemia gene (together with one perfectly normal beta-chain gene). This situation can very closely with mild iron-deficiency anemia. However, persons with thalassemia minor have a normal blood iron level. No treatment is necessary for thalassemia minor.
Thalassemia major: The child born with thalassemia major has two genes for beta thalassemia and no normal beta-chain gene. This causes a striking deficiency in beta chain production and in the production of Hb A. Thalassemia major is, therefore, a serious disease.
At birth the baby with thalassemia major seems entirely normal. This is because the mosyt of the hemoglobin at birth is still fetal hemoglobin (Hb F). Hb F has two alpha chains (like Hb A) and two gamma chains (unlike Hb A). It has no beta chains so the baby is protected at birth from the effects of thalassemia major.
Ok that is all. Every thing in the lab looks really very easy as loading and unloading the sample from the machine. So I go and find some more information about the test. I hope it is helpful!!!
Enjoy reading^_^

SIP

2007-09-06T07:29:00.000-07:00

Helloo everyone! I'm here to answer Ms Chew's question for me..

Sigificance of Nitrite dipstick test in Urine FEME
The presence of nitrite in the urine indirectly indicates that there is nitrite-releasing bacteria present. This test depends on the conversion of nitrate to nitrite by nitrate reductase, an enzyme usually produced by gram-negative bacteria. A positive nitrite test is shown by a pink colouration in the nitrite test region on the test strip, indicating bacterial infection. Not all kinds of bacteria releases nitrite. However, if bacteriuria is significant but WBCs are absent when viewed microscopically, the bacteria formed may be due to contamination because of wrong collection or handling method. Hence, bacteria should not be reported.

That's all folks!

Cheong Shu Hui
TG02

2007-08-31T23:33:00.000-07:00

Hi Ms Chew... i read up on some biochemical tests avaliable in my lab... so here they are:

The Indole Test

The organism is inoculated into tryptone broth. Indole positive bacteria such as Escherichia coli produce tryptophanase, an enzyme that cleaves tryptophan, producing indole and other products. When Kovac's reagent is added to a broth with indole in it, a dark pink color develops. The indole test must be read by 48 hours of incubation because the indole can be further degraded if prolonged incubation occurs. The acidic pH produced by Escherichia coli limits its growth.

The Citrate Test

The citrate test utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate as its sole carbon and energy source. Simmon's media contains bromthymol blue, a pH indicator with a range of 6.0 to 7.6. Bromthymol blue is yellow at acidic pH's (around 6), and gradually changes to blue at more alkaline pH's (around 7.6). Uninoculated Simmon's citrate agar has a pH of 6.9, so it is an intermediate green color. Growth of bacteria in the media leads to development of a Prussian blue color (positive citrate). Enterobacter and Klebsiella are citrate positive while E.coli is negative.

Triple sugar Iron (TSI) - & Hydrogen sulfide production (H2S)

Examines fermentation of glucose, lactose, and sucrose and checks if hydrogen sulfide is produced in the process. A pH indicator will change the color of the media in response to fermentation. Where the color change occurs in the tube will indicate what sugar or sugars were fermented. The presence of a black color indicates that H2S was produced. To inoculate, use a needle to stab agar and then uses a loop to streak the top slated region.

SLANT COLOR:
Interpretation
RED
Does not ferment either lactose or sucrose
YELLOW
Ferments lactose and/or sucrose

BUTT COLOR/CONDITION:
Interpretation
RED
No fermentation of glucose
YELLOW
Some fermentation of glucose has occurred, acid has been produced
GAS FORMED
Seen as cracks in the agar, bubbles, or the entire slant may be pushed out of the tube.
BLACK
H2S has been produced

Motility test

This test can be used to check for the ability of bacteria to migrate away from a line of inoculation thanks to physical features like flagella. To perform this test, the bacterial sample is inoculated into motility media using a needle. Simply stab the media in as straight a line as possible and withdraw the needle very carefully to avoid destroying the straight line. After incubating the sample for 24-48 hours observations can be made. Check to see if the bacteria have migrated away from the original line of inoculation. If migration away from the line of inoculation is evident then you can conclude that the test organism is motile (positive test). Lack of migration away from the line of inoculation indicates a lack of motility (negative test result).

Oxidation - fermentation tests (OF test)

This biooxidation test is used to decide the oxidative or fermentative metabolism of a carbohydrate or its non-utilization.

A. OF Media (green colored)
Use for Gram-negative rods.
yellow = positive green or blue = negative

B. CTA Media (orange colored)
Use for Gram-positive cocci.
yellow = positive orange or red = negative

Nitrate test

This is a test used to detect the presence of nitrite. Some facultative bacteria convert nitrate to nitrite by removing an oxygen from nitrate using the enzyme nitratase.

Interpretation:
After incubation, the broth must be turbid. A clear broth indicates that your organism did not grow and cannot be tested. Add several drops of 2% acetic acid to the broth culture. Shake gently. A positive reaction is shown by production of a dark color.

2007-08-25T09:29:00.000-07:00

Name :Dorene Chen

Hello people, it's my turn to write blog again.

To date, I have been to biochemistry (done blog), haematology and serology department.

Serology

The tests I will be going through are:

1)AMC (Anti microsomal/thyroid peroxidase antibodies = same thing but different name)
2)ATG (Antibodies to ThyroGlobulin).

3)ANF slide (Anti nuclear Factor)

AMC/ ATG testing ( methods are the same so i join them together =D)
AMC/ATG testing tests for presence of autoimmune antibodies. It uses gelatin particles sensitized with human Thyroglobulin (Tg) or thyroid peroxidase (TPO) antigen for agglutination. serum containing specific antibodies will react with gelatin particle to form a smooth mat of agglutinated particles in the microtitration plate (aka "ELISA" plate) testing tests for

Autoimmune thyroid disease is autoimmune disease of the thyroid gland in which the patient's own immune system attacks his own thyroid. In patients suffering from this disease may secrete anti thyroglobulin and Anti microsomal antibodies against their own thyroid antigen (thyroglobulin, microsomal/thyroid peroxidase antigen are found in thyroid).

Thyroglobulin (Tg) is a protein found and produced by thyroid and T3, T4 are produced from this protein. T3, T4 are hormones that aids in transporting protein, controlling metabolism and they affect almost every process in the body.

Microsomal/ thyroid peroxidase (TPO) is an enzyme in thyroid that aids in the function of producing T3, T4 through oxidising iodide compounds in blood and to incorporate the oxidized iodide compounds into T3, T4.

Method (uses multi-well/microtitration plate aka "ELISA" plate):

Note: the picture above shows only the reagents to add in one row.
1)add 50µL of sample diulent to 1st well. Add 75 µL to 2nd-6th well.
2) add 10µL of positive control to first well. add 10 µL of sample to other row in the multiwell plate.
3) Do serial dilution by taking 25µL of suspension from 1st well into 2nd well. pipette and mix well. From 2nd well, pipette another 25 µL to 3rd. repeat until 6th well. discard away the 25µL from the 6th well.
4 ) Add 25µL unsensitized particles ( these particles will not cause any reaction, act as control) only to the 2nd well of the control.
5) Add 25 µL sensitized particles (these particles cause agglutination when antibodies is present) to the other well.
6) Incubate for 3 hours.

Positive result: Form large ring (dark purple color background with light purple ring shape in the center cause by agglutination). For very strong reaction, the dark purple color background disappears completely.

Negative result: Button shaped (Purple color throughout)

2007-08-17T19:48:00.000-07:00

Name: Teng Cassandrea
Laboratory: Biochemistry

Neonatal serum bilirubin

o Breakdown of hemoglobin>>globin + heme
o Globin + iron >>re-utilized, leaving porphyrin portion of heme
o Breakdown of heme occurs in reticuloendothelial system
o Porphyrin ring opened up to form biliverdin
o Biliverdin>>reduced to bilirubin
o Bilirubin>>transported in plasma to liver>> conjugated with glucuronic acid
o Conjugated bilirubin>>further broken down in the intestines by intestinal bacteria>>urobilinogen
o Various reduction products of bilirubin may be absorbed from intestine>>returned to liver for re-excretion
o Any overload or blockage of this system raises levels of plasma total bilirubin

At least 80% of plasma bilirubin is unconjugated or indirect. When bilirubin in the blood is excessive, it deposits in the tissues, which causes jaundice. It may be caused by:
o Production of bilirubin more than the normal liver can excrete (e.g. hemolytic disease).
o Failure of the damaged liver to excrete the bilirubin produced in normal amounts (e.g. hepatitis).
o Obstruction of the excretory ducts in liver.

