• Morphology/classification
• Virulence factors and pathogenesis flowchart
• Clinical features
• Systematic laboratory diagnosis: specimens, timing, collection, transport, microscopy, culture/media, colony characters, biochemical tests, serology/antigen/molecular tests, susceptibility testing, and animal inoculation where applicable
• Brief treatment, prevention, and vaccines

Topic map

1. Tuberculosis
2. Meningitis
3. Syphilis
4. HIV/AIDS
5. Malaria
6. Enteric fever
7. Vibrio cholerae
8. Sterilization
9. Hypersensitivity
10. Classical complement pathway
11. Antigen-antibody reactions
12. Immunoglobulins
13. Poliomyelitis
14. Rabies
15. Gonorrhoea
16. Urinary tract infection

• Major bacteriology: TB, meningitis, enteric fever, cholera, gonorrhoea, UTI
• Virology: HIV, polio, rabies
• Parasitology: malaria
• Immunology: hypersensitivity, complement, antigen-antibody reactions, immunoglobulins
• Infection control: sterilization

Tuberculosis (TB)

Definition

1. Causative organisms

Mycobacterium tuberculosis complex (MTBC)

• M. tuberculosis - commonest cause in humans
• M. bovis
• M. africanum
• M. caprae
• M. microti

Morphology

• Slender, straight or slightly curved bacilli, about 2-4 µm long
• Non-motile, non-capsulated, non-sporing
• Aerobic
• Acid-fast due to lipid-rich cell wall containing mycolic acid
• Appears as red bacilli on Ziehl-Neelsen stain against a blue background
• May show beaded appearance and serpentine cords in culture

2. Virulence factors

3. Pathogenesis

Flowchart

• Healing with fibrosis and calcification, or
• Latent infection, or
• Progressive primary TB, or
• Reactivation/post-primary TB, commonly in lung apices

Granuloma

• Central caseous necrosis
• Epithelioid cells
• Langhans giant cells
• Peripheral lymphocytes and fibroblasts

Post-primary/reactivation TB

4. Clinical features

Pulmonary TB

• Cough for more than 2 weeks, with or without sputum
• Fever, often low-grade and evening rise
• Night sweats
• Weight loss, anorexia, fatigue
• Hemoptysis
• Chest pain and breathlessness in advanced disease

Extrapulmonary TB

• Lymph node TB: painless cervical lymphadenopathy, sinus formation
• Pleural TB: pleural effusion
• TB meningitis
• Miliary TB: fever, weight loss, hepatosplenomegaly, millet-seed lesions
• Bone and joint TB: Pott disease of spine
• Genitourinary TB: sterile pyuria, dysuria
• Abdominal TB: ascites, intestinal obstruction
• Cutaneous TB

5. Laboratory diagnosis of tuberculosis

1. Importance of laboratory diagnosis

• Confirm TB disease
• Detect infectious pulmonary cases
• Identify M. tuberculosis complex
• Detect rifampicin and other drug resistance
• Guide appropriate treatment
• Differentiate TB from non-tuberculous mycobacteria and other respiratory infections

2. Etiological agents

3. Specimens

4. Specimen collection

a. Optimal timing

• Collect sputum before starting anti-tubercular treatment.
• For routine pulmonary TB testing, an early-morning sputum sample may have a higher yield, but a good-quality spot sample is also acceptable under current programmatic practice.
• For urine TB: collect early-morning urine on 3 consecutive days.
• For blood culture in suspected disseminated TB: collect before treatment.

b. Correct type of specimen

• Deep expectorated sputum for pulmonary TB
• CSF for TB meningitis
• Aspirate/biopsy for lymph nodes, bone, or other extrapulmonary sites

c. Avoid contamination

• Do not collect saliva, postnasal secretion, or nasopharyngeal mucus as sputum.
• Use a clean, wide-mouthed, sterile, leak-proof container.
• Avoid formalin for samples intended for culture or CBNAAT. Formalin-fixed tissue can be used only for histopathology.

d. Procedure for sputum collection

1. Explain and demonstrate deep cough technique.
2. Ask patient to rinse mouth with clean water.
3. Collect in an open, well-ventilated area, away from others.
4. Patient takes deep breaths and coughs deeply from chest.
5. Collect 3-5 mL of mucoid or mucopurulent sputum.

