(a) (i) Describe the morphological characteristics of *Cryptococcus neoformans* on microscopic examination. Discuss the…

(a)(i) Cryptococcus neoformans: morphology and pathogenesis of cryptococcosis

Morphology. It is a round to oval yeast, 4 to 10 μm, with narrow-necked (narrow-based) budding and no hyphae in tissue. It has a thick polysaccharide capsule. In India ink preparation of CSF the capsule is seen as a clear halo around the yeast against a black background; mucicarmine stains the capsule red, and silver stains show the yeast. On culture (Sabouraud agar at 37 °C) it gives mucoid colonies; it is urease positive and produces melanin from phenolic substrates (brown colonies on bird-seed agar).

Pathogenesis. Infection is by inhaling desiccated yeast or spores from soil and pigeon droppings, giving a small, often silent, lung focus. In people with defective cell-mediated immunity (HIV with low CD4 count, transplant recipients, steroid use) the yeast spreads through the blood and reaches the meninges and brain, causing meningoencephalitis and cryptococcomas; skin, bone and prostate may be involved. Virulence factors are the capsule (glucuronoxylomannan), which resists phagocytosis, depletes complement and suppresses inflammation; melanin, which protects against oxidants; urease; and the ability to grow at 37 °C.

(a)(ii) Meningococcaemia, laboratory diagnosis of meningococcal meningitis, and vaccines

Definition. Meningococcaemia is bloodstream infection with Neisseria meningitidis, causing fever and a petechial or purpuric rash, and in severe cases septic shock with adrenal haemorrhage (Waterhouse-Friderichsen syndrome).

Laboratory diagnosis. CSF is turbid, with many neutrophils, low glucose and high protein. Gram stain of CSF shows Gram-negative, kidney-shaped diplococci, often inside neutrophils. Culture of CSF and blood on chocolate agar or Thayer-Martin medium in 5 to 10% CO₂ gives oxidase-positive colonies that ferment glucose and maltose; this is confirmatory. Antigen detection in CSF by latex agglutination, and PCR, are useful when antibiotics were given before sampling. Smear from petechiae may also show organisms.

Vaccines. Plain polysaccharide vaccines (bivalent A and C, quadrivalent A, C, W, Y) give short protection and no herd effect. Conjugate vaccines (MenACWY, MenA conjugate used in the African meningitis belt, and pentavalent A, C, W, Y, X) give longer protection and reduce carriage. Serogroup B vaccines (Bexsero, Trumenba) are protein-based. Vaccination is advised for outbreaks, Hajj pilgrims, asplenia and complement deficiency.

(b)(i) Injury, hurt and grievous hurt, and secondary markings on a fired bullet

Definitions as per the IPC (now the Bharatiya Nyaya Sanhita, BNS, in brackets): Injury (section 44 IPC; BNS section 2(14)): any harm whatever illegally caused to any person in body, mind, reputation or property. Hurt (section 319; BNS 114): causing bodily pain, disease or infirmity to any person. Grievous hurt (section 320; BNS 116): only the following kinds of hurt: emasculation; permanent loss of sight of either eye; permanent loss of hearing of either ear; loss of any member or joint; destruction or permanent impairing of the powers of any member or joint; permanent disfiguration of the head or face; fracture or dislocation of a bone or tooth; and any hurt that endangers life, or causes severe bodily pain, or makes the person unable to follow ordinary pursuits for 20 days.

Secondary markings. When a bullet is fired from a rifled barrel, primary markings are the class characteristics from the rifling: the number, width, depth and twist of lands and grooves. Secondary markings are the fine individual striations along the lands and grooves, produced by tiny manufacturing imperfections, wear and metal fouling in the barrel. No two barrels give identical striations, so they are the "fingerprint" of a particular weapon. Comparing them under a comparison microscope with test bullets from the suspected weapon identifies the weapon.

(b)(ii) Types of finger impressions and DNA fingerprinting

Types of finger impressions: latent (invisible, from sweat, developed with powder or chemicals), patent (visible, made with blood, ink or dirt) and plastic (moulded in soft material like wax, soap or putty). Patterns are arches (plain, tented), loops (ulnar, radial), whorls and composites.

DNA fingerprinting (Alec Jeffreys, 1985). It identifies a person from variable, non-coding repeat sequences of DNA, which differ between individuals (identical twins excepted). Method: DNA is extracted from blood, semen, hair roots or bone; the classical procedure cut it with restriction enzymes, separated the fragments by gel electrophoresis, transferred them by Southern blotting and hybridised them with labelled probes to show VNTR bands. Modern practice amplifies short tandem repeats (STRs), usually 13 to 20 loci, by PCR and reads them by capillary electrophoresis; Y-STR and mitochondrial DNA are used for male lineage and old or degraded samples. Interpretation: matching of all loci with random match probabilities of one in billions identifies a person; a mismatch excludes. Uses: identifying culprits in rape and murder cases, paternity and kinship, disaster victims, missing persons and exonerating the innocent. Contamination of samples and chain of custody must be guarded.

(c)(i) Mechanism of action of potassium-sparing diuretics

They act on the late distal tubule and collecting duct principal cells, where sodium enters through epithelial sodium channels (ENaC) under aldosterone, and potassium is secreted into the lumen because of the negative luminal potential. Aldosterone antagonists (spironolactone, eplerenone) block the intracellular mineralocorticoid receptor and prevent aldosterone-induced synthesis of ENaC and Na⁺-K⁺-ATPase. Direct ENaC blockers (amiloride, triamterene) block the channel on the luminal side. Sodium entry falls, the lumen becomes less negative, so potassium and hydrogen ion secretion are reduced, giving mild natriuresis (about 2% of filtered sodium) with potassium retention. Hyperkalaemia is the main danger, and spironolactone also causes gynaecomastia. They are used with thiazides or loop diuretics, in heart failure, cirrhotic ascites and primary hyperaldosteronism.

(c)(ii) Zidovudine

Zidovudine (AZT) is a thymidine analogue nucleoside reverse transcriptase inhibitor; once phosphorylated to the triphosphate it terminates the viral DNA chain. Indications: HIV infection as part of combination antiretroviral therapy; prevention of mother-to-child transmission (in pregnancy, labour and the infant); post-exposure prophylaxis; and, with interferon, adult T-cell leukaemia. Adverse effects: bone marrow suppression (anaemia with macrocytosis and neutropenia, the commonest), myopathy, lactic acidosis with hepatic steatosis, headache, nausea, nail pigmentation and lipoatrophy. Haemoglobin and counts are monitored.