Group: Laboratory Medicine

Subject: General Microbiology and Parasitology (Paper-II)

Q-1-a) Name the diseases / lesions produced by Staphylococcus (Jan-2021)

Diseases/lesions produced by staphylococcus:

Cause	System involed	Lesions
1.Direct invasion or Inflammatory	a. Skin disease	Impetigo, furuncles, carbuncles, paronychia, cellulitis, folliculitis and hydradenitis suppurativa, surgical wound infections, eyelid infections (blepharitis), postpartum breast infections (mastitis).
	b. Septicemia (sepsis)	Sepsis can originate from any localized lesion, specially wound infection, or as a result of intravenous drug abuse.
	c. Heart	Infective endocarditis on normal or prosthetic heart valves.
	d. Bones and joints	Osteomyelitis (in children) – Septic arthritis
	e. Lung	Pneumonia in post-operative patients or following viral respiratory infection.
	f. CNS	Meningitis, Brain abscess
2. Toxin mediated	– Food poisoning (Gastroenteritis) [Caused by Enterotoxin]Toxic shock syndrome [Caused by Toxic shock syndrome toxin]Scalded skin syndrome [Caused by Exfoliatin]

Q-1-b) What is a capsule? Name some capsulated bacteria. Give advantage and disadvantage of capsule. (Jan-2021)

Capsule:The capsule is a gelatinous layer covering the entire bacterium, situated outer to the cell wall.

Name of capsulated bacteria according to composition:

1. Polysaccharide capsule: (most of the capsulated bacteria)

Example:

•  Neisseria meningitides 
• Streptococcus pneumonia
• Klebsiella pneumonia
• Haemophilus influenzae
• Bordetella pertussis
• Some strains of staphylococcus.

2. Polypeptide capsule:

 Example:

• Bacillus anthracis

3. Hyaluronic acid capsule:

Example:

• Streptococcus pyogens.

Advantage of capsule:

• Protection from desiccation. The capsule helps to prevent the bacteria from drying out, which is important for survival in harsh environments.
• Protection from antibiotics. The capsule can make it more difficult for antibiotics to reach the bacterial cell wall, which can make the bacteria more resistant to treatment.
• Protection from the immune system. The capsule can help the bacteria to evade the immune system by preventing the immune cells from recognizing and destroying them.
• Attachment to surfaces. The capsule can help the bacteria to attach to surfaces, such as the lining of the gut or the teeth, which can help them to colonize and cause infection.

Disadvantage of capsule:

Disadvantage of a capsule is that it can make it more difficult for the bacteria to spread through the bloodstream. This is because the capsule can make the bacteria more sticky, which can help them to adhere to other surfaces. This can make it more difficult for the bacteria to travel through the bloodstream and cause infection in other parts of the body.

Q-1-c) Draw and label stages of E.H. Write down the intestinal and extra intestinal lesion of E.H.(Jan-2020,21)

A.Intestinal lesions:

• Acute intestinal lesions-Ulcers
• Chronic intestinal lesions-Scar, ulcers

B.Extra intestinal or metastatic lesions:

• Amoebic liver abscess
• Amoebic lesions at other sites like lung.pleura,pericardium.

Q-1-d) Define immunity. What are the beneficial harmful effects of immunity. (Jan-2020,21)

Immunity: 

Recently, Immunity is defined as-

“ all those physical mechanisms that endow the animal with the capacity to recognize materials as foreign to itself and to neutralize, eliminate or metabolize them.”

Beneficial effects of immunity:

a) Prevention of many diseases by vaccination, eg-measles vaccine, polio vaccine etc. 

b) Following clinical & sub-clinical infections, there is production of immunoglobulins which gives

protection to the host. The majority of the beneficial effects are unseen.

Harmful effects of immunity:

1. Hypersensitivity conditions like asthma,rheumatic fever,glomerulonephritis etc.

      b) Autoimmune diseases like rheumatoid arthritis, SLE (Systemic lupus erythematosus),    autoimmune haemolytic anaemia etc.

Q-1-e) Discuss the growth requirements of bacteria. (Jan-2021)

Bacterial growth requirement:

A) Nutritional factors:

1. Source of energy/Essential elements: Carbohydrates, proteins, lipids & nucleic acid etc. 
2. Growth factor: Bacteria can not synthesize these factors and therefore must be provided for their growth & multiplication.

