Microbiology

Classification and General Features

Bacterial Cell Wall

Lipopolysaccharide and Endotoxin

LPS has three parts — lipid A, a core polysaccharide, and the O-specific side chain → lipid A is the toxic moiety; the O side chain is the O antigen used in serotyping (as in Salmonella and E. Coli O157) → On bacterial lysis, lipid A is released and binds TLR4 on macrophages → Massive release of TNF-alpha, IL-1 and IL-6 → Fever, hypotension, capillary leak, disseminated intravascular coagulation → septic shock and multi-organ failure

Other Structures and Their Significance

• Capsule — usually polysaccharide (but polypeptide (D-glutamate) in Bacillus anthracis). It is antiphagocytic and therefore a major virulence factor; demonstrated by the quellung reaction and by negative staining. Capsular polysaccharide is the basis of the pneumococcal, meningococcal and Hib conjugate vaccines
• Flagella — organs of motility, made of flagellin; the H antigen. Arrangements: monotrichous, lophotrichous, amphitrichous and peritrichous. Motility is demonstrated by hanging drop or in semisolid medium
• Fimbriae (common pili) — short, numerous, for adhesion to host cells; a virulence factor in uropathogenic E. Coli and gonococci. sex pilus — long, one or two per cell, mediates conjugation and the transfer of resistance plasmids
• Spores — dormant, highly resistant forms produced by Bacillus and Clostridium; contain dipicolinic acid with calcium and very little water, which is the basis of their heat resistance. They are the reference standard for sterilisation
• Plasmids — extrachromosomal circular DNA carrying resistance and virulence genes, transferable between organisms
• Cell membrane — site of respiration and transport; sterols are absent except in mycoplasma

Bacterial Morphology and Arrangement

• Arrangement reflects the plane of division and whether daughter cells separate — division in one plane with cells remaining attached gives chains; division in several planes gives clusters. It is a genuinely useful identifying feature on a Gram film

Applied Aspects

• The peptidoglycan wall is absent in man, which is why penicillins and cephalosporins have such a wide therapeutic index — their only serious hazard is allergy
• Mycoplasma has no cell wall, so beta-lactams are useless against it; this is why atypical pneumonia requires a macrolide or tetracycline
• Endotoxin survives autoclaving, so intravenous fluids and devices must be tested for pyrogens (the LAL test) as well as for sterility — a sterile solution can still cause rigors
• Gram-negative sepsis remains a leading cause of death, and the cytokine cascade explains why killing the organism with an antibiotic can transiently worsen the patient through massive endotoxin release
• Capsular vaccines fail in infants unless conjugated to a protein carrier, because polysaccharide alone provokes a T-independent response without memory
• The Gram stain remains one of the most valuable and cheapest investigations in medicine — available within minutes, it guides initial antibiotic choice long before culture results
• L-forms and spheroplasts are cell-wall-deficient variants produced by penicillin or lysozyme; they can revert and may explain some relapses after apparently adequate treatment
• Fimbriae explain why urinary infection is so common in women — uropathogenic E. Coli adhere by P fimbriae to uroepithelial receptors, and the short urethra shortens the journey
• Motility is a useful bedside laboratory clue — the darting "fish in a stream" motility of Vibrio cholerae in a hanging drop, abolished by specific antiserum, has been used for rapid diagnosis in outbreaks
• Antigenic structure underlies serotyping — the O (somatic), H (flagellar) and K or Vi (capsular) antigens are the basis of the Kauffmann-White scheme for Salmonella and of E. Coli serotyping such as O157:H7
• Rickettsiae and chlamydiae are bacteria, not viruses, despite being obligate intracellular organisms — they have both DNA and RNA and a cell wall, and they respond to antibiotics, which viruses do not

Definitions

Physical Methods — Heat

The Autoclave in Detail

• Principle — water boils at 100°C at atmospheric pressure; raising the pressure raises the boiling point, so saturated steam at 15 lb/in² reaches 121°C
• All air must BE removed — this is the single most important requirement. Air is a poor conductor, and a pocket of trapped air is cooler than the surrounding steam, so items within it are not sterilised. Modern autoclaves use a pre-vacuum to remove it
• Steam must be saturated and must condense on the load, releasing latent heat and wetting the surface; superheated or wet steam both perform poorly
• Loading matters — packs must not be overpacked and must allow steam to penetrate; the holding time begins only when the whole load has reached temperature
• Not suitable for oils, powders, greases (steam cannot penetrate), or heat-labile plastics and endoscopes