Neonatal physiological jaundice is common in newborns due to an immature fetal liver. The liver cannot conjugate all the bilirubin produced because of deficient enzymes. This jaundice however, disappears as the liver matures and enzymes function improves.

Upon receiving the patient samples, the samples are spun down so that the serum can be obtained for an accurate reading. The samples are that loaded into the REICHERT UNISTAT Bilirubinometer using a curvette. The readings are the recorded onto the request form and left to be verified.

G6PD screening test

o G6PD>>key enzyme in hexose monophosphate pathwayàrequired for formation of NADPH.
o NADPH>>essential for maintaining integrity of erythrocyte membrane
o G6PD deficiency may be the cause of hemolytic disease of newborn
o Drug-induced hemolytic anemia is most commonly associated with erythrocyte deficiency of G6PD.
o Deficiency is due to presence of G6PD enzyme that is present in young cells but rapidly disappears with cell aging.
o Disease is transmitted as an X-linked recessive.
o Full-expression occurs also in females who are homozygous for the gene; intermediate expression occurs in the heterozygous female carrier
o Fluorescent spot test is used to screen for G6PD deficiency
o NADPH produced in the reaction fluoresces under long-wave UV-light
o If there is a marked deficiency of G6PD or if this enzyme is lacking entirely, no fluorescence will be observed.

Samples for G6PD screening are usually cord blood for routine screening of newborn. The samples should always be received in EDTA tubes to prevent blood from clotting. Four types of control are used for this test; intermediate, blank, deficient and positive. The positive control is taken from the patient sample and is changed everyday. First, 100 microlitres of buffer is aliquoted into labeled tubes. This is followed by 5 microlitres of the patient’s blood. It is mixed left to incubate for 10 minutes. After the incubation, the samples are placed on filter paper and left to incubate for another 10 minutes. Over-incubating will cause false results; hence a timer must always be set. The filter paper is then read under the UV-light.

If the results are weak or negative, the test must be repeated and the sample must be checked for blood clots. If the blood is clotted, the nurse must be contacted to take a fresh sample of blood in an EDTA tube.

Urine FEME

o Kidneys maintain the internal environment by selectively excreting or retaining various substances accordingly to specific body needs
o They consist of a number of smaller functional units>>urinary tubules or nephrons
o Nephrons open to large collecting ducts, which open into a ureter.
o The two ureters run backward to open into a urinary bladder
o One of the main functions of the kidneys is the removal of waste products such as urea, uric acid, and creatinine from the body
o They also regulate volume and composition of body fluids and maintain correct osmolality, ion concentrations, and acid-base status of body
o First step in the production of urine is filtration of plasma passing through kidneys.
o Filtration occurs from glomerular capillaries into Bowman's capsule to form tubular fluid
o Glomerular filter prevents plasma proteins from passing into nephrons, but is permeable to all other plasma constituents (such as ions, glucose, amino acids, urea, etc)
o Thus filtration in the kidney is essentially non-selective — substances, which the body needs to retain, are filtered, as well as those substances, which need to be excreted.
o Urine analyzed to detect body disturbances>>endocrine or metabolic abnormalities, in which kidneys function normally but excrete abnormal amounts of abnormal amounts of metabolic end products specific for a particular disease.
o Analysis is also done to detect for intrinsic conditions that may adversely affect kidneys or urinary tract.
o Diseased kidneys cannot function normally in regulating volume and composition of body fluids, and in maintaining body homeostasis
o Consequently, substances normally retained by kidney or excreted in small amounts may appear in urine in large quantities, or substances normally excreted may be retained.
o Structural elementsàRBCs, WBCs, epithelial cells from urinary tract, and cast from diseased kidneys may appear in the urine
o Crystals, bacteria, yeast, protein, nitrite, glucose, ketone bodies, urobilinogen and bilirubin can also be found in the urine
o Different types of crystals:
§ pH<7 Amorphous nitrate
§ pH>or= 7 Amorphous phosphate
§ Calcium oxalate
§ Uric acid
§ Triple phosphates
§ Ammonium nitrate
§ Di-calcium phosphate
§ Calcium carbonateCalcium sulphate

Haematology

2007-08-11T08:07:00.000-07:00

Hello everyone! I've been attached to the haematology department for the past 7 weeks. There is 1 test that I'll like to share with you'll.

D-dimer Test (coagulation test)

The specific degradation of fibrin(i.e. fibrinolysis) is the mechanism responding to fibrin formation. Plasmin is the fibrinolytic enzyme that degrades fibrin into D-dimer. Hence, its appearance in the plama indicates that the fibrinolytic system is working in response to coagulation activation.

Coagulation Activation Plasmin
Fibrinogen -------------->Fibrin------------>D-dimer

D-dimers are crossed-linked degradation products of fibrin. D-Dimers are often associated with deep venous thrombosis(DVT), disseminated intravascular coagulation(DIC) and embolisms.
Hence, D-dimer can serve as a screening test for DVT.

Test Principle
D-Dimer test is a rapid latex agglutionation slide test that uses mouse monoclonal antibodies for semi-quantitative determination of D-dimer in the human plasma. The latex particles in the D-dimer reagent are coated with mouse anti-human D-dimer monoclonal antibodies. If the patient's plasma contains D-Dimes, it will cause agglutination when mixed with the latex particle suspension.

Test Procedure
1) The patient's plasma sample is checked against the name on the lab test request form.
-Neat Report-
2) 20uL of patient's plasma is pipetted into 1 circle on the black test card.
3) 20uL of D-dimer reagent is pipetted into the same circle on the test card.
4) The 2 drops on the test card are mixed using a mixing rod.
5) The test card is manually rotated for 2 mins in a manner such that the liquid swirls around the entire test circle.
6) When 2 min is up, check for agglutioination (i.e.bluish-white dots).
7) If no agglutination is observed, it is reported as D-dimer level<0.5ug/ml>

If agglutination is observed, proceed to semi-quantitative determination of D-dimer using serial dilution.

-Semi Quantitative Determination-
1) 5 test circles are labelled as 1:2, 1:4, 1:8,1:16 and 1:32.
2) 20uL of D-dimer reagent is added to all the 5 test circles.
3) Similarly, 20ul of buffer is added to all the 5 test circles.
4) 20ul of patient's plasma is added to only the test circle labelled 1:2.
5) The patient's plasma is serially diluted with the buffer in the remaining test circles.
6) The reagent in the test circles are mixed using a mixing rod, in the following sequence, from 1:32 to 1:16 to 1:8 to 1:4 to 1:2.
7)The test card is manually rotated with 2 mins.
8) When 2 min is up, check for agglutination.
9) Plasma D-dimer levels are obtained by multiplying the dilution number by 0.5ug/ml. E.g. If 1:8 is the highest dilution that produces agglutination, the plasma D-dimer level=0.5ug/ml x8= 4ug/ml. Hence, plasma D-dimer level is reported as >4ug/ml.

Maybe this diagram cn help you'll to understand better..

Finally..That's the end of my post. Have fun reading it ahh.

Cheong Shu Hui
TG02
0503277F

(Sasi's) Q and A

2007-08-10T10:07:00.000-07:00

Q1. Refer to Najib’s

Answer: The persistence of crystals in the urine can lead to inflammation of the mucus lining of the bladder and urethra. This can cause clinical signs identical to urinary tract infection, or the subsequent compromised state of the bladder mucosa lead to urinary infections. Chronic urinary crystals can also form bonds with one another, leading to the formation of stones within the bladder. Stone cause severe inflammation of urinary bladder, leading to blood in the urine, pain, and chronic infections. Kidney stones can obstruct the urinary passages and cause severe pain, and bleeding to the patient.Treatment of urinary crystals is accomplished through prescription diets that stablize the urine pH and eliminate dietary factors that lead to crystal formation. Small bladder stones can be treated by dissolution with the appropriate prescription diet like u had mentioned in your question. However, larger stones usually have to be removed surgically, and the patient has to placed on a good prescription diet to prevent their recurrence. Calcium oxylate stones cannot be dissolved with diet and have to be removed surgically. However, effective diets exist to prevent their recurrence. If the crystals do not pass out of the body naturally, the physician may use shock wave treatment. In this treatment, a shock wave focused on the stone from outside the body disintegrates it.