                        i) Essential growth factors: Vitamins e.g. PABA, thiamin, riboflavin, nicotinic acid. pyridoxine, folic acid, cyanocobalamin etc.

                        ii) Essential trace elements / Minerals: Potassium, calcium, magnesium, iron, copper, zinc, manganese etc.

                        iii) Accessory factors for some bacteria: e.g. Factor V (NAD) & Factor X (heme).

B) Environmental factors:

 i. Water: it is essential for all metabolic pathway of bacteria, as almost 80% of cell constituent is

made up of water.

ii. Oxygen: according to O₂ requirement organisms are-

Obligate aerobe-e.g. Pseudomonas

Obligate anaerobe.g. Clostridium

Facultative anaerobe- e.g. Staphylococci, E.coli

Aerotolerant anaerobe

 Microaerophilic – e.g. Neisseria

iii. Carbon dioxide: certain bacteria requires more CO₂ (5-10%) for their growth e.g.- Neisseria

gonorrhoeae, Brucella abortus etc.

 iv. Temperature: bacteria are divided according to temperature requirement into following groups-

Psychrophilic (15-20°C) – ego Saprophytes,

Mesophilic (30-37°C) – ego Most of the pathogenic bacteria.

Thermophilic (50-60°C) – ego Bacillus stearothermophilus.

v. pH of the Medium:

 Most bacteria grow best in pH (7.2-7.4)

Acidophils – Grow in highly acid media (4-4.5) e.g.- Lactobacilli 

Alkalophils – grows in highly alkaline media (8-9) ego V. cholerae

vi. Light.

vii. Osmotic pressure / Salt concentration.

Q-2-a) Name some opportunistic fungal infections with examples. (Jan-2021)

Opportunistic fungi/mycoses:

The fungal infections which does not occur in an immuno-competent person, but occur when the body is immunocompromised, are called opportunistic mycoses.

Example:

Opportunistic fungal agents	Diseases
Candida albicans	Different forms of candidiasis
Cryptococcus neoformans	Cryptococcosis
Aspergillus fumigatus	Aspergillosis
Mucor	Mucor mycosis/phycomycosis
Pneumocystis carinii / P. jiroveci	Pneumonia in immunocompromised patients

Q-2-b) Define virus with example. Write examples of DNA virus and RNA virus. (Jan2021)

Virus:

Viruses are particles composed of an internal core containing either DNA or RNA (but not both) covered by a protective protein coat.

Example: Hepatitis B virus,Pox virus etc.

 On the basis of type of nucleic acid:

A. DNA viruses:

Example:

•  Poxvirus.
•  Herpes virus.
• Hepadna virus
• Adenovirus.
• Papova virus.
• Parvo virus.

(For easy remember- PHHAPP)

B. RNA viruses:

Example:

• Myxo virus. 
•  Rhabdo virus.
• Corona virus.
• Picorna virus.
• Toga virus.
• Arena virus.
• Reo virus.
• Retro virus.

(For easy remember- MRCP,TARR)

Q-2-c) Write down the WHO safety code of practice for microbiology laboratory. (Jan-2021)

Code of Safe Laboratory Practice :- 

1. Mouth pipetting or specimens should be avoided.

2. keeping the laboratory clean.

3. Work spaces should be decontaminated everyday.

4. Washing and sterilization of reusable specimens. containers, needles, syringes, lancets, slides, coven. St glasses, pipettes ate by appropriate procedures.

5. Hand should be washed by using chemical disinfectants such as 5%, Phenol or Lysol, after handling infectious material.

6. Wearing an apron and safe footwear, use of protective gloves, goggles, face shield, dust mask, eyewash bottle.

7. Laboratory doors should be kept closed when work is in progress.

8. Eating, drinking, storing food and smoking should not be permitted in the laboratory.

9. Control of noise and other causes of loss of concentration.

10. Proper maintenance and routine cleaning of equipment.

11. Safe handling and storage of chemicals and reagents.

12. Regulations covering the safe packing and transport of specimens.

Q-2-d) Give the life cycle of Hookworm (AD). (Jan-2021)

Answer:

Life cycle of Ancylostoma duodenale:

•  Infective form: Filariform larva
• Portal of entry: Skin
• Migration: Through lungs.
• Site of location: Small intestine.

Life cycle:

(a) Host: Man is the only definitive host. No intermediate host.