Sterilisation Control

• Bacterial spores are used because they are the most resistant form of life to the process being tested; if the spores are dead, everything else is

Other Physical Methods and Chemical Agents

• Radiation — non-ionising (UV, 254 nm) causes thymine dimers but has very poor penetration, so it disinfects surfaces and air (operating theatres, safety cabinets) and cannot sterilise a wrapped object; it also damages the eyes and skin. ionising (gamma rays, electron beam) penetrates deeply and is "cold sterilisation" used industrially for disposable syringes, gloves, catheters, sutures and plastics
• Filtration — membrane filters of 0.22 micrometre pore size for heat-labile fluids (sera, antibiotic solutions, vaccines); viruses and mycoplasma pass through. HEPA filters remove particles above 0.3 micrometre from air in operating theatres, laminar flow cabinets and isolation rooms
• Gaseous sterilisation — ethylene oxide for heat-sensitive items (endoscopes, prosthetic heart valves, plastics); it is an alkylating agent, effective but highly toxic, mutagenic, carcinogenic and explosive, and requires prolonged aeration afterwards. Alternatives are hydrogen peroxide plasma and formaldehyde vapour
• Chemical disinfectants — glutaraldehyde (2%) for endoscopes, being sporicidal with prolonged contact; hypochlorite for surfaces and spills, and sporicidal, which is why it is used against Clostridioides difficile; 70% alcohol, chlorhexidine, povidone-iodine for skin
• Spaulding classification — critical items entering sterile tissue or the vascular system require sterilisation; semi-critical items contacting mucous membranes (endoscopes) require high-level disinfection; non-critical items touching intact skin require low-level disinfection

Choosing a Method

Applied Aspects

• Use a biological indicator regularly, not merely tape; autoclave tape shows only that the pack met steam, not that sterilisation was achieved
• Perform the Bowie-Dick test daily on a vacuum autoclave; failure of air removal is the commonest cause of sterilisation failure
• Clean before you disinfect — organic matter (blood, pus, faeces) inactivates hypochlorite and quaternary ammonium compounds and shields organisms; this is the most frequently neglected step
• Endoscopes are a recognised vehicle of transmission, and outbreaks have been traced to inadequate cleaning of channels; the process is manual, tedious and easily short-cut
• Prions resist all routine methods — standard autoclaving does not destroy them, and extended cycles (134°C for 18 minutes), sodium hydroxide, or disposal by incineration are required
• Reuse of single-use devices is common in India for reasons of cost, and is a real infection risk; where it is unavoidable, validated reprocessing is essential rather than informal cleaning
• Boiling is disinfection, not sterilisation, and the distinction is frequently misunderstood in smaller facilities where boiled instruments are treated as sterile
• Check the expiry and integrity of sterile packs; a pack that is damp, torn or has been stored badly is no longer sterile whatever the indicator shows
• Disinfectant solutions can themselves become contaminated — outbreaks of Pseudomonas and Burkholderia have been traced to diluted or topped-up containers, which is a real hazard where cost drives dilution

Bacterial Nutrition and Growth Requirements

• Nutritional requirements — a carbon source, nitrogen, inorganic ions, water, and for some organisms specific growth factors that they cannot synthesise
• Classification by carbon and energy source — autotrophs use carbon dioxide; heterotrophs require organic carbon, and all medically important bacteria are heterotrophs
• Oxygen requirement — obligate aerobes (Pseudomonas, Mycobacterium); obligate anaerobes (Clostridium, Bacteroides), which lack catalase and superoxide dismutase and are killed by oxygen; facultative anaerobes (most Enterobacteriaceae, staphylococci), which grow either way; microaerophilic (Campylobacter, Helicobacter); and capnophilic organisms requiring extra carbon dioxide (Neisseria, Haemophilus, Brucella)
• Temperature — most pathogens are mesophiles growing optimally at 37°C; psychrophiles and thermophiles are of food and environmental importance
• PH — most grow best near neutrality (7.2 to 7.6); Vibrio cholerae prefers alkaline conditions (pH 8.5), which is exploited in alkaline peptone water, and lactobacilli tolerate acid

The Bacterial Growth Curve

Lag phase — no increase in number; the cells are metabolically very active, synthesising enzymes and adapting; the cell is at its largest → log (exponential) phase — growth at a constant maximum rate; cells are uniform, most metabolically active and most susceptible to antibiotics and disinfectants; this is the phase used for Gram staining and sensitivity testing → stationary phase — nutrient exhaustion and accumulation of toxic products; division equals death; spores, exotoxins and secondary metabolites are formed here → decline (death) phase — the number of viable cells falls; involution forms appear