e. Adequate quantity

• Sputum: preferably 3-5 mL
• CSF: as much as safely possible, because TB bacilli may be scanty
• Aspirate or tissue: adequate material for microscopy, culture, NAAT, and histopathology

f. Labelling

• Patient name and identifier
• Age/sex
• Specimen type and site
• Date and time of collection
• Test requested
• Treatment history and suspected drug resistance, if relevant

5. Transport

• Send specimen to laboratory promptly.
• Use a sterile, leak-proof, screw-capped container.
• If delay is unavoidable, keep specimen refrigerated at 2-8°C where feasible.
• Follow triple packaging and biosafety regulations for referral samples.

6. Macroscopic appearance

Sputum

• Preferred: thick, purulent, mucopurulent, blood-stained, or caseous particles
• Unsatisfactory: clear, watery saliva

CSF in TB meningitis

• Usually clear or slightly opalescent
• On standing, may form a delicate cobweb clot, in which bacilli may be concentrated.

7. Microscopy

A. Ziehl-Neelsen (ZN) stain

1. Make a thin smear from purulent portion of sputum.
2. Air-dry and heat-fix.
3. Stain with strong carbol fuchsin and heat gently.
4. Decolorize with acid-alcohol.
5. Counterstain with methylene blue.

• Simple, cheap, rapid
• Detects highly infectious smear-positive pulmonary TB

• Less sensitive in paucibacillary disease, children, HIV infection, and extrapulmonary TB
• Cannot distinguish M. tuberculosis from non-tuberculous mycobacteria
• Cannot detect drug resistance

B. Fluorescent microscopy

• Auramine-O or auramine-rhodamine stain
• AFB appear bright yellow-green against dark background
• Faster screening because smears can be examined at lower magnification
• More sensitive than conventional ZN microscopy in many settings

C. Concentration methods

8. Culture

A. Solid media

B. Liquid culture

• Faster detection, often about 1-3 weeks
• Better recovery in paucibacillary samples
• Useful for drug-susceptibility testing

• Expensive
• Higher contamination risk
• Requires specialized laboratory facilities

9. Identification and biochemical reactions

10. Molecular diagnosis

A. CBNAAT/GeneXpert MTB/RIF

1. DNA of M. tuberculosis complex
2. Resistance to rifampicin by detecting mutations in the rpoB gene

• Sputum
• Bronchoalveolar lavage
• Gastric aspirate
• CSF
• Lymph-node aspirate
• Tissue and selected extrapulmonary specimens, according to validated protocols

• Rapid, usually within about 2 hours
• High sensitivity and specificity
• Detects TB and rifampicin resistance simultaneously
• Minimal hands-on processing
• Useful in smear-negative disease and extrapulmonary TB

• Does not provide a full drug-susceptibility profile
• Rifampicin resistance should trigger further resistance testing
• May remain positive after successful treatment because DNA from dead bacilli can persist
• Cost and equipment requirements

B. Truenat MTB/MTB Plus and MTB-RIF Dx

• Chip-based real-time PCR system
• Detects MTBC and rifampicin resistance
• Used in decentralised settings in India

C. Line probe assay (LPA)

• PCR amplification followed by hybridization with probes
• Detects MTBC and mutations conferring resistance
• First-line LPA can identify resistance to rifampicin and isoniazid
• Second-line LPA can assess fluoroquinolone resistance and resistance to selected second-line drugs

D. Targeted next-generation sequencing

• Can detect mutations associated with resistance to multiple anti-TB drugs
• Mainly used in referral laboratories

11. Drug-susceptibility testing (DST)

Purpose

A. Phenotypic DST

• Proportion method on LJ medium
• Absolute concentration method
• Resistance-ratio method
• Automated liquid-culture DST, for example MGIT 960

B. Genotypic DST

12. Serology and antigen detection

Serology

Tuberculin skin test (TST/Mantoux test)

• Detects delayed type IV hypersensitivity to purified protein derivative (PPD).
• Indicates TB infection or sensitization, not necessarily active disease.
• May be positive after BCG vaccination or exposure to non-tuberculous mycobacteria.
• Can be false negative in severe illness, malnutrition, HIV infection, or recent viral infection.

