PART B - Applied Aspects of Microbiology

Q1. Hepatitis B & Hepatitis C: Transmission, Universal Precautions & Vaccination (10 marks)

A) Modes of Transmission

• Sexual contact - most common route in adults in developed countries
• Percutaneous (parenteral) - needlestick injuries, IV drug use, contaminated instruments
• Perinatal (vertical) - mother to newborn during delivery (most common route globally in high-endemic areas)
• Blood transfusion - less common now due to screening
• Close household contact - through sharing razors, toothbrushes, minor skin abrasions
• HBV is stable on environmental surfaces for at least 7 days and is 50-100x more infectious than HIV

• Parenteral route (IV drug use) - single most important route today
• Blood and blood products - reduced greatly by screening
• Needlestick injuries in healthcare workers (risk ~3%)
• Sexual transmission - less efficient than HBV; mainly in high-risk groups (MSM, multiple partners)
• Perinatal transmission - occurs in ~5% of births from HCV-positive mothers
• No vaccine is available for HCV; prevention relies entirely on precautions

B) Universal Precautions

1. Gloves - wear when handling blood, body fluids, mucous membranes, or non-intact skin
2. Mask + Eye protection - use when splashes or aerosols are possible
3. Protective gowns/aprons - wear and remove before leaving work area
4. Needles - use only disposable needles; discard directly into puncture-resistant sharps containers without recapping
5. Decontamination - clean work surfaces with 0.5% sodium hypochlorite (1:10 bleach)
6. Hand hygiene - wash hands before and after patient contact, after removing gloves
7. Autoclaving/ethylene oxide gas - for sterilization of metal instruments
8. No mouth pipetting - do not eat, drink, or smoke in work areas

C) Vaccination

• Available since 1982
• Originally plasma-derived (HBsAg from carriers); now replaced by recombinant DNA vaccine (yeast-derived HBsAg)
• Highly effective - reduces HBV infection significantly
• Schedule: 3 doses (0, 1, 6 months) for adults; at birth + 2 more doses for infants
• HBIG (Hepatitis B Immune Globulin) - used for post-exposure prophylaxis and in newborns of HBsAg-positive mothers (within 12 hours of birth)
• Vaccination of health care workers is recommended along with monitoring of anti-HBs levels
• In high-endemic areas, vaccination of newborns is the cornerstone of control

• No vaccine available for HCV
• Prevention depends entirely on standard precautions, harm reduction programs (needle exchange), and blood product screening

Q2. UTI: Etiological Agents, Pathogenesis, Clinical Manifestations & Laboratory Diagnosis (10 marks)

A) Etiological Agents

• Escherichia coli - accounts for ~80% of uncomplicated UTIs
• Klebsiella pneumoniae
• Proteus mirabilis - associated with struvite stones
• Pseudomonas aeruginosa - common in catheter-associated and nosocomial UTIs
• Enterobacter spp.

• Staphylococcus saprophyticus - second most common in young sexually active women
• Enterococcus faecalis
• Staphylococcus aureus - in bacteremic seeding of kidney

• Candida spp. - in immunocompromised or catheterized patients

• Chlamydia trachomatis, Neisseria gonorrhoeae - urethritis

B) Pathogenesis

1. Colonization of the periurethral area (especially in women - short urethra) with gut flora
2. Ascent up the urethra into the bladder (most common route)
3. Virulence factors of E. coli include: Type 1 fimbriae (mannose-sensitive) bind to uroplakin receptors; P-fimbriae (mannose-resistant) allow upper tract invasion; Hemolysin, aerobactin (iron acquisition), K antigen (anti-phagocytic capsule)
4. Host defenses overcome: normal voiding, urine osmolality, Tamm-Horsfall protein, secretory IgA
5. Infection may remain in the bladder (cystitis) or ascend to kidneys via ureters (pyelonephritis)
6. Hematogenous route - less common; occurs in S. aureus, Candida bacteremia