Culture Media — Classification

• MacConkey agar is both selective and differential — the bile salts and crystal violet select against gram-positives, while lactose with neutral red differentiates fermenters from non-fermenters. It is the single most useful plate in a routine laboratory

Methods of Anaerobic Culture

• Anaerobic jar (McIntosh-Fildes) with a gas-generating sachet producing hydrogen and carbon dioxide, and a palladium catalyst; an indicator (methylene blue or resazurin) confirms anaerobiosis
• Anaerobic chamber or glove box — the most reliable, allowing plates to be handled without oxygen exposure
• Robertson cooked meat medium — the meat particles absorb oxygen and provide reducing substances; also indicates saccharolytic (pink meat) or proteolytic (black, foul-smelling) activity
• Thioglycollate broth — contains a reducing agent and permits both aerobic and anaerobic growth in one tube
• Pre-reduced media and prompt transport matter as much as the incubation method; anaerobes die during transit, which is the commonest reason they are not isolated

Identification of Bacteria from Culture

• Colony morphology — size, shape, edge, elevation, consistency, pigment and odour. Pseudomonas produces greenish pyocyanin with a characteristic fruity smell; Proteus swarms over the plate
• Haemolysis on blood agar — alpha (partial, greenish — viridans streptococci, pneumococcus); beta (complete, clear — Streptococcus pyogenes, Staphylococcus aureus); gamma (none — enterococci)
• Gram stain of the colony, and motility by hanging drop
• Biochemical reactions — catalase (separates staphylococci, positive, from streptococci, negative); coagulase (separates S. Aureus from other staphylococci); oxidase (positive in Pseudomonas, Vibrio and Neisseria; negative in Enterobacteriaceae); urease, indole, citrate, and the triple sugar iron reaction
• Serotyping with specific antisera for Salmonella, Shigella and E. Coli; and MALDI-TOF mass spectrometry, which identifies an isolate from its protein fingerprint within minutes where available

Measurement of Bacterial Growth

Applied Aspects

• Collect the specimen before antibiotics are given — a single dose can sterilise blood cultures and leave the patient without a diagnosis for the rest of the illness
• Choose the right medium and tell the laboratory what you suspect; mycobacteria, anaerobes, fungi and fastidious organisms all need specific media and prolonged incubation that will not be set up routinely
• Use a transport medium for swabs that cannot reach the laboratory promptly, and never allow a specimen for anaerobic culture to be exposed to air
• Interpret growth in the context of the site — a positive culture from a normally sterile site is significant; growth from sputum, wound swabs or urine may be colonisation or contamination
• Automated blood culture systems have transformed practice, giving results in hours rather than days, though cost limits availability in India
• Quality control of media, reagents and incubators is what makes laboratory results trustworthy, and is invisible until it fails
• Blood culture bottles must be filled to the marked volume; volume is the single greatest determinant of yield, and an under-filled bottle commonly explains a negative result in an obviously septic patient
• Incubate for long enough — brucella, HACEK organisms, fungi and mycobacteria all require extended incubation that will not happen unless the laboratory is told what is suspected
• Growth of three or more organisms from urine indicates contamination, not polymicrobial infection, and the specimen should be repeated rather than treated
• Interpret a colony count alongside the pyuria and the symptoms; bacteriuria without symptoms is usually not treated, except in pregnancy and before urological instrumentation
• Media must be quality-controlled with known organisms before use, since a batch that fails to support growth produces false negatives that nobody detects
• Selective media reduce sensitivity as well as raising specificity, so a non-selective plate is inoculated alongside where the organism is uncertain
• Automated blood culture systems have transformed turnaround, flagging positives within hours rather than days, though their cost limits availability in much of India
• Prolonged incubation of a "negative" culture is worthwhile when the clinical picture is convincing; slow growers are lost when plates are discarded at 48 hours by protocol
• Store media correctly — blood agar deteriorates, selective agents lose potency and plates dry out; the commonest cause of a laboratory "not growing anything" is the medium, not the specimen
• Record the antibiotic history on the request form, since prior therapy explains a sterile culture from an infected site and changes how the result should be read