(b) Stages: The various stages of life cycle are described below:

Stage-1: Passage of eggs from the infected host: The eggs, containing segmented ova with four blastomeres, are passed out in the faeces of the human host.

Stage-2: Development in soil: Egg→ Rhabditiform larvae → Molts twice → Filariform larva (infective stage).

Stage-3: Entrance into a new host: by penetration of skin, after cast off their sheaths.

Stage-4: Migration: Filariform larva in subcutaneous tissue→ Migrates through lymphatics or small venules →Venous circulation → Right heart→→ Pulmonary circulation → Alveolar space → Migration to bronchioles→ Trachea→ Larynx→ Epiglottis→ Pharynx → Swallowed in small intestine, during migration or on entering the esophagus, a third moulting occurs.

age-5: Localization and lying of eggs: The growing larva settle down into small intestine → Fourth moulting occurs→ Sexual maturation of larva→ Adult worm→→ Female adult worm lay egg→ The cycle is repeated→ Time interval skin penetration and appearance of egg in faeces is about 6 weeks.

Q-2-e) How will you diagnose the malarial parasite in the laboratory? (Jan-2021)

Laboratory diagnosis of malaria:

Principle:

Diagnosis of malaria depends on demonstration of malarial parasite in the peripheral blood film, detection of antibody or antigen from blood, and also by nucleic acid based techniques.

Steps:

1) Specimen collection: Blood.

2) Microscopic examination: Leishman (or Giemsa) stained smear of thick and thin blood films.

•  Thick smear – to detect the presence of parasite.
• Thin smear-to detect the species of parasite.

3) Culture: rarely done where there arrives difficulties in detecting the parasite.

4) Antigen detection: Immunochromatographic ‘dipstick’ tests, also called rapid diagnostic tests (RDTs).

5) Serological tests: Does not detect current infection, but rather measures past experience. 

• IFAT-for detection of antibody in chronic malaria.
• ELISA.

6) Blood count: Leucopenia with monocytosis (15-20%)

7) Nucleic acid based technique: PCR. (Polymerase Chain Reaction)

Q-3-a) How can you diagnose hepatitis B in a laboratory? (Jan-2021)

Answer:

Sero Markers of HBV infection:

1) Detection of Ag in the serum:

• HBs Ag
• HBe Ag

2) Detection of Ab in the serum:

• Anti HBc IgM 
• Anti HBs IgG
• Anti HBe IgM

Interpretation of serological marker:

Status	HbsAg	HBsAb	HBeAg	HBeAb	HBcAb
Active HBV	+	–	+	–	IgM
Chronic HBV (high infect)	+	–	+	–	IgG
Chronic HBV (low infect)	+	–	–	+	IgG
Recovery	–	+	–	–	IgG
Vaccinations	–	+	–	–	–

Clinical importance/significance:

• HBsAg is the indication of active, chronic infection and convalescent carrier.
• HBeAg in blood indicates active replication of virus and the patient is highly infectious.
• HBsAb in blood indicates person with immunity following infection.
• HBcAb IgM in blood indicates the surest marker of active infectious. 
• HBe Ab IgM in blood indicates recovery and low transmissibility.

Q-3-b) What is hypersensitivity reaction.Classify hypersensitivity reaction with example. (Jan-2021)

Hypersensitivity:

Hypersensitivity may be defined as inappropriate or excessive immune response to an antigenic stimulus in a pre-sensitized host leading to tissue damage.

Classification of  hypersensitivity reactions:

A. Coombs and Gel (combined) classification:

•  Type I or anaphylactic hypersensitivity
• Type II or cytotoxic hypersensitivity
•  Type III or immune complex mediated hypersensitivity
• Type IV or delayed or cell-mediated hypersensitivity 
• Type V or stimulatory hypersensitivity.

B. On the basis of onset of action: 

•  Immediate (requires minutes to hours) e.g. Types I, II, III & V
•  Delayed (requires hours to days) e.g. Type-IV

C. On the basis of mechanism of action: 

• Antibody mediated, e.g. Types I, II, III & V. 
•  Cell mediated, e.g. Type-IV hypersensitivity.

Q-3-c) Define and classify sterilization. How can you sterilize glasswares,sharp articles,dressing and rubber catheter. (Jan-2021)

Sterilization : 

Sterilization is the process by which all viable microorganisms including spores are killed or eliminated.