Interferon-gamma release assays (IGRA)

• Detect interferon-gamma released by T cells in response to antigens such as ESAT-6 and CFP-10.
• More specific than TST in BCG-vaccinated persons.
• Used mainly to detect latent TB infection.
• Cannot reliably differentiate latent infection from active TB disease.

13. Animal inoculation

6. Drug-resistant tuberculosis

Definitions

Causes of acquired resistance

• Inadequate or irregular treatment
• Wrong drug combination
• Incorrect dose or poor-quality drugs
• Poor adherence
• Interrupted drug supply
• Failure to perform DST
• Inadequate absorption or drug-drug interactions

7. Treatment - brief

Drug-susceptible TB

• H - Isoniazid
• R - Rifampicin
• Z - Pyrazinamide
• E - Ethambutol

• Intensive phase: 2 months HRZE
• Continuation phase: 4 months HR

Important adverse effects

MDR/RR-TB

• Requires an all-oral, DST-guided regimen with second-line drugs.
• Current regimens may use bedaquiline, linezolid, fluoroquinolones, clofazimine, cycloserine and other agents according to resistance pattern and national guidelines.
• Never add a single drug to a failing regimen.

8. Prevention and control

• Early case detection and rapid molecular diagnosis
• Complete effective treatment with adherence support
• Contact tracing and screening of household contacts
• Cough etiquette and masks for infectious patients
• Adequate ventilation and avoidance of overcrowding
• Preventive treatment for eligible contacts and persons with latent TB infection
• HIV testing and antiretroviral therapy where indicated
• BCG vaccination

9. BCG vaccine

Full form

Nature

• Live attenuated vaccine prepared from Mycobacterium bovis
• Given intradermally

Dose and site

Schedule

• At birth or as early as possible, according to the national immunization schedule.

Local reaction

Protection

Contraindications

• Severe immunodeficiency
• Symptomatic HIV infection or infants with known severe immunodeficiency, according to local policy
• Immunosuppressive therapy
• Pregnancy is generally a contraindication to live vaccines

Complications

• Local ulceration
• Regional lymphadenitis
• Suppurative lymphadenitis
• Osteitis, rarely
• Disseminated BCG infection in severe immunodeficiency

Last-minute revision points

1. Specimen of choice for pulmonary TB: good-quality sputum.
2. Stain: Ziehl-Neelsen, showing red AFB on blue background.
3. Culture medium: Lowenstein-Jensen medium.
4. Culture colony: rough, tough, buff.
5. CBNAAT: detects MTBC and rifampicin resistance in about 2 hours.
6. MDR-TB: resistance to isoniazid + rifampicin.
7. Current XDR-TB: MDR/RR-TB + fluoroquinolone resistance + bedaquiline and/or linezolid resistance.
8. BCG: live attenuated M. bovis vaccine, intradermal at birth.
9. BCG prevents mainly: severe childhood TB, especially TB meningitis and miliary TB.
10. TST/IGRA: indicate infection/sensitization, not proof of active TB.

Meningitis

Definition

1. Types and causative organisms

2. Clinical features

Common symptoms

• Fever
• Severe headache
• Vomiting
• Neck stiffness
• Photophobia
• Altered sensorium
• Seizures
• Positive Kernig sign and Brudzinski sign

In infants

• Poor feeding
• Irritability
• High-pitched cry
• Bulging anterior fontanelle
• Hypothermia or fever
• Seizures

Meningococcal meningitis

• Petechial or purpuric rash
• Septicemia
• Shock
• Disseminated intravascular coagulation may occur

3. Laboratory diagnosis of meningitis

1. Importance

• Identify the etiological agent
• Differentiate pyogenic, tuberculous, and viral meningitis
• Start targeted antimicrobial therapy
• Detect drug resistance where relevant
• Prevent spread, particularly in meningococcal disease

2. Specimens

• CSF is the specimen of choice
• Blood for culture, especially in bacterial meningitis
• Serum for antigen detection or serology in selected cases
• Skin lesion aspirate/scraping in meningococcemia, if present
• Other specimens as indicated, such as sputum in TB or blood in disseminated fungal infection

3. Collection of CSF

Procedure

• Collect CSF by lumbar puncture using strict aseptic precautions.
• Usually collect in 3 sterile, labelled tubes:
  1. Biochemistry and serology
  2. Microbiology: Gram stain, culture, antigen detection, molecular tests
  3. Cell count and differential count

Important points

• Collect before antibiotics, whenever possible.
• Send immediately to laboratory. Do not refrigerate CSF suspected to contain N. meningitidis or H. influenzae, since they are delicate organisms.
• Collect adequate volume. A larger volume is especially important in suspected TB or fungal meningitis because organisms may be scanty.
• In suspected raised intracranial pressure, focal neurological deficit, papilledema, or severe altered consciousness, assess safety before lumbar puncture.