C) Clinical Manifestations

D) Laboratory Diagnosis

1. Urine collection - midstream clean-catch specimen (preferred); catheter specimen if needed
2. Urinalysis:
   • Pus cells (pyuria): >10 WBCs/mm³ on microscopy
   • Bacteria on Gram stain
   • Nitrite test (positive for Gram-negatives)
   • Leukocyte esterase (positive)
   • Hematuria, proteinuria may be present
3. Urine culture (gold standard):
   • Significant bacteriuria: ≥10⁵ CFU/mL in midstream clean-catch
   • Lower counts significant in symptomatic women (≥10² for E. coli)
   • Culture on blood agar and MacConkey agar
   • Identify organism + antimicrobial sensitivity testing (AST)
4. Blood culture - in suspected pyelonephritis or urosepsis
5. Imaging - USG/CT for structural abnormalities, stones, obstruction in complicated UTI

Q3. HIV: Clinical Manifestations & Modes of Transmission (10 marks)

A) Modes of Transmission

1. Sexual contact (most common worldwide):
   • Unprotected receptive anal intercourse - highest risk
   • Vaginal intercourse - both male-to-female and female-to-male
   • Risk increased by other STIs (genital ulcers)
2. Parenteral (blood):
   • Sharing needles/syringes among IV drug users
   • Needlestick injuries in healthcare workers (~0.3% per exposure)
   • Blood transfusion and blood products (now rare with screening)
   • Organ/tissue transplantation
3. Vertical (mother-to-child):
   • In utero (transplacental) - across all trimesters, risk higher in 3rd trimester
   • Intrapartum - during delivery (most common)
   • Postnatal - through breast milk
   • Overall risk ~25% without intervention; reduced to <2% with antiretrovirals

B) Clinical Manifestations - Four Stages

• Resembles infectious mononucleosis
• Fever, pharyngitis, dysphagia, lymphadenopathy
• Maculopapular rash, fatigue, myalgia, arthralgia
• Hepatosplenomegaly, leukopenia
• Self-limiting; most patients do not seek care
• High viral load; seroconversion occurs within 3-6 months

• Lasts 5 to >15 years
• Patient is a carrier; may have persistent generalized lymphadenopathy
• CD4 count slowly declining (normal >500/mm³)

• CD4 count 200-500/mm³
• Constitutional symptoms: weight loss >10%, fever, night sweats, chronic diarrhea
• Oral candidiasis (thrush), oral hairy leukoplakia
• Herpes zoster, recurrent bacterial infections
• Has NOT yet developed AIDS-defining illness

• CD4 count <200/mm³ (diagnostic criterion for AIDS)
• Opportunistic infections (OIs):
  • Pneumocystis jirovecii pneumonia (PCP) - most common OI in US
  • Toxoplasmosis (brain abscess)
  • Cryptococcal meningitis
  • CMV retinitis
  • MAC (Mycobacterium avium complex)
  • Cryptosporidium diarrhea
  • Tuberculosis (reactivation)
• AIDS-defining malignancies:
  • Kaposi's sarcoma (HHV-8)
  • Non-Hodgkin's lymphoma
  • Invasive cervical carcinoma
• Neurological: HIV encephalopathy (AIDS dementia complex), peripheral neuropathy

Q4. Window Period (5 marks)

• In HIV, it is the period after exposure when the person is infected and infectious but tests negative on standard antibody-based ELISA
• During this window, viral replication is occurring but sufficient antibody has not yet been produced to be detectable
• With HIV: window period is approximately 3-12 weeks with 3rd/4th generation assays (detecting both antigen p24 and antibody), and up to 6 months with older 2nd generation antibody-only tests
• Nucleic Acid Testing (NAT/PCR) shortens the window period to ~10-33 days by detecting viral RNA directly
• The window period is critical in blood transfusion safety - a donor tested during this period may screen negative yet transmit infection
• It is also relevant for HIV testing in clinical practice - a negative test during the window period must be repeated after the window has passed