Classification of sterilization : 

1. Physical 
2. Chemical 
3. Filtration 

A.Physical method : 

1. Heat
   • High Heat
     • Dry Heat
       • Hot air oven 
       • Incineration
     • Moist Heat
       • Boiling
       • Steaming
         • With Pressure
           • Autoclave
         • Without Pressure
           • Tyndallization
           • Koch’s Steamer
       • Pasteurization
         • Holding Method
         • Flash Method
   • Low Heat
     • Water Bath
     • Vaccine Bath
2. Radiation
   • X-ray
   • Gamma ray
   • UV ray

B.Chemical method : 

1. Alcohols
2. Phenols.
3. Ethylene oxide
4. Detergents
5. Dettol
6. Acids and alkalis
7. Formaldehyde
8. Glutaraldehyde
9. Oxidizing agents

C.Filtration method : 

1. Bark field
2. Chamber land
3. Membrane filter
4. Hepa filter

Sterilization method	Materials to be sterilized
Hot-air-oven	Glass wares
5% Cresol(Lysol)	Sharp articles
Autoclave	Dressing
Ionizing radiation	Catheter

Q-3-d) Write down the steps of urine culture. What are the common organisms isolated? (Jan-2021)

Ans: Step of urine culture-

• The patient should clean the genital area with soap and water before providing the urine sample.
• The sample should be collected midstream, meaning that the first part of the urine stream is discarded and the middle part is collected. This is to avoid contamination of the sample with bacteria from the skin.
• The urine sample should be collected in a sterile container.
• The urine sample is transported to the laboratory and inoculated into a culture medium. The culture medium is a nutrient-rich substance that allows bacteria to grow.
• The culture medium is incubated at a warm temperature, typically 37 degrees Celsius. This is the ideal temperature for bacteria to grow..
• The culture is checked for the growth of bacteria after 24-48 hours. If bacteria are present, they will be identified and the antibiotic susceptibility of the bacteria will be determined.

Common organism isolated in culture media:

• Staphylococcus aureus 
• Streptococcus pyogenes 
• Escherichia coli
• Pseudomonas aeruginosa 
• Klebsiella pneumoniae

Q-3-e) Write  the steps of ZN staining. (Jan-2021)

Ans: 

Acid Fast Bacillus (AFB) eg-mycobacterium tuberculosis is stained by Ziehl-Neelsen (ZN) staining.

Instrument:

• Glass slide
• Cotton
• Platinum loop/wire loop
• Test tube with holder
• Staining rack 
• Spirit lamp 
• Match
• Microscope

Reagent: 

• Carbol fuchsin Solution
• 20%  H₂So₄
• Methylene blue / Malachite green
• Distilled water 
• cedar wood oil.

Specimen: Sputum

Steps:

1. clean the slide and make it greeze-free.

2. Make a thin, uniform film with a sterilized loop.

3. Dry the film in air.

 4. Fix the film by slowly passing the slide 3-4 times through a flame.

5. Heat carbol fuchsin in a test tube till fumes appear.

6. Cover the slide with fuming carbol fuchsin and Keep for 5 minutes.

7. wash with water.

8. Decolourize with 20% sulphuric acid or acid alcohol until only a faint pink colour remains.

9. wash with water. 

10. Counterstain with methylene blue for 20-30 seconds.

11. wash with water and dry in the air.

Examine: Examine under oil-immersion of the microscope.

Q-4-a) Write down some organisms that can be differentiated in MacConkey’s agar media. (Jan-2021)

Organisms  can be differentiated in MacConkey’s agar media as lactose fermenters and non-lactose fermenters bacteria-

Lactose fermenters:

• Escherichia 
• Klebsiella 
• Enterobacter 
• Arizona 
• Serratia 
• Citrobacter

Non-lactose fermenters:

• Salmonella 
• Shigella 
• Proteus 
• Morganella 
• Hafnia 
• Providencia

Q-4-b) What is chocolate agar media?Name some solidifying agents and indicators used in culture media.Jan-2021

 Chocolate agar media:

Chocolate agar is a type of enriched growth medium used for isolating and cultivating fastidious bacteria, such as Haemophilus influenzae and Neisseria species. It is a variant of blood agar that contains red blood cells that have been lysed by slowly heating to 80°C. This releases nutrients from the red blood cells that are essential for the growth of fastidious bacteria.