Q2. Refer to Dorene's

Answer:
Common Causes of Urine Discolouration

Brown:
Pathologic causes: Bile pigments,myoglobin
Food and drug causes: Levodopa, metronidazole, nitrofurantoin, some antimalarial agents

Brownish-black:
Pathologic causes: Bile pigments, melanin, methaemoglobin
Food and drug causes: Cascara, levodopa, methyldopa, senna

Green or blue:
Pathologic causes: Pseudomonal UTI, biliverdin
Food and drug causes: Amitriptyline, indigo carmine, IV cimetidine,IV promethazine, methylene blue, triamterene

Orange:
Pathologic causes: Bile pigments
Food and drug causes: Phenothiazines, phenazopyridine

Red:
Pathologic causes: Haematuria, aemoglobinuria, myoglobinuria, porphyria
Food and drug causes: Beets, blackberries, rhubarb Phenolphthalein, rifampicin

Yellow:
Pathologic causes: Concentrated urine (orange to gold in dehydration)
Food and drug causes: Carrots, Cascara

(fyi) Turbidity: Cloudy urine may be due to excess phosphate crystals precipitating in alkaline urine, which is of no clinical significance. However, it can also be seen in pyuria secondary to infection, chyluria (usually secondary to filariasis), hyperuricosuria secondary to a diet high in purine-rich foods, lipiduria and hyperoxaluria. Dorene, hope the above information would have cleared your doubts and also the part on Turbidity.

Q3. Refer to Lizzie's

Answer:
Whenever a Myco test is done, we compare the results to the positive control results. Yes, there is sure a chance for a false positive result to occur if the wells were subjected to agitation.
- Basically, a definite compact button in center of well with a smooth round outer margin indicates a negative result. A definite large ring with firmly agglutinated particles spread within the ring indicates a postive result.
- A specimen showing (-) with Unsensitized particles (at 1:20 final dilution) but demonstrating (+) or more with Sensitized particles (at 1:40 final dilution) is interpreted as positive. The end antibody titre is determined as the final dilution giving a (+) pattern.
- Regardless of the reading of the reaction pattern with Unsensitized particles, a specimen showing (-) with Sensitized Particles (at 1:40 dilution) is interpreted as negative.

Q4. Refer to Sally's

Answer:
hemolytic anemia is a disorder characterized by the premature destruction of red blood cells (rbcs) by the body's natural defenses against invading organisms. Hence, an elevated level of agglutinin will result in the premature destruction of rbcs. Normally, the red blood cells have a life span of approximately 120 days before they are destroyed by the spleen. In individuals with hemolytic anemia, the red blood cells are destroyed prematurely and the rate of production of new cells in the bone marrow can no longer compensate for their loss. The severity of the anemia is determined by the length of time that the red blood cells survive and by the rate at which the bone marrow continues to create new red blood cell production.

(fyi) The immune hemolytic anemias are classified according to the optimal temperature at which the antibodies act to destroy red blood cells. As their names imply, cold antibody hemolytic anemia occurs at temperatures of approximately 0 to 10 degree celsius (temperatures colder than normal body temperature), while warm antibody hemolytic anemia (WAHA) occurs at temperatures of 37 degrees celsius or higher.

Q5. Refer to Jia Hao's

Answer: There are two common types of hCG tests. A qualitative hCG test detects if hCG is present in the blood. A quantitative hCG test (or beta hCG) measures the amount of hCG actually present in the blood. First of all, you have to understand the purposes of both the tests. A qualitative hCG test is done via the commercial test strips to detect if a woman is pregnant in the first place. Quantitative hCG test is done to guage roughly how long has it been since the woman got pregnant.

In my experience at the lab, the manual testing method using the commercial strip is sensitive enough a method to see if the woman is pregnant. This is done at the Microbiolody Lab. On the other hand, at the Serology lab, we carry out the quantitative hCG test (or beta hCG) whichmeasures the amount of hCG actually present in the blood using the Immunolite Analyzer. For that, we do use the patients' serum for testing. This, like I mentioned, is used to roughly guage how long has the woman been pregnant.

(fyi) hCG levels during pregnancy:

hCG levels in weeks from LMP (gestational age) :
3 weeks LMP: 5 - 50 mIU/ml
4 weeks LMP: 5 - 426 mIU/ml
5 weeks LMP: 18 - 7,340 mIU/ml
6 weeks LMP: 1,080 - 56,500 mIU/ml
7 - 8 weeks LMP: 7, 650 - 229,000 mIU/ml
9 - 12 weeks LMP: 25,700 - 288,000 mIU/ml
13 - 16 weeks LMP: 13,300 - 254,000 mIU/ml
17 - 24 weeks LMP: 4,060 - 165,400 mIU/ml
25 - 40 weeks LMP: 3,640 - 117,000 mIU/ml
Non-pregnant females: <5.0>

- These numbers are just a guideline-- every woman’s level of hCG can rise differently. It is not necessarily the level that matters but rather the change in the level.

- In about 85% of normal pregnancies, the hCG level will double every 48 - 72 hours. As you get further along in pregnancy and the hCG level gets higher, the time it takes to double can increase to about every 96 hours.

- A single hCG reading is not enough information for most diagnoses. When there is a question regarding the health of the pregnancy, multiple testings of hCG done a couple of days apart give a more accurate assessment of the situation.

Q6. Refer to Vino's

Answer:

Upon the reasearch I have done, IgM type antibodies are stated as the most common type of cold agglutinin. However, there was this another antibody I came across named the Donath Landsteiner antibody. The Donath Landsteiner antibody is an IgG autoantibody that binds to red cells in the cold and fixes complement. Lysis occurs when cells are warmed to 37°C. Presence of the antibody is diagnostic for paroxysmal cold haemoglobinuria.

Specimen is collected into 2 plain tubes, one at 37°C and one at 4°C since cold agglutinins cause red blood cells to clump only at temperatures lower than 37 degree celsius. Supernatant serum examined for evidence of red cell lysis, comparing a tube incubated at 4°C then 37°C with a tube maintained at 37°C. No lysis in either tube indicates a negative result.

Q7. Refer to Royston's

Answer: Actually, as mentioned by my Lab personnel, controls not tallying is a rare occurrence as both the positive and negative controls are commercially prepared by manufacturers who have already run internal Qc for those controls before marketing them. However, in the event whereby the controls results do not tally with the previous day's, the controls will be run again. A different pair of positive and negative controls would be run this time. If despite that, the controls do not tally with the previous day's, the analyzer has to be calibrated again. Calibration refers to the process of determining the performance parameters of an artifact, instrument, or system by comparing it with measurement standards. Calibration assures that a device or system will produce results which meet or exceed some defined criteria with a specified degree of confidence. For example, my lab personnel from the microbiology lab mentioned that the photometer of the urine analyzer is blanked and restarted in the calibration process.

Hope Ive answered your questions. Feel free to clarify any more doubts of yours pertaining to my answers especially. Thank you once again for reading my posts! :)

Sasi

0503804g

Tg02

Cytogenetics

2007-08-06T05:46:00.000-07:00

Unstimulated BM/ bone core/ leukaemic blood culture setup

Purpose: To establish haematopoietic cell cultures grown in culture flasks for cytogenetic analysis of haematological disorders.
BM is ideal specimen for chromosome studies in leukaemia. When BM aspirate is unsuccessful or is a dry tap, bone core or leukaemic blood may be used instead.
Blood may be taken in a sodium heparinized vacutainer .

Overnight culture set up (24- hour culture set up)

1.For marrow collected in sodium heparin vacutainer tube, pipette half the amount of BM aspirate into the Direct harvest Medium for direct harvest.

2.Obtain the recent FBC result from haematological lab and record in the BM culture worksheet.

3.Use the WBC as a guide to determine the number of drops of BM into the culture media.

4.Thaw one flask of complete RPMI 1640 culture media in a 37oC water bath for 20 mins

5.Place the specimen tube in bucket (a container to hold the tubes during centrifugation), screw tight the lid cover and centrifuge at 1500 rpm for 10 mins

6.Pipette the buffy coat out of specimen tube with a sterile 3 ml plastic transfer pipette into a sterile centrifuge tube.

**Buffy Coat > it is a layer of white cells which can be used to determine the cell lineage pathway

7.Sometimes, in order to assess the cellularity of the BM specimen (to determine the number of drops of buffy coat into the centrifuge tube). Smear and staining is also done and observes under the microscope (similar to our BBANK smear and stain). If there are more white cells we call it hyperplastic and low WBCs, we call it as hypoplastic. In case of hyperplastic, we only use ½ or 1 drop of the buffy coat ( > 1 drop will result in increased growth cells leading to insufficient space for growth). Hypoplastic BM, we will add about 3-4 drops.