Q5. Aseptic Collection of Urine Sample (5 marks)

Midstream Clean-Catch (Most Common Method)

1. Patient education - explain the procedure clearly
2. Handwashing - patient washes hands thoroughly with soap and water
3. Genital cleansing:
   • Females: Separate the labia with one hand; cleanse from front to back with antiseptic wipes (3 separate wipes: left, right, center)
   • Males: Retract foreskin; cleanse the urethral meatus with antiseptic wipe
4. Begin voiding - pass the first part of urine (first 10-20 mL) into the toilet (this flushes urethral flora)
5. Collect midstream - without stopping the stream, collect the middle portion of urine (~30-50 mL) in a sterile, wide-mouthed, leak-proof container
6. End of void - allow remaining urine to pass into toilet
7. Labeling - label the container immediately with name, date, time
8. Transport - transport to lab within 2 hours, or refrigerate at 4°C for up to 24 hours (refrigeration prevents bacterial overgrowth)

Other Methods

• Catheter specimen - used in unconscious, uncooperative, or post-surgical patients; reduces contamination but carries infection risk
• Suprapubic aspiration (SPA) - gold standard for anaerobic culture; needle passed suprapubically into a full bladder; any bacterial growth is significant

Q6. Opportunistic Parasitic Infections (5 marks)

Major Opportunistic Parasitic Infections

Diagnosis

• Stool microscopy (modified AFB stain for Cryptosporidium, Isospora), PCR, biopsy
• CT/MRI brain for Toxoplasma
• BAL (bronchoalveolar lavage) + silver stain for PCP

Management

• Treat the underlying immunosuppression (ART for HIV)
• Specific antiparasitic drugs (TMP-SMX for PCP, pyrimethamine + sulfadiazine for Toxoplasma, nitazoxanide for Cryptosporidium)
• Prophylaxis in high-risk patients (CD4-count guided)

Q7. Steps Involved in Blood Collection for Culture (5 marks)

Equipment Needed

• Sterile gloves, antiseptic (70% alcohol + povidone-iodine or chlorhexidine), aerobic and anaerobic blood culture bottles, sterile syringe/butterfly needle, tourniquet, adhesive bandage

Procedure

• Collect before antibiotic administration if possible
• Ideally collect during a fever spike or chill when bacteremia is highest

• Collect 2-3 sets from different sites (at least 2 sites, 30-60 minutes apart for subacute cases; simultaneously for acute sepsis)
• Each set = 1 aerobic + 1 anaerobic bottle
• Volume: 8-10 mL per bottle in adults (minimum 5 mL); 1-3 mL in pediatrics
• Larger volume significantly increases yield

• Select a suitable vein (antecubital fossa preferred)
• Apply tourniquet
• Cleanse the skin:
  1. Wipe with 70% isopropyl alcohol (30 seconds), let dry
  2. Apply povidone-iodine 1-2% or chlorhexidine gluconate 2% in concentric circles from center outward; allow to dry for 60-90 seconds (do not fan or blot - this is critical)
  3. Do not re-palpate the vein after cleaning; if re-palpation needed, wear sterile gloves

• Wipe the rubber septum of each blood culture bottle with 70% alcohol and allow to dry

• Perform venipuncture with a sterile needle
• Collect the required volume of blood
• Inject aerobic bottle first, then anaerobic (so any air entering goes into aerobic bottle)
• Mix gently by inversion

• Label each bottle immediately: patient name, date, time, site of collection, set number
• Transport to laboratory within 1-2 hours at room temperature
• Do not refrigerate blood culture bottles (cold inhibits organism growth)
• Culture bottles are incubated in automated systems (BACTEC, BacT/ALERT) at 35-37°C, monitored continuously for CO₂ production

• Avoid collecting from IV lines or sites with active infection/cellulitis
• If IV line culture is required, change needle before injecting into culture bottle
• Maintain strict aseptic technique throughout