Name some solidifying agents:

1. Agar (sea weed extract)
2. Egg whole
3. Gelatin
4. Potatoes
5. Serum
6. Tomato powder

Name some indicators:

1. Phenol red
2. Bromothymol blue
3. Methyl red
4. Turbidity indicator

Q-4-c) What is a vaccine? Classify vaccine with example.Jan-2021

Vaccines:

Vaccines are immuno-biological substances which are introduced to the body of a person or other animals to produce immunity against a specific disease.

Classification of vaccines with example: Vaccines may be–

1. Live attenuated vaccine:

 Bacterial:

• BCG vaccine (for tuberculosis).
• Typhoid oral.
• Plague.

Viral:

•  Oral polio vaccine.
• Yellow fever. 
•  Rubella
•  Mumps
• Influenza

Rickettsial:

• Epidemic Typhus.

2. Inactivated or killed vaccine:

 Bacterial:

• Pertussis vaccine (for whooping cough).
•  Cholera vaccine.
•  Typhoid vaccine. 
• Plague vaccine.

Viral:

• Salk vaccine (Polio).
• Rabies.
• Influenza. 
• Hepatitis B.

3. Toxoids:

Bacterial:

• Tetanus toxoid-Tetanus.
•  Diphtheria toxoid – Diphtheria.

4. Extracted cellular fractions:

• Meningococcal vaccine. 
• Pneumococcal vaccine.

5. Combination.

• DPT-Diphtheria, Pertussis, Tetanus. 
• MMR-Measles, Mumps, Rubella.
• DT.
• DP.
• DPT with typhoid vaccine. 
• DPTP (DPT + inactivated polio).

6. Recombinant vaccine: 

More recent preparations are subunit vaccine and recombinant vaccine, eg recombinant hepatitis-B vaccine.

Q-4-d) Define antigen of antibody.Name different  antigen, antibody reaction with example. (Jan-2021)

Antigen: 

Any substance which binds with antibodies is called antigen. It may be immunogenic or non-immunogenic.

Antibody: 

An antibody is a type of globulin (Immunoglobulin) produced in response to an antigen with which it reacts specifically.

Antigen-Antibody reaction: 

The specific combinations of antigens and antibodies giving rise to observable reaction are termed as antigen- antibody- reaction.

Name different  antigen, antibody reaction with example.Jan-2020 এর 2(B)নং দেখুন।

Q-4-e) Give the morphological classification of microorganism with example. (Jan-2021)

Micro-organisms:

They are small organisms and are either microscopic or submicroscopic creatures.

Classification of microorganisms:

Microorganism:

A. Cellular

1. Eukaryotes

• Protozoa
• Fungi
• Algae

2. Prokaryotes (Bacteria)

1. Extracellular (Most of the bacteria)
2. Intracellular (Rickettsiae,Chlamydiae)

B. Acellular (Virus)

    (Obligate intracellular)

Q-5-a) Write short note on:COVID-19 vaccine. (Jan-2021)

Ans:

The COVID-19 vaccine is a safe and effective way to protect yourself from the coronavirus disease 2019 (COVID-19). The vaccine helps your body develop antibodies that fight the virus, so you are less likely to get sick if you are exposed to it. The vaccine can also help prevent serious illness, hospitalization, and death from COVID-19.

The COVID-19 vaccine is recommended for everyone 6 months of age and older. It is especially important for people who are at high risk of serious illness from COVID-19, such as older adults and people with underlying health conditions.

The COVID-19 vaccine is safe and effective. The most common side effects are mild and go away on their own within a few days. These side effects may include pain, redness, and swelling at the injection site, as well as fatigue, headache, muscle pain, chills, fever, and nausea.

Here are some additional things to keep in mind about the COVID-19 vaccine:

• You will need two doses of the vaccine, spaced at least 3 weeks apart.
• You may need a booster shot after 6 months or more.
• The vaccine is not 100% effective, but it is the best way to protect yourself from COVID-19.
• Even if you are vaccinated, you should still wear a mask, social distance, and wash your hands often to help prevent the spread of the virus.

Q-5-b) Write a short note on: Hot air oven. (Jan-2021)

Hot air oven:

Hot air oven is the best apparatus for dry heat sterilization.It is a double walled metal chamber which is electrically heated.The fan or turbo blower fixed inside the chamber assists the uniform circulation of hot air.