4. Macroscopic appearance of CSF

5. Microscopy

A. Gram staining

B. Ziehl-Neelsen stain

• Used in suspected TB meningitis.
• AFB may be detected in centrifuged CSF deposit.
• Sensitivity is low because TB meningitis is usually paucibacillary.

C. India ink preparation

• Used for suspected cryptococcal meningitis.
• Shows round budding yeast cells with a broad clear halo due to capsule.
• Especially useful in immunocompromised patients, including HIV infection.

D. KOH mount

• May be used for fungal elements in suspected fungal meningitis.

6. Culture

A. Pyogenic meningitis

B. TB meningitis

• Culture on Lowenstein-Jensen medium.
• Liquid culture such as MGIT can detect growth earlier.
• Growth is slow, often taking weeks.

C. Fungal meningitis

• Sabouraud dextrose agar.
• Cryptococcus neoformans produces creamy colonies.
• Urease test is positive.

7. Biochemical identification

8. Antigen detection

Latex agglutination test

• Streptococcus pneumoniae
• Neisseria meningitidis
• Haemophilus influenzae type b
• Group B streptococci
• E. coli K1

Cryptococcal antigen test

• Detects cryptococcal capsular polysaccharide antigen in CSF or serum.
• Highly useful in cryptococcal meningitis.
• Can be detected by latex agglutination or lateral-flow assay.

9. Molecular diagnosis

PCR/NAAT

• N. meningitidis
• S. pneumoniae
• H. influenzae
• Enteroviruses
• HSV
• TB meningitis
• Cryptococcal and other fungal infections in selected laboratories

CBNAAT/GeneXpert for TB meningitis

• Performed on CSF.
• Detects M. tuberculosis DNA and rifampicin resistance.
• A positive result is highly useful.
• A negative result does not exclude TB meningitis because CSF may contain very few bacilli.

10. Antibiotic susceptibility testing

• Perform on bacterial isolates from CSF or blood culture.
• Important for pneumococci, Gram-negative bacilli, H. influenzae, and meningococci where indicated.
• For TB, molecular and phenotypic drug-susceptibility testing is performed.
• Results guide definitive therapy.

4. CSF findings: pyogenic vs TB vs viral meningitis

One-line differentiation

• Pyogenic: neutrophils, high protein, low glucose, turbid CSF.
• TB: lymphocytes, very high protein, low glucose, cobweb clot.
• Viral: lymphocytes, normal glucose, clear CSF.

5. Treatment - brief

6. Prevention

• Vaccination against H. influenzae type b, pneumococcus, and meningococcus
• Early diagnosis and appropriate antimicrobial therapy
• Chemoprophylaxis for close contacts of meningococcal meningitis cases:
  • Rifampicin
  • Ciprofloxacin
  • Ceftriaxone
• Respiratory droplet precautions in suspected meningococcal infection
• BCG vaccination helps prevent severe childhood TB, including TB meningitis

Last-minute revision points

1. Specimen of choice: CSF.
2. Most important test: Gram stain and culture of CSF.
3. Meningococcus: Gram-negative kidney-shaped diplococci, oxidase positive, glucose and maltose fermentation.
4. Pneumococcus: Gram-positive lancet-shaped diplococci, optochin sensitive, bile soluble.
5. H. influenzae: needs X and V factors.
6. TB CSF: lymphocytes, high protein, low glucose, cobweb clot.
7. Viral CSF: lymphocytes, normal glucose, clear fluid.
8. Cryptococcus: India ink positive and cryptococcal antigen positive.
9. CBNAAT on CSF: detects TB DNA and rifampicin resistance.
10. Meningococcal contacts require chemoprophylaxis.