8.After which, the buffy coat is then transferred to a RPMI 1640 culture media and placed in the CO2 incubator for overnight.

**For the steps are similar for 48-, 72- hour culture too. Interleukins are added before placing the culture flasks into the CO2 incubator.

LMQA

1.Safety of all lab staff is very important especially when handling the samples

2.It is also very important to maintain the temperature of refrigerator, incubator, freezer and other lab equipment as all these will have an effect on the sample.

3.All equipment is calibrated everyday early in the morning and is recorded. All QC forms are reviewed periodically by QC officer to ensure the equipment functions and perform according to specification

4.QC forms must be used for recording the performance check and maintenance on each piece of equipment

5.All documented QC forms are filled

Vinodhini

TGO2

CYTOGENETICS ( BONE MARROW)

2007-08-05T05:49:00.001-07:00

Hi Guys!!! This is already the sixth week of our attachment and here i am to tell you and share with you ppl abt my experience in CYTOGENETICS lab. Til today, i am enjoying myself working in this lab. There are three different stations in cytogenetics lab. One is prenatal (lab) where you will handle amniotic fluid samples, bone marrow (lab) where you will get to know more about haematological diseases and finally FISH which is actually aneuploidy screening of prenatal specimen. Currently i am under BM in short for bone marrow lab. I have lots to say but I will be covering the basics of cytogenetics, procedures, etc.

What is Cytogenetics?

Cytogenetics is the study of chromosome strucutre, pathology, function and behaviour. Chromsomes are best studied at mitotic or meiotic metaphase. Studies such as FISH methods may utilize interphase cells.

The type of sample and culture techniques used in our lab:

Basically we receive bone marrow, bone core and sometimes perpheral blood for chromosome analysis. We receive both local and regional samples. For local cases (the day you collect the bBM from the patient and the day you receive the sample is the same date) we have to perfom two different cultures. One is Direct Harvest and the other one is either one of these, 24-, 48-, 72- hour culture with or without mitogen (growth factors) depending on the diagnosis of the disease. As for Regional cases (if the date of collection of specimen is different from the date we receive is different) we can only set up only one or two (24-, 48-, 72- hour) cultures but not Direct Harvest. The reason for this will be explained shortly!

What is Direct Harvest and 24-, 48-, 72-hour culture??

Direct harvest: This is done to capture only spontaneously dividing cells (both abnormal and normal ones) upon arrival of the sample.

Culture: Cells are cultured for the study of non-spontaneouls dividing components or cells. The type of culture (whether it is 24-, 48-or 72- hour) depends on the diagnosis of the disease. E.g in my lab we usually perform 24-hour culture (also known as unstimulated culture due to the absence of mitogen = growth factor) for myeloid disorders one such example would be acute myeloid leukaemia (AML). As for lymphoid disorders, we will usually do a 48-hour culture with Interleukin-2 (which is actually a growth factor) and this is known as stimulated culture due to the addition of interleukin. One e.g. of lymphoid disorder would be Acute lymphoblastic leukaemia( ALL). As for 72-hour culture, we will usually do this for Myeloma cases (disease) as the cells will grow very slowly. Interleukin-6 is added for Myeloma cases. As you guys know the interleukins are produced in the body and these are required for certain abnormal clones or cells to grow (in the body itself). This is the reason why we add interleukins (to provide in vivo environment).

What i am trying to say is some abnormal cells or clones will grow under different conditions. Certain cells gorw very rapidly and this can be captured only in Direct Harvest culture. However, there are also cells that grow very slowly (1 to 3 days) and in order to capture these cells after you cultured them ( providing the in vivo environment for the cells to grow).

The procedures and the purpose of these procedures:

1) Harvesting: The first step in most harvest procedure is to arrest cells in the stage in which they must be in for standard cytogenetic analysis: Metaphase or prometaphase. In order to arrest cells under metaphase stage we have to add Colcemide. Colcemide will stop all the cells at metaphase stage for analysis. The effect of colcemid is to prevent spindle fiber formation, which would normally pull the sister chromatids to opposite poles for incorporation into two daughter cells. It also causes the chromosomes to condense, a process which is accentuated by increasing the time of exposure and concentration.

2) Hypotonic treatment: The second major step in harvesting is treament with a hypotonic saline solution to increase the cell volume so that the chromosome can find or have sufficient space to spread out ( to get beautiful metaphase to analysis) *i will try to get photos to show you ppl wat i mean by "beautiful metaphase"*

*Note: the above two solutions are prepared in our lab and both solutions can be found in one tube *

3) Fixation: The third constant feature of chromosome harvesting is the fixation of the cells. This process removes water from the cells and preserves them, hardening membranes and chromatin and in some way, preaparing the chromosome for banding (staining) procedure.
Fixative used: 3:1 methanol acetic acid. Fixative will cause the cells to be stronger ( harden the cell membrane). Cells are then kept in the fridge for slide making. During the fixative process, red cells present in the sample are also removed thus giving us a clear suspension. Basically fixation strengthens the cells and also gives a clear suspension for slide making. ( Fixation = washing of cells)

4) Slide Making: Once cells have been well fixed, they are then dropped onto glass slides and dried using specific condotions for optimal chromosome spreading and morphology.
**Theory of air drying: As the fixative evaporates, the layer of fixative becomes thinner and the meniscus pushes down on the top of the cell, enlarging the area of the cell and pressing the metaphase between the upper and lower membranes, spreading them out. We want the chromsomes to spread so that suring analysis we wont get chromsomes that are clumped together thus not allowing us to analyse them clearly.

After slide making, the slides are then stained using the Wrights and Giemsa stain. This is known as GTG banding. the G bands by trypsin using Giemsa and WRight's stain. Giemsa/Trypsin banding invloves the treatment with a proteolyitc enzymes trpysin and subsequent staining with a combination of Romanowsjy dye (Giemsa and Wright's stain). Dark bands generally replicate their DNA in late S phsase, contain A + T rich DNA, appear to contain relatively few active genes and differ from light ands in terms of protein composition. THe G bands by this technique allows the identification of individual chromosomes and structural rearrangements.

After staining, the slided are then covered with the coverslip (using DPX) and dried for about 30 mins and the slides are ready for microscopic analysis.

Guys there is more to tell you but i dun want to bombard you guys with so much of info.. I try to summarize as much as i can. So if there is anything u are not sure abt please feel free to ask me.. As for the photos i will try to put them up asap so that u will noe wat am trying to say about metaphase chromosomes etc etc.. Alrite?? And sorry if this is too long!!! I try to shorten but i couldn. I think i have to do another posting to tok about LMQA. I will do it asap ok! SOrry for the late posting too!!

Have great week ahead ppl!

Vinodhini
TGO2
0503171A

Pictures of urinary casts and other urinary cellular components

2007-07-29T19:21:00.002-07:00

Spermatozoa

Renal Epithelial cells

Yeast cells (notice the budding)

Cellular cast

Granular cast

Hyaline cast

Sasi
Tg02
0503804g

Urinary Crystals pictures and brief infor (sasi's)

2007-07-29T19:21:00.001-07:00

Uric Acid crystals

Calcium Oxalate crystal (look out for the 'X' )

Triple phosphate crystal (coffin like)

Crystal formation in urine can be caused:

- By an augmentation of concentration beyond the supersaturation capacity. This situation is mostly the result of a decreased dilution like in a case of insufficient water intake.

- By a decreased supersaturation capacity. This situation could be caused by a decrease in inhibitors concentration, a neutralization of these inhibitors, by some electrolytes, or a by pH change.

- By the presence of crystals with a promoter effect on the crystallization of another species. Crystallization of calcium oxalate promoted by amorphous urates is a good example of this phenomenon.