Principle: Temperature needed in a hot air oven is 160 degree celsius  for one hour.Spore are also killed at this temperature.To guard against temperature variations air should be made to circulate by a suitable fan.

Uses of hot air oven:

Hot air oven is used for sterilization of-

1. Sterilization of glassware,such as all glass syringes,test tubes,petri dishes,pipettes,flasks and instruments like forceps,scissors,knives,scalpels etc.
2. Sterilization of powders,fats,oils and greases which are impermeable to moist heat.
3. Sterilization of cotton swabs.

Advantages of hot air oven:

1. Glass wares like petri dishes, swab sticks, and glass syringes can be sterilized. 

2. Powder, fats, grease etc. can be sterilized.

3. Sharp cutting surgical instruments can be sterilized.

Disadvantage of hot air oven:

1. Need more temperature

2. Time consuming

3. Plastic materials can not be sterilized in this process.

4. Natural and synthetic fibers cannot be sterilized.

5. Destroy the constituents of normal media.

Q-5-c) Write a short note on: Fumigation. (Jan-2021)

Ans:

In microbiology, fumigation is used to disinfect laboratory equipment and facilities, as well as to control the growth of microorganisms in the air and on surfaces. The most commonly used fumigants in microbiology are formaldehyde, ethylene oxide, and methyl bromide.

Here are some of the safety precautions that should be taken when using fumigants in microbiology:

• Always wear personal protective equipment (PPE), such as a respirator, gloves, and goggles, when working with fumigants.
• Follow the manufacturer’s instructions carefully when using fumigants.
• Fumigation should only be performed by trained personnel.
• The area to be fumigated should be well-ventilated before anyone enters.

Q-5-d)  Write a short note on: Tuberculin test. (Jan-2021)

Tuberculin Test (Mantoux test):

It is a hypersensitivity test that depends on delayed hypersensitivity reaction, which develops in individuals.

Purpose (use) of the test:

i) Aid to diagnosis. 

ii) For contact tracing.

iii) For post-reaction check up.

Reagent:

Tuberculin reagent is a purified protein derivative (PPD) of mycobacteria. ‘WHO-recommended tuberculin’ is “2TU PPD Rt 23′ ‘ diluted by Tueen80.

Technique of injection:

0.1 ml volume of tuberculin is slowly injected on the flexor aspect of the fore-arm intradermally.

Measurement of reaction:

The test is examined after 3 days and is carefully palpated for any induration.

Interpretation of the test:

.

• If induration is 10 mm or more: Positive (+ve).
• If induration is less than 10 mm: Negative (-ve)

Limitation of tuberculin test: 

          A. Tuberculin test may be positive other than active tuberculosis in-

                                                   i. Past TB

                                                   ii. Previous vaccination.

           B.Tuberculin test may be negative in spite of having TB (false negative) in-

• Severe tuberculosis (25% of cases negative) 
•  Extremes of age (newborn & old age)
•  Malnutrition
• HIV (if CD4 count <200 cells/ml)
• Immunosuppressive drugs
• Malignancy
• Sarcoidosis etc.

Q-5-e) Write short note on : AIDS. (Jan-2021)

AIDS: (Acquired Immuno Deficiency Syndrome) 

It is a syndrome complex, which is caused by human immunodeficiency virus (HIV)

Lab diagnosis of AIDS:

Principle: 

AIDS is diagnosed by serological test for antibody detection against HIV. Electron microscope examination is done in a reference laboratory. Cell culture is done for research purpose, Nucleic acid based techniques are used in special cases.

Specimen collection:

 Blood for serological test & culture.

Procedure:

 (a) Isolation of virus – HIV can be cultured from lymphocytes in peripheral blood or infected people.The most sensitive virus isolation technique is to co-cultivate. The test sample with uninfected, mitogen stimulated peripheral blood mononuclear cell (PBMC) which takes 7-14 days.

(b) Detection of viral antigen – ELISA. 

(c) Detection of antibody – ELISA confirmed by western blot and ICT, RIA, IFT.

(d) Detection of viral nucleic acid – PCR.

 (e) Immune function test – by counting CD4+ number. e.g. CD4+ cell count below 200/mm³ indicates cell mediated immunity severely suppressed & the patient is suffering from opportunistic infection.

(5) Non-specific tests-

• Raised ESR.
• Leucopenia.
• Lymphopenia.
• Reversal CD4 & CD8 cell ratio.
• Functional abnormality in macrophage & B cell.