Syphilis

Definition

1. Morphology

• Thin, delicate, tightly coiled spirochete, about 6-15 µm long
• Has regular corkscrew-like spirals
• Motile by axial filaments or endoflagella
• Too thin to be seen clearly by ordinary light microscopy or Gram stain
• Demonstrated by:
  • Dark-ground microscopy
  • Direct fluorescent antibody test
  • Silver impregnation stains
  • PCR/NAAT

2. Pathogenesis

Important pathogenetic features

• Organism has an outer membrane with few exposed antigens, allowing immune evasion.
• It causes endarteritis and perivascular inflammation.
• Tissue damage in late syphilis is largely immune-mediated.

3. Stages and clinical features

A. Primary syphilis

Incubation period

Clinical features

• Chancre: single, painless, clean, indurated ulcer at site of inoculation
• Common sites: genitalia, cervix, anus, oral cavity, lips
• Base is clean, with scanty serous discharge
• Regional lymph nodes: discrete, firm, painless, non-suppurative
• Heals spontaneously in 3-6 weeks even without treatment

B. Secondary syphilis

Clinical features

• Fever, malaise, headache, sore throat
• Generalized, non-tender lymphadenopathy
• Symmetrical, non-itching maculopapular rash
• Rash characteristically involves palms and soles
• Mucous patches in mouth, pharynx, or genital area
• Condylomata lata: moist, broad, flat, highly infectious lesions in anogenital folds
• Patchy “moth-eaten” alopecia
• May have hepatitis, nephritis, meningitis, uveitis

C. Latent syphilis

• No clinical manifestations
• Serological tests remain positive

Types

• Early latent syphilis: infection acquired within the preceding 1 year
• Late latent syphilis: infection for more than 1 year or of unknown duration

D. Tertiary syphilis

1. Gummatous syphilis

• Gummas are granulomatous destructive lesions.
• May affect skin, bone, liver, and other organs.

2. Cardiovascular syphilis

• Syphilitic aortitis
• Ascending aortic aneurysm
• Aortic regurgitation
• Coronary ostial narrowing

3. Neurosyphilis

• Asymptomatic neurosyphilis
• Meningovascular syphilis
• General paresis of the insane
• Tabes dorsalis
• Optic atrophy
• Argyll Robertson pupil: accommodates but does not react to light

4. Congenital syphilis

Transmission

A. Early congenital syphilis

Features

• Stillbirth, prematurity, low birth weight
• Snuffles: blood-stained nasal discharge
• Generalized lymphadenopathy
• Hepatosplenomegaly
• Jaundice
• Anemia and thrombocytopenia
• Maculopapular or bullous skin rash, especially involving palms and soles
• Osteochondritis and periostitis
• Pseudoparalysis of Parrot due to painful bone lesions
• Meningitis

B. Late congenital syphilis

Hutchinson triad

1. Hutchinson teeth: notched, peg-shaped upper central incisors
2. Interstitial keratitis
3. Eighth cranial nerve deafness

• Saddle nose
• Frontal bossing
• Rhagades around mouth
• Mulberry molars
• Saber shins
• Clutton joints: painless bilateral knee effusions

5. Laboratory diagnosis

1. Importance

• Confirm syphilis
• Identify stage of infection
• Screen antenatal patients and prevent congenital syphilis
• Detect neurosyphilis
• Monitor response to treatment
• Detect reinfection or treatment failure

2. Etiological agent

• Treponema pallidum subspecies pallidum

• T. pertenue - yaws
• T. endemicum - endemic syphilis or bejel
• T. carateum - pinta

3. Specimens

4. Collection and transport

• Collect lesion exudate before local antiseptics or antibiotics.
• Clean chancre gently with sterile saline.
• Avoid bleeding while collecting serous fluid.
• Send immediately for dark-ground examination because motility decreases on delay.
• Collect venous blood aseptically in a plain tube for serum.
• CSF should be collected by lumbar puncture under aseptic precautions and sent promptly.

5. Direct detection methods

A. Dark-ground microscopy

• Rapid
• Useful in early primary and secondary syphilis

• Needs fresh specimen and trained observer
• Not useful for oral lesions
• Less useful in late syphilis

B. Direct fluorescent antibody test

• Uses fluorescent-labelled antibody against T. pallidum
• Can detect organisms in lesion material or tissue

C. Silver staining

• Warthin-Starry and related silver stains may demonstrate spirochetes in tissue sections.