Usual urine crystals found in Urine:
Alkaline pH
Amorphous phosphates
Triple phosphates

Ammonium biurates

Calcium phosphates
Calcium carbonates

Acid pH

Amorphous urates
Uric acid

Calcium oxalates

Cystine

Sasi

Tg02

0503804g

Microbiology and Serology

2007-07-29T19:21:00.000-07:00

Hi everyone,
Sasi here reporting from one of the private labs in Singapore. The first three weeks of my attachment I was attached to the Microbiology department. I basically had to run urine analysis via the URISYS 2400 urinalysis analyzer. The purpose of UAN (Urine Analysis) is for the semi-quantitative determination of pH, leukocytes, nitrites, protein, glucose, ketone bodies, urobilinogen, bilirubin, and erythrocytes in urine. Urine test strips are used to measure certain constituents in urine which signify renal, urinary, hepatic and metabolic disorders. In the mornings, I would start off by running the controls for the machine by loading the thawed controls. Then, I would record down both the positive and negative control results and make sure they tallied with the previous days' control results. In the afternoon, majority of the urine samples would begin to arrive. The urine specimen must be collected in a clean, dry container, either plastic or glass without preservatives. Once the specimens were received, they were labelled with the appropriate barcodes and loaded into the machine. When loading the samples, I had to make sure the sequence number on the monitor matched the first sample that was going to be loaded so that the results can be easily tracked down (via the sequence number) in the future in case of an emergency. This whole process of running the samples had to be done fast and accurately in order for the other follow up processes to take place. One of the follow up steps was to centrifuge the samples, print out the results of the Una, literally highlight the abnormal results and hand them over to the microscopy section of where urine FEME (Full examination microscopic examination)will be conducted.

The urine analysis results include:
- a description of color and appearance
- Specific Gravity- This detects ion concetration of the urine. Small amounts of protein or ketoacidosis tend to elevate results of the specific gravity.
- pH- pH of healthy individuals is usually between 5 and 6.
- glucose
- ketone bodies
- protein
- urobilinogen
- RBC number
- WBC number

The numbers and types of cells and/or debris present can bring about a great detail of information and may suggest a specific diagnosis.

Eosinophiluria - associated with allergic interstitial nephritis, atheroembolic disease
RBC casts - associated with glomerulonephritis, vasculitis, malignant hypertension
WBC casts - associated with acute interstitial nephritis, exudative glomerulonephritis, severe pyelonephritis
(heme) granular casts - associated with acute tubular necrosis
crystalluria -- associated with acute urate nephropathy (or "Acute uric acid nephropathy", AUAN)
calcium oxalate - associated with ethylene glycol toxicity

Also, the presence of urinary casts presence under microscopy evaluation hold significance as diagnostic and prognostic indicators of kidney disease. They are cylindrical and they are generated in the small tubules and collecting ducts of the kidney, and they generally maintain their shape and composition as they pass the lower parts of the urinary system.

Some common casts I spotted during my experience included:

Hyaline casts: Hyaline casts are solidified Tamm-Horsfall mucoprotein secreted from the tubular epithelial cells of individual nephrons. Low urine flow, concentrated urine, or an acidic environment can contribute to the formation of hyaline casts and thus, they may be seen in normal individuals in dehydration or vigorous exercise. They are cylindrical and clear.

Granular casts: They can result either from the breakdown of cellular casts, or the inclusion of aggregates of plasma proteins (eg, albumin) or immunoglobulin light chains. Depending on the size of inclusions, they can be classified as fine or coarse, though the distinction has no diagnostic significance. Their appearance is generally more cigar-shaped and of a higher refractive index than hyaline casts.

Epithelial cell casts: They are formed through inclusion or adhering of desquamated epithelial cells of the tubule lining. Cells can adhere in random order or in sheets, and are distinguished by large, round nuclei and a lower amount of cytoplasm. These can be seen in acute tubular necrosis and toxic ingestion, such as from mercury, diethylene glycol, or salicylate. Cytomegalovirus and viral hepatitis are organisms that can cause epithelial cell death as well.

In addition, I also did manual pregnancy testing(UPT- Urine Pregnancy testing) using a commercial kit called StanbioQuStick test kit. Its intended use was for the visual qualitative detection of the hormone hCG in urine. Human chorionic gonadotropin(hCG) is a glyco protein hormone secreted by the developing placenta after fertilization. When the test strip is placed into a vessel of urine, the urine migrates upward, transporting the coloured reagent onto the surface of the dye particles by immuno-reaction. Visual formation of one line is read as negative and two coloured lines represent a positive result.

I also observed how stool samples are tested using commercial test kits, for example to test for fecal occult blood. Also, for amoeba identification in the stool sample, the sample had to be stained with normal saline solution and iodine before microscopy examination. Besides, I also had the chance to observe how urine streaking was done on the culture plate. Basically, it was different from the streaking we normally do in school. In the micro lab, they streak it in the form of an inverted christmas tree.

Ok this marks the end of my micro lab experience. Feel free to ask me questions.

The following 3 weeks after microlab, I was attached to the serology department which I am in till now. Its the same lab Yeng Ting( from Med bankers group) was attached to in her first 3 weeks. Hence, the techniques she has posted in her blog are exactly the same as what I am required to do as well. So, please kindly refer to her blog. However, apart from what she has wrote about, I want to highlight another two particular tests I did.
One of them is is the WWF test. Not world wrestling federation or watsoever ah.. It is called the 'Widal Weil Felix ' test.
This test measures the level of warm or cold agglutinins in blood. Agglutinins are antibodies that cause the red blood cells to gather together. Cold agglutinins are active at cold temperatures. Warm agglutinins are active at normal body temperature.
These antibodies can cause a hemolytic anemia. This occurs when the body destroys its own red blood cells. Distinguishing between warm and cold agglutinins can help understand why the hemolytic anemia is occuring and directs therapy.
Basically, there will be 12 reagents present. These reagents help test for the different types of salmonella. In our lab, this is the main focus for WWF.
I will have to pipette out 40 microlitres of serum 12 times onto 12 test circles and also a drop of each reagent 12 times onto the same 12 test circles and let them mix on a rotator for 1 min. The timing is crucial as after 1 min, the results may be false positive.
If there is agglutination present, titre must be done.

Normal Values:
Warm agglutinins: no agglutination in titres at or below 1:80
Cold agglutinins: no agglutination in titres at or below 1:16

Abnormal Values mean:

Elevated levels of cold or warm agglutinins can cause hemolytic anemia. Some patients with moderately elevated levels will have no hemolysis and most likely won't require therapy.
The presence of warm agglutinins may occur with:
- Infections, including brucellosis, rickettsial disease, salmonella infection and tularemia Lymphoma
- Lymphoma
- Systemic lupus erythematosus
- Medications, including methyldopa, penicillin, and quinidine

The presence of cold agglutinins may occur with:
- Infections, especially Mycoplasma pneumonia. Also after viral, staphylococcal, and malaria infection.
- Cancer, including lymphoma and multiple myeloma
- Systemic lupus erythematosus

The other test I did was the mycoplasma pneumoniae test using the SERODIA-MYCO commercial test kit. It is designed for the sole purpose of detecting anti-Mycoplasma pneumoniae. Mycoplasma pneumonia is an infection of the lungs caused by Mycoplasma pneumoniae. Symptoms include fever, dry cough, sore throat, headache, chest pain etc.

Test method:
1. 100 microlitre of diluent in the first well.
2. 25 microlite of diluent in the 2nd-6th well.
3. 25 microlitre sample into the first well and mix and carry over 25 microlitre onto the subsequent wells (till the 6th well) and then discard the last 25 microlitre.
4. Add a drop of unsensitized cells to the 2nd well.
5. Add a drop of sensitized cells to the 3rd, 4th, 5th and 6th well.

Results:
If there is a distinct button formed in the middle of the well, it is negative. If there is no distinct button but a cloudy pinky suspension, its is positive. You have to note till which well this suspension forms and record the titre accordingly.

Alright, this again marks the end of my experience at serology department. Feel free to ask me questions. Thank you for reading!

Sasi
Tg02
0503804g

Q & A

2007-07-27T06:58:00.000-07:00

Hey guys, before I answer any question, I would like to apologise about one mistake that I have made. I mentioned PMS in my previous post I said that it stands for Personal Security Manager. Actually it means Process System Manager. Sorry about that.

Why is G6PD commonly done on newborns and what are the symptoms involved?
G6PD deficiency is an inherited condition due to gene defects. As this condition is silent (not much symptoms) and G6PD also plays an important part in metabolic pathway of RBCs (anaerobic respiration), an early detection will allow both patient and the doctor to know what are the thing that patient should avoid such as medication, certain strenuous exercise and contact with certain stuff or even infection so as to prevent the worsening of the condition.
Basically the patient behaves like any normal person. They do not display any symptoms unless they touches some things or took certain medications that they should avoid. Only then will they display symptoms of one who has hemolytic anaemia, example difficulty in breathing, paleness, tiredness, rapid heartbeat. However, once the trigger is removed or resolved, the symptoms of G6PD deficiency usually disappear fairly quickly, typically within a few weeks. Because we want to avoid this symptoms from happening, some hospitals actually include G6PD as a test in a newborn package.
Examples of some things that G6PD-deficient patients should avoid: aspirins, moth ball (contains napthalene that is harmful, can cause RBCs destruction in patients).