D. PCR/NAAT

• Detects T. pallidum DNA from lesion exudate or tissue.
• Useful when direct microscopy is unavailable.
• PCR assays are usually laboratory-developed rather than universally commercially available.

6. Serological diagnosis

A. Nontreponemal tests

Examples

• VDRL: Venereal Disease Research Laboratory test
• RPR: Rapid Plasma Reagin test

Principle

Uses

• Screening
• Estimating disease activity
• Quantitative reporting of titre
• Monitoring treatment response
• Detecting reinfection or treatment failure

Interpretation

False-positive VDRL/RPR

Prozone phenomenon

B. Treponemal tests

Examples

• TPHA: T. pallidum hemagglutination assay
• TPPA: T. pallidum particle agglutination assay
• FTA-ABS: fluorescent treponemal antibody absorption test
• Treponemal EIA/CLIA
• Rapid treponemal tests

Principle

Uses

• Confirmation of a positive screening test
• Treponemal-test-first screening algorithms

Limitation

VDRL/RPR vs TPHA/FTA-ABS

7. Testing algorithms

Traditional algorithm

Reverse sequence algorithm

8. Neurosyphilis diagnosis

• Meningitis
• Stroke in young person
• Cranial nerve palsy
• Visual or auditory symptoms
• Tabes dorsalis
• Cognitive or psychiatric changes

Tests

• Serum nontreponemal and treponemal tests
• CSF cell count and protein
• CSF-VDRL
• CSF FTA-ABS/TPPA in selected cases

9. Congenital syphilis diagnosis

• Maternal infection and adequacy/timing of maternal therapy
• Clinical examination of newborn
• Quantitative maternal and neonatal VDRL/RPR titres, preferably by the same test and laboratory
• CBC, platelet count
• CSF-VDRL, CSF cell count, and protein where indicated
• Long-bone radiographs
• Dark-field microscopy, fluorescent antibody testing, or PCR of lesion/placental tissue where available

7. Treatment - brief

Early syphilis

• Benzathine penicillin G 2.4 million units IM, single dose

Late latent syphilis or latent syphilis of unknown duration

• Benzathine penicillin G 2.4 million units IM once weekly for 3 doses
• Total dose: 7.2 million units

Tertiary syphilis without neurosyphilis

• Same regimen as late latent syphilis, after excluding neurosyphilis.

Neurosyphilis, ocular syphilis, or otosyphilis

• Aqueous crystalline penicillin G intravenously for 10-14 days, according to guideline-based dosing.

Pregnancy

• Penicillin is the only proven treatment that adequately treats both mother and fetus.
• Penicillin-allergic pregnant patients should be desensitized and then treated with penicillin.

Penicillin allergy in non-pregnant adults

Jarisch-Herxheimer reaction

• Acute fever, chills, headache, myalgia, and transient worsening of lesions after treatment.
• Caused by rapid destruction of spirochetes.
• Occurs especially in early syphilis.
• It is not a penicillin allergy.

8. Prevention

• Safe sexual practices and consistent condom use
• Early diagnosis and treatment
• Screening in pregnancy
• Test and treat sexual partners
• Screen for HIV and other STIs
• Follow-up with quantitative VDRL/RPR titres
• Partner notification and counselling

Last-minute revision points

1. Organism: Treponema pallidum, a motile spirochete.
2. Primary lesion: painless, indurated chancre with painless lymphadenopathy.
3. Secondary rash: non-pruritic, commonly involves palms and soles.
4. Most infectious lesions: chancre, mucous patches, condylomata lata.
5. Direct diagnosis: dark-ground microscopy of lesion exudate.
6. Screening and follow-up: VDRL/RPR.
7. Confirmation: TPHA/TPPA or FTA-ABS.
8. Treponemal tests remain positive after successful treatment.
9. MDR not relevant to syphilis: penicillin remains the standard treatment.
10. Hutchinson triad: Hutchinson teeth + interstitial keratitis + eighth-nerve deafness.
11. Drug of choice: benzathine penicillin G.
12. In pregnancy: treat with penicillin after desensitization if allergic.

HIV/AIDS

Definition

1. Causative agent and types

• Family: Retroviridae
• Subfamily: Lentivirinae
• Types:
  • HIV-1: common worldwide and more virulent
  • HIV-2: mainly in West Africa and India; lower transmissibility and slower progression

Modes of transmission

1. Sexual contact
2. Blood and blood products
3. Shared contaminated needles/syringes
4. Mother-to-child transmission: pregnancy, delivery, breastfeeding
5. Occupational exposure: needle-stick injury, mucosal exposure

2. Structure of HIV

Components

Genes

3. Pathogenesis

Flowchart

Mechanisms of CD4 cell depletion

• Direct virus-induced cytopathic effect
• Syncytium formation
• Apoptosis/pyroptosis of infected and bystander cells
• Cytotoxic T-cell-mediated killing of infected CD4 cells
• Chronic immune activation and exhaustion
• Destruction of lymphoid tissue architecture

Important cells infected

• CD4 T lymphocytes
• Monocytes/macrophages
• Dendritic cells
• Microglial cells of CNS

4. Clinical stages of HIV infection

1. Acute HIV infection

Features

• Fever
• Sore throat
• Rash
• Lymphadenopathy
• Myalgia
• Headache
• Oral ulcers
• Diarrhea
• Aseptic meningitis in some patients

2. Asymptomatic/clinical latency stage

• Patient may remain asymptomatic for years.
• Persistent generalized lymphadenopathy may occur.
• HIV continues to replicate.
• CD4 count gradually declines.

3. Symptomatic HIV disease

• Weight loss
• Persistent fever
• Chronic diarrhea
• Oral candidiasis
• Recurrent bacterial infections
• Herpes zoster
• Tuberculosis
• Recurrent respiratory infections

4. AIDS

AIDS-defining illnesses

• Pneumocystis jirovecii pneumonia
• Esophageal candidiasis
• Cryptococcal meningitis
• Toxoplasmosis of brain
• Disseminated tuberculosis
• Cytomegalovirus retinitis
• Recurrent bacterial pneumonia
• Chronic cryptosporidiosis
• Kaposi sarcoma
• Invasive cervical carcinoma
• Non-Hodgkin lymphoma
• Progressive multifocal leukoencephalopathy

5. Opportunistic infections according to CD4 count

6. Laboratory diagnosis

1. Importance of laboratory diagnosis

• Detect HIV infection early
• Prevent transmission
• Identify mother-to-child transmission risk
• Assess immune status by CD4 count
• Measure viral load
• Monitor response to ART
• Diagnose HIV in infants born to HIV-positive mothers
• Screen blood donors safely

2. Specimens

• Serum or plasma: antibody/antigen tests
• Whole blood: rapid tests, CD4 count
• EDTA plasma: HIV RNA viral load
• Dried blood spot: useful in infant testing and remote settings
• CSF: selected cases with neurological disease
• Blood culture/other specimens: for suspected opportunistic infections

3. Specimen collection and transport

• Collect blood aseptically using a sterile needle and labelled tube.
• Serum: plain tube.
• Plasma and CD4 count: EDTA anticoagulated blood.
• Label clearly with patient details, date, specimen type, and test requested.
• Transport promptly according to laboratory protocol.
• Maintain confidentiality and obtain informed consent as per testing policy.

4. Antibody detection tests

A. ELISA/EIA

• Detect anti-HIV antibodies.
• Third-generation assays detect IgM and IgG antibodies.
• Fourth-generation tests detect both:
  • HIV-1/2 antibodies
  • p24 antigen

B. Rapid/simple tests

• Immunochromatographic assay
• Dot immunoassay
• Particle agglutination assay

• Rapid
• Simple
• No sophisticated equipment required
• Useful in ICTC and peripheral settings

5. Antigen detection

p24 antigen

• Capsid protein of HIV.
• Appears in blood before antibodies develop.
• Useful in early infection and in fourth-generation HIV assays.
• May become undetectable after seroconversion because it forms antigen-antibody complexes.
• May reappear in advanced disease when viral replication is high.

6. Molecular diagnosis

HIV RNA PCR

• Detects plasma viral RNA.
• Used for:
  • Early diagnosis during window period
  • Viral load measurement
  • Monitoring ART response
  • Detecting treatment failure

HIV DNA PCR

• Detects proviral DNA.
• Important for diagnosis in infants born to HIV-positive mothers because maternal IgG antibodies cross the placenta.
• Antibody tests are unreliable in infants below 18 months.