Why G6PD occurs mostly in males than females?
G6PD is inherited as an X-linked recessive trait. There is a mutation in teh X chromosome in which will affect the production of G6PD for metabolism. For females, it is very rare to find a complete deficient case as it has to have mutation in both X chromosomes. Even if they have only just one defect X chromosome, they will only be a carrier. However, for males, just a defect in the X chromosome and they will inherit this deficiency.

What is the metabolic pathway that G6PD is involved in?
The metabolic pathway is happening inside RBC. G6PD is an enzyme that catalyses the conversion of glucose-6-phosphate and NADP to gluconate-6-phosphate and NADPH. NADPH is important because it actually yield 3ATP. Therefore patients with G6PD deficiency cannot carry out anaerobic respiration.

What is the principle of the test and what cause the blood mixture to fluoresce?
The test makes use of the metabolic pathway mentioned above. The reagent actually contains glucose-6-phosphate, NADP and some other components. When the patient's blood contain G6PD, it will be able to convert the NADP to NADPH. It is teh NADPH that cause the fluorescence under UV.

Is there a difference in the intensity of fluorescence between the 1st, 2nd and 3rd drops?
Yes, there is! The 1st drop is always lighter because of the short reaction time. By the 2nd and 3rd drop, clear fluorescence should be seen.

To Zahirah,
Yes, the Neg control is a commercial blood. It is specially prepared by the manufacturer such that it contains no G6PD. If it is positive, we will definitely change it and use a new one. We will check on teh amount of reagents left in the analyser two times a day. Therefore, we have never met with a situation where there is not enough reagent. Furthermore, we also put in extra set of reagents for those tests that are commonly requested such as HIV, renal analytes.
The purpose of centrifuging is because these chemistry analytes are found in the serum and so centrifuging will separated the serum from the blood cells.

To Jue Xiu,
Modular P and E run all sorts of tests. Basically it anything that are chemistry. For example, lip profile (LDL, HDL, Cholesterol), renal screen (Na, K), HIV, HBs Ag, Anti-Hepatitis A Virus). It should be similar to the test you done in your lab just that we use different machine.

Sally

Pictures

2007-07-24T06:16:00.000-07:00

As promised, this is the pictures that I have taken with permission. It is not very clear so pls pardon me.

This is the absorbent paper that i'm refering to. And you can see the three drops on each circle.

This is the same piece of paper seen under UV light. If you noticed, there is no fluorescence seen on the first circle (Neg control). However, there is flourescence on the second circle (with patient's blood). And the intensity of the fluorescence is from light to brighter clockwise direction.

Sally

Biochemistry/ Immunology

2007-07-21T23:05:00.000-07:00

Hey guys, this is Sally. I'm sorry for this late posting but I'm sure it's worth the wait because I have lots to share. But before I start, let me give you a brieg introduction on what I'm doing. The hospital I'm working in has only one lab and it is divided into different sections such as Admin, Haematology, Biochemistry/Immunology, Microbiology and Cytology/Histology. Needless to say, I'm posted to the Biochemistry/Immunology section and will be staying there for the next few months.

Basically, I get to work with all analysers and machines almost everyday. 95% of my work are automated and so what I simply have to do is to learn on the theory of the tests and the procedures involved in the operating and maintenance of these big machines. Everyday, we receive as many as 500 blood samples from patients and they will request for tests such as renal screen (K, Cl, Na...), lipid profile (glucose, HDL, LDH, triglycerides...), liver test (AST, ALP, creatinine...), HIV and many more. Then, we have to centrifuge the samples (which comes in gel tubes) at 3000rpm for 10 min before loading into the main analysers called Modular P & E. These Modulars will then test for the quantity of the requested analytes using the serum in the tubes. We do not have to order the test manually. This is because the Modulars are linked to the PSM (Personal Security Manager) and the PSM is further linked to the LIS. So when the Modulars scan the barcode label on the tube, the PSM will transmit the requested tests for the particular tube from the LIS to the Modulars and they will straightaway know what tests to do.

After reading this, you may think that my work is much relaxed but it is not like what you think because we have to be extremely careful about this machines. We have to do daily checks and maintenance to make sure that there is sufficient reagents and we run controls many times a day. Furthermore, we always have to check results generated to ensure that they are correct and making sense and even before loading, we have to be able to think quick, because sometimes doctor may send in only a plain tube for many tests and these tests are performed using different machines. If the sample is an urgent one, we still have to remember to load the tube at another machine after it finished processing at the previous one. Sometimes, when the workload is heavy, we may forget about it. So, my supervior will contantly remind us to make sure we did not leave out on any tubes.

The other tests are either manually performed or the preparation procedures are more tedious and they are microalbumin (qualitative) dip stick test, blood gases test, homocysteine, anti-nuclear antibody, rubella, anti-dsDNA test, G6PD test and anti-thyroglobulin test. A few of them are scheduled tests because there are less frequently requested.

Today, I chose to focus on G6PD test because this is a test that I get to perform everyday and I have found alot of information on it.

What is G6PD and what is the purpose of the test?

G6PD is glucose-6-phosphate dehydrogenase and it is an enzyme involved in the metabolic pathway of RBCs. G6PD test is usually performed on newborns and it is to test for any G6PD deficiency. It is an inherited condition which means that unless one of your parents have this condition, if not you will not have this deficiency at all. This condition is mostly seen in males than females.

Procedures of the G6PD test

Take two small cups and label one of them "Neg" and the other with the baby's lab request no. Note: Neg means negative control

Then take 100ul of G6PD reagent into both cups.

For the Neg control, add 2.5ul of the negative control "blood" and mix thoroughly by pipetting up and down.

Same for the patient's cup, add 2.5ul of baby's blood into the cup and mix thoroughly.

Then on a piece of absorbent paper, label with the word Neg and patient's lab request no.

Add one drop of each mixture on the piece of absorbent paper.

Repeat step 7 every 5 min for two times.

Finally, incubate in the oven at 35 degree celsius for 5 min and observe under long wave UV light.

Pls refer to the above drawing. I will try to take the pic of the real test. But for now, you just have to understand what i'm drawing.

Results

For the Neg control, there should not be any fluorescence seen. However, if you see flourescence on the test using the baby's blood, it indicates that the baby is having the enzyme, G6PD and so he/she is not suffering from G6PD deficiency.

This test is very simple and easy to perform but we must always ensure that before reading the results under the UV light, the absorbent paper must be dry. If not, this may result in false positive result. Simply, you won't see any fluorescence on the patient's test even when he/she is not suffering from this deficiency and you may mistaken the patient as suffering from G6PD deficiency and this can be serious. Therefore, adequate drying is important!

Hope that by now, you have learnt something.

If you have read carefully, I have left out on a few points for you to ask me so please feel free to ask if you are not clear.

Sally, TG02

0503315D

urinalysis/routineµbiology

2007-07-17T05:25:00.000-07:00

Hi~liuqian here~
First of all ,I am sorry abt the late posting！in my attachment place , we have chance to go every lab and rotate per 2 weeks. This is the 3rd week already ,so I ve already attached to 2 labs, urinalysis/ non-routine and micro lab.
First, I say something about the urinalysis/ non-routine. The tests running there are urine FEME, CSF analysis, urine osmolality and urine phase contrast.

URINE FEME:
This is the most common test running in this lab. It is ordered by doctor for urinary tract infection.
Procedure:
A: chemical testing of urine analytes
1. Mix the urine to be tested by inverting the sample several times before opening the sample cap
2. Completely immerse al reagent areas of the strip
3. Record the result of PH, Albumin, Sugar, Ketone, and blood.
B: microscopic examination
Microscopic examination of urine sediment is examined unstained.
1. Mix the urine sample and transfer to the KOVA glasstic slide chamber with plastic pipette
2. Quantitate and examine all cells under microscope
3. For low cell count samples count the total cells of 4 complete quadrants of the
counting rids
4. For high cell count samples count 10 small grids within different quadrant

CSF FLUID
This test is ordered by doctor for meningitis.
The things we are looking for in CSF analysis:
1. appearance
2. cell counts with differential
3. glucose
4. total protein
5. smears for Gram’s stain and AFB stain
6. Indian ink preparation for encapsulated crytococcus

URINE PHASE CONTRAST
Urine phase contrast is a method of distinguish glomerular bleeding from causes of haematuria by examining the appearance of RBC in urine.
Procedure:
1. centrifuge 10 or 12 ml of urine for 10 mins at 2000rpm
2. discard 9 or 11 ml of supernatant
3. resuspend 1 ml of sediment and charge into the KOVA chamber
4. examine for cast, epithelial cells, micro-organism, crystals at200X
5. quantitate casts, EC, RBC and WBC at 400 X
6. roughly calculate the ratio of ISO to DIS(ISO RBC is very shine and round under the phase contrast dark background microscope while DIS RBC is dark and variable in shape.