Viral load

• Measures copies of HIV RNA per mL of plasma.
• Best marker of viral replication and response to ART.
• A falling or undetectable viral load indicates response to treatment.

7. CD4 count

Importance

• Indicates degree of immunosuppression.
• Helps assess risk of opportunistic infections.
• Used for baseline evaluation and monitoring where indicated.

Normal CD4 count

Interpretation

Methods

• Flow cytometry
• Point-of-care CD4 testing in selected settings

7. Window period

• The person may be infectious.
• Antibody tests can be negative.
• Repeat testing after the appropriate interval is required following a high-risk exposure.

8. NACO HIV testing strategy - exam answer

Strategy I

• Used for screening blood and blood products.
• A single highly sensitive test is used.
• Reactive units are discarded and never transfused.
• It is not used to diagnose HIV infection in an individual.

Strategy II

• Used for surveillance purposes.
• Two different tests are used sequentially.

Strategy III

• Used for diagnosis of HIV infection in individuals.
• Three different tests are performed sequentially.
• If all three tests are reactive, the person is reported HIV-positive.
• If tests are discordant, the report is inconclusive and repeat testing after the appropriate interval is advised.

Important points

• Testing should be voluntary, confidential, and accompanied by pre-test and post-test counselling.
• A reactive screening test alone does not establish a diagnosis.
• HIV-positive persons should be linked promptly to ART services and evaluated for TB and other co-infections.

9. Treatment - brief

Antiretroviral therapy (ART)

Principle

• Two nucleoside/nucleotide reverse transcriptase inhibitors, plus
• One integrase strand transfer inhibitor

Common first-line regimen

• TDF: tenofovir disoproxil fumarate
• 3TC: lamivudine
• DTG: dolutegravir

Classes of antiretroviral drugs

10. Post-exposure prophylaxis (PEP)

Indications

• Needle-stick injury with potentially HIV-infected blood
• Splash of blood/body fluid to mucosa or non-intact skin
• Sexual assault
• Condom breakage or unprotected sexual exposure with a known or high-risk HIV-positive source
• Sharing needles

Immediate first aid

Needle-stick injury

• Wash immediately with soap and running water.
• Do not squeeze the wound aggressively.
• Do not scrub, bleach, or apply irritant chemicals.

Splash to eye/mouth

• Flush immediately with large amounts of water or saline.

PEP regimen

• Start as soon as possible, ideally within 2 hours.
• Do not delay while awaiting source test results if exposure is significant.
• PEP should generally not be started after 72 hours.
• Continue for 28 days.

• HIV test
• Hepatitis B and hepatitis C assessment
• Renal and liver function tests as appropriate
• Pregnancy test where relevant

11. Prevention

• Safe sexual practices and condom use
• Screening of blood donors
• Use of sterile disposable needles and syringes
• Universal precautions for healthcare workers
• Prevention of mother-to-child transmission through ART
• Early ART for all HIV-positive individuals
• Treatment of sexually transmitted infections
• HIV testing and counselling
• PEP after significant exposure
• Pre-exposure prophylaxis (PrEP) for persons with substantial ongoing HIV risk

Last-minute revision points

1. HIV is an enveloped RNA retrovirus of the Lentivirus group.
2. gp120 binds CD4 and CCR5/CXCR4; gp41 causes fusion.
3. p24 is the capsid antigen.
4. Key enzymes: reverse transcriptase, integrase, protease.
5. HIV causes progressive CD4 T-cell depletion.
6. Most important OI in India: tuberculosis.
7. CD4 <200/µL: risk of Pneumocystis jirovecii pneumonia.
8. CD4 <100/µL: toxoplasmosis and cryptococcosis.
9. CD4 <50/µL: CMV retinitis and disseminated MAC.
10. Fourth-generation tests detect p24 antigen + HIV antibodies.
11. HIV DNA/RNA PCR is required to diagnose HIV in infants below 18 months.
12. NACO Strategy III uses three sequential different reactive tests for diagnosis.
13. First-line ART commonly: TDF + 3TC + DTG.
14. PEP: start immediately, preferably within 2 hours, maximum 72 hours, and continue for 28 days.