I attached to micro-lab after 2 weeks in urine bench. Basically doctor sent specimen to micro-lab to find out which bacteria causes the infection. So there are different types of specimen, urine, blood, fluid etc.
The specimens are divided into stool, urine, and blood and miscellaneous.

STOOL for food handler------selenite broth------after 16-18h 35 dgree-------salmonella-shigella plate

sceen for fecal pathogen---salmonella-shigella plate+ MacConkey plate+Campylobater plate

Note: if watery stool.must +blood agar+ alkaline peptone+triosulphate citrate bile salt sucrose agar----------------subculture alkaline peptone water to thiosulphate citrate bile sucrose agar

Load the BLOOD vials into BACTEC
Blood specimen normally sent in vials, aerobic or anaerobic. The media contained in the vials consist of soybean casein digest broth with resin and 0.05% w/v sodium polyenrthol sulphate(SPS). Bactec culture vials that are equipped with a sensor for the detection of CO2. Growth of microorganism within the vial results in the production of CO2, which reacts with a dye in the sensor. Fluorescence of the sensor proportional to the CO2 production is measured by a photo-detector. Vial that exeed a pre-determined threshold level will be flagged by the BACTEC instrument as “positive”.
STERILE FLUID:
Blood agar Blood agar
+ Chocolate agar +MacConkey agar +
CDC anaerobic agar + Cooked meat

&
Cooked meat must be
Subcultured after 24h
Incubation
Subculture into chocolate agar
+ CDC anaerobic agar

NON-STERILE FLUID:

Blood agar + Chocolate agar Chocolate agar + MacConkey agar

Finally i finish writing . Enjoy reading:)

Liu Qian

0503935i

TG02

2007-07-07T21:45:00.000-07:00

Department : Clinical Chemistry/Biochemistry

Name:Dorene Chen
Admin no":0504156A
class: TG02

I was assigned to work in Quest Laboratories, department Clinical Chemistry/Biochemisty for the 1st 3 weeks of attachment. The department is almost fully automated except for a few tests like OGTT (Oral Glucose Tolerance Test), HbA1c (Glycosylated Haemoblobin) and Drug 5/7. For the first week, my duty is to run HbA1c using the machine. for the second week, i learnt how to test OGTT and Drug 5/7 using testing kits.

HbA1c testing :

EDTA tube is used to test for HbA1C as the whole blood is needed to test for glycoslyated haemoglobin.

Before loading sample into machine (ADVIA 1650 - Bayer) bubbles are removed to prevent the probes from taking in the bubbles. This is because the machine only required 10 microlite and difference in small volume of blood will alter the results.

the sample is then loaded into the machine and results will show in 30 mins time.

Ranges :========================Actions to be taken:
<4.5% =========Low=============Rerun test in 1:41 dilution
4.5% to 8%======Reference Range====Record results
8% to 12%====== Moderate High======Rerun sample in 1:41 dilution
12% to 15%======High============ Reun sample in 1:51 dilution
>15%==========Very high=========Rerun sample in 1:81 dilution

Dilution of the samples during rerun has to be done manually.. eg: in 1:41 dilution, 400 microlite of denatured water and 10 microlite of sample are added and incubate at room temperature for 10 mins. diluted samples will then be loaded into machine for analysis.

Prinicple of HbA1C:
Hba1c is an diagnostic tool to control, check for diabetes.

when glucose levels are persistantly high, glucose will enter RBC and form an irreversible complex with haemoglobin, to form glycosylated haemoglobin (HbA1C). this form of haemoglobin will remain in RBC until RBC dies. (RBC has a life span of 120 days).

Calculating % of HbA1c in RBC will therefore determine the glocose level that always reside in the blood. In diabetes patient, % of HbA1c is usually high. A normal level of HbA1c will show that diabetes patient has a good control of the disease.

Principle of ADVIA 1650 (Hba1C)

Uses immunoassay method. The machine first uses probe to extract 10 microlite of sample into curvette. 400 microlite of denatured water is then added.

This water causes cell lysis to allow contents in RBC to be exposed and used for experiment (sample will change from red to brown in color).

Latex coated specific antibody is then added into the curvette and this ab will bound to random glucose. An agglutinator is then added and it will bound to Ab to produce scattered light that increase the absorbance.

If there is presence of Hba1c in blood, Hba1c will competes with the agglutinator to bound to Ab and this reduces the amount of agglutinator bound to Ab which in turn reduces the scatted light and absorbance.

The amount of absorbance recorded will reflect the amount of Hba1c.

OGTT test

OGTT (Oral Glucose Tolerance Test) tests for the amount of glucose present in urine sample. Usually 3 different urine samples are needed : fasting, 1st hour, 2nd hour. Sometimes, (i dunno why) only fasting and 2nd hour are received but we can still use it to find the results. Combur test kit (dip stick) is used to detemine glucose level.

The process is quite simple: just dip the stick into urine and the colour of the reagent in stip stick will change immediately. there will be different color for different glucose level. However, the results is valid for only 3o seconds so we have to read the results immediately. This is because the reagent will react with the surroundings and change the intensity of the color.

Taken from : www.tierklinik.de/medizin.php?content=00151 , www.notfalllabor.de/Tests/20-0-10131-597-0-0-0-K.html

fasting urine : the "reference range" of glucose is usually normal or 1+ and ketone neg
1st hour urine : glucose is 4+ to 3+ and ketone neg
2nd hour urine : glucose 3+ to 2+ and ketone neg.
ketone should not be present in urine.

Eg: reference range of fasting urine is <20mg/dl, color="#ff0000" size="4">Drug 5/7 test
tests for THC (marijuana) , COC (cocaine), OPI (opiates), MET (methamphetamine), AMP (Amphetamine)

on the test kit, there are 2 lines (control and test line). The "control line" must always show when doing the test (this is to show result is accurate if no control line is seen, result is considered invalid). For the "test line" presence of this line will show neg result of this drug while absence of this line will show positive result for this drug. This test is based on chemical reactions that the reagent used will formed a colorless product with the specifc drug tested.

Visible Control line (C), visible Test line (T) ==== Negative

Visible Control line (C), no Test line (T)======= Positive

No Control line (C), no Test line (T) ==========Invalid test (rerun)

Btw, urine sample is used for this test.. don't know why they want to do this test too.. well, that's all I had learnt in the 1st 2nd week =)

HISTOPATHOLOGY/LMQA

2007-06-30T22:43:00.000-07:00

Student: TENG CASSANDREA

Topic: Histology/ LMQA

On my first day of work I was posted to the histology laboratory where they dealt with tissue processing. The scope of the laboratory’s work consisted of:

Receiving of specimens
Tissue processing by tissue processor
Embedding
Paraffin sectioning
Frozen sectioning
Staining by autostainer or manual special staining
Cover slipping and labeling
Immunohistochemistry

Some of the procedures that I observed were:

Receiving specimens

Check the information on the requisition form against labels on the specimen bags and bottles to check if they match (reduces error).

Requisition forms come in pairs; yellow and white forms.

Yellow forms are kept in case of error (easy traceability).

When no errors detected, the forms are signed by the lab tech that received the samples(acountability).

Specimens kept in order in rack and request information are manually entered into database (LIS).

Paraffin sectioning

For good sections, must operate microtome at constant rate.

Must not section to fast as tissue may become damaged or even ‘disappear’ (too much tissue is cut off).

Freezing the block will enable thinner sections.

Levels of sectioning (1, 2, 3) reflects the depth of sectioning; the deeper the tissue is cut, the higher the level.

Working out bubbles and folds in section is done in a 45 degrees Celsius water bath.

After fishing for the sections, the slides with sections are placed into an 85 degrees Celsius oven for 15 minutes to enhance tissue adherence to slide.

Hard tissues like fibroid and bone tissues are softened first by softlen or de-cal solution.
Water bath cannot be too hot as tissues may ‘disperse’.

Request for full face (FF)à tissue is initially not fully exposed when sectioned hence a request for the ‘fuller’ view of tissue.

Sometimes ribbons have holes during sectioning. This is due to poor processing; wax did not penetrate through the tissue.

Frozen sectioning

o Rapid diagnosis of tumors and carcinomas using the cryocut.
o For patients still on operation table (OT).
o Uses rapid H&E method as report has to be submitted in 20 minutes.
o Uses the O.C.T compound; O.C.T. compound is an embedding medium for frozen tissues to ensure Optimum Cutting Temperature.
o Sometimes surface is not leveled so cryomold is used.

Embedding

o Use different size of molds depending on size of tissue
o During embedding, tissue must be orientated in such a way that gives FF; the inner and outer layer must also be seen (e.g. ovarian cyst)
o Once wax is added to mold and tissue is embedded, leave in ice; this aids in easy removal of block from mold.
o Colored paper is embedded at the back of the block (traceability of medical technologist (MT) that did embedding)
When embedding, tissues are pressed down to ensure that tissue is leveled. this ensures that not only one part of the tissue is exposed first when sectioning.
o Scanty tissues like endothelial tissues are placed in gel and between sponges or filter bag to ensure that tissues are not lost during processing.
o Sponges are not used for big tissue as it covers the cassette holes making it difficult for the wax to penetrate the tissues(poor embedding).

Lab automation (LMQA)

Some equipment in the laboratory are:

Autostainer
Autocoverslipper
Bondmax ( immunohistochemistry)
Shendon Celsior (tissue processor)

These equipments help increase the laboratory’s productivity rate which in turn increases efficiency of laboratory.

Some practices that I observed (LMQA/QC):

Counter-checking of label on slides with printed labels and paraffin block(check morphology on slides and in block to match)

After staining, counter-check slides with a control to see if its up to standard.

Control run in autostainer before patient sample to check if H&E is of quality.

Staining manual consist of policies and procedures (policy number ,revision date, effective date, approved by, applies to)

Daily updating of QC checklist (waterbath, oven, autostainer, tissue processor, etc)

Lab safety aspect

The hospital that I’m posted to is well equipped with safety gears and has many safety measures installed in case of an emergency. Some examples are:

Safety blankets
Eye wash and shower stations
Separate chemical stores
Fire trigger points
Emergency exits
Radiation safety (isotopes;lazers)
Bio-safety (infectious material;BSC)
Electrical safety
PPE (googles,chemical-resistant gloves, masks, lab coats, chemical boots)
Laboratory safety training (training checklist; lab safety talks and videos)

So this was what i did in my first week...oh and i watched an autopsy also... damn bloody..!! lol... anyways i'll keep you guys updated... cheers..!

2007-05-13T23:01:00.000-07:00

So far we have narrowed down our 10 diseases to 4 possible infections. This includes :

Rat bite fever

An infectious disease which develops after having been bitten or scratched by an infected rat or rodent.

Cat Scratch Disease

This disease is trasmitted when a healthy individual has been scratched by an infected cat. This is especially so when playing with kittens.

Leptospirosis

This disease can be transmitted through dogs or rodents. The bacteria involved is called Leptospira. It usually occurs in tropical country.

Typhus

It can be transmitted by fleas, using cats, dogs, rats as a reservoir. The bacteria involved is called R. Typhi.

In general, we felt that these diseases are worth looking into as their symptoms were similar to the symptoms developed by the eldery man. These diseases also had a very good route for transmission compared to the other diseased.

2007-04-24T20:21:00.000-07:00

Medical Microbiology

Case Study 1:

A 85 year-old male was found to be staying alone in one-room flat. It is found that his mattress was soiled with human excreta and urine. The premise is infested with cockroaches, fleas and rats. It is known to the neighbours that the elderly man has the habit of feeding stray dogs and cats in the neighbourhood,

The elderly man was referred to a nursing home and presented with high fever,rash and general weakness.

How would you approach this situation in order to provide final diagnosis of this suspected microorganism(s)?

Web sites:

1. Rashes caused by allergic reactions to cockroaches http://www.the-piedpiper.co.uk/th2.htm

2. Lassa Fever caused by infected rodents or ratshttp://72.14.235.104/search?q=cache:64Ub4_XuY5AJ:www2.ncid.cdc.gov/travel/yb/utils/ybGet.asp%3Fsection%3Ddis%26obj%3Dviral_hemorrhagic.htm+illnesses+caused+by+contact+with+human+excreta&hl=en&ct=clnk&cd=1&gl=sg

3. Cat scratch diseasehttp://familydoctor.org/024.xml#2

4. Cholera http://www.medicinenet.com/cholera/article.htm

5. Typhoid http://en.wikipedia.org/wiki/Typhoid

6. Cryptosporidiosis http://www.dhpe.org/infect/crypto.html

7. Dogs and cats (possible illnesses)http://www.kidshealth.org/parent/infections/skin/pet_infections.html

8. Human excreta and Urine- possible pathogens http://www.sulabhenvis.in/pages/database_detail.asp?id=46

9. Diseases From Stray Cats and Dogs http://www.medicinenet.com/script/main/art.asp?articlekey=52173

10. Cat Scratch Fever http://animalpetdoctor.homestead.com/Zoonotics.html

About Us

2007-04-24T18:59:00.000-07:00

Hi we are ALsubs from PG02... Our group name was inspired by our group's confusion..-laughs- We didnt know what subject this blog was for so we decided to named it ALsubs, the abbreviation for all subjects... -smiles- Our group consists of Sally, Sasi, Liu Qian, Dorene, Vino, Shu Hui and Cassandrea...

This is our final year and all of us are nervous... Yet also kinda excited... We are prepared for any challenges ahead and are ready to give our best... No worries, with a group like this, everythings going to be okay... -smiles-

We the members of ALsubs
Pledge ourselves to work harder,
And help each other,
To perform well,
To share our knowledge,
And broaden our horizon.

Our interests:

Cassandrea
Hi ppl... My name is Cass and I'm 20 this year... Kinda excited that its my final year in TP... one last huddle and im off to work...-smiles- Anyways, i have an interest in biology. Fascinated by how our body works..:) Apart from being a science geek, i like to write... But strangely, i don't read as much... that accounts for my bad spelling... -laughs- Anyways, good luck to everyone... cheers...

Dorene
The subjects that interest me most are Diseases and blood related subjects. I am amazed that things so microscopic can affect our life. I enjoyed listening to Chinese/Korean pop music :P, though I don't really understand the langauge well ^^. I like reading Harry Potter books and watched drama serials by downloading them in the net (opps!).. well, that, about me..

Liu Qian
Hi, i am more interested in the things related to diseases and how we can prevent ourselves from these diseases. i hope i can work in a clinical diagnosis lab in the future. My interests are watching drama and movies, listen to the music and tranvel. i hope one day in the future i can tranvel around the word and use my knowledge to help others~

Sally
Hi! I am more interested in clinical stuff. I enjoyed Basic Pharmacology very much and hope to be able to deal with the discovery of human diseases and drugs and all that. My interests are reading, singing, and meeting up with friends for chit chatting.

Sasi
Heya! Im Sasi and Im turning 22 this year. This is my 3rd year in Tp and so far, Ive learnt alot about what lifesciences. The subjects have been fairly interesting and I have learnt to adapt well to the different challenges posed to me. Ive had my fair share of fun as well. My interests include music, non-fiction books, chilling out with friends and drawing. I hope to further my interest in the diagnostic aspects covered in my course.

Shu Hui
Yo! I'm somehow more interested in doing microscopic work, such as Basic Microbiology where we learn how to identify bacteria and classify them. As for my future, maybe i'll work for a few years to earn more $$ 1st(haha..) before deciding whether I 'll further my studies.Yup. Hmm..what are my interests?? Do I have any at all??!?!! Heex. It'll be shopping with best buds, having heart-to-heart talks and reading during my free time. =P

Vino
My interest is to obtain a degree in Biomedical Science and work in that field for few years before applying for M.D (MBBS) as a postgraduate in GMS.
In my spare time, i read magazines and books. I love watching movies especially in theatres. hmmm Love watching TV programmmes like.. "friends", "prison break" and
"ER" etc... thats abt it... will fill up this later.. byeeee