Bacterial Genome and Variation

• The bacterial genome is a single circular double-stranded DNA molecule with no nuclear membrane and no introns; it is haploid, so any mutation is immediately expressed
• Extrachromosomal elements — plasmids (autonomous circular DNA, often carrying resistance or virulence genes; the F factor confers the ability to conjugate); transposons ("jumping genes" that move between DNA molecules and can carry resistance genes); and integrons, which capture and express gene cassettes and are a major vehicle for multidrug resistance
• Variation arises by mutation or by gene transfer, and the distinction matters clinically: mutation is vertical and slow, gene transfer is horizontal and can spread resistance between species overnight

Mechanisms of Gene Transfer

Mechanisms of Drug Resistance

Genetic Techniques in Diagnosis

• Polymerase chain reaction (PCR) — amplifies a target sequence; rapid, extremely sensitive, and detects organisms that are non-cultivable, slow-growing or already suppressed by antibiotics. Real-time PCR quantifies, as in HIV and hepatitis viral load
• CBNAAT / GeneXpert — a cartridge-based automated PCR that detects Mycobacterium tuberculosis and rifampicin resistance within about 2 hours; it has transformed tuberculosis diagnosis in India
• Line probe assay — detects mutations conferring resistance to isoniazid, rifampicin and second-line drugs
• Nucleic acid hybridisation and DNA probes; whole genome sequencing for outbreak investigation and resistance prediction
• Limitations of molecular methods — they detect nucleic acid, not viability, so they may remain positive after successful treatment; they are prone to contamination giving false positives; and they do not provide an isolate for full sensitivity testing

Bacterial Mutation and its Consequences

• Mutation is a spontaneous heritable change in the base sequence, occurring at a rate of roughly 10-6 to 10-9 per cell division; it is random and not induced by the drug
• Because bacteria are haploid, a mutation is expressed immediately, without the masking that a second allele would provide
• Types — point mutation (substitution), frameshift (insertion or deletion), and deletion of larger segments
• Clinical consequences — single-step high-level resistance to rifampicin (rpoB), streptomycin and fluoroquinolones (gyrA), which is exactly why these drugs must never be used alone in tuberculosis
• The mathematics of combination therapy — if resistance to one drug arises at 10-6 and to another at 10-8, an organism resistant to both arises at 10-14, which exceeds the number of bacilli in any lesion. This is the whole rationale for multidrug regimens in tuberculosis, leprosy and HIV
• Phenotypic variation — changes in expression without genetic change, such as capsule loss on subculture or the smooth-to-rough colony transition

Genetic Basis of Vaccines and Diagnostics

• Recombinant vaccines — the gene for a protective antigen is expressed in yeast or another host: the hepatitis B surface antigen vaccine and the HPV vaccine are made this way, avoiding any need to handle the pathogen
• Live attenuated organisms are produced by repeated subculture or by deliberate deletion of virulence genes, which gives a more stable attenuation than the classical method
• MRNA and viral vector vaccines deliver the genetic instructions for an antigen rather than the antigen itself, and the platform can be redirected to a new pathogen very quickly — as the COVID-19 vaccines demonstrated
• Molecular diagnosis — PCR and real-time PCR, CBNAAT detecting M. Tuberculosis and rifampicin resistance within about 2 hours, line probe assays, and whole genome sequencing for outbreak tracing and resistance prediction
• Molecular typing — pulsed-field gel electrophoresis, multilocus sequence typing and sequencing have largely replaced phage typing where they are affordable, and allow an outbreak strain to be traced between wards, hospitals and countries
• CRISPR was discovered as a bacterial immune system against phages, storing fragments of previous invaders to recognise them again; it is now the basis of genome editing — a reminder that fundamental microbiology repeatedly yields tools nobody was looking for
• Genetically engineered organisms produce many therapeutic proteins — human insulin, growth hormone, erythropoietin, interferons and clotting factors — which removed the risks of animal and cadaveric sources
• Antibiotic resistance genes are used as markers in genetic engineering, which has itself raised concern about their environmental spread
• Resistance carries a fitness cost in most organisms, so reducing antibiotic use allows sensitive strains to re-establish; resistance is therefore reversible in principle, which is the strongest argument for stewardship
• The gut is the great mixing vessel — commensals and pathogens exchange plasmids there under the selective pressure of whatever antibiotic the patient is taking, which is why an antibiotic given for a chest infection can select urinary resistance
• Environmental and agricultural use contributes substantially, and resistant organisms reach humans through food, water and effluent; this is a public health problem beyond clinical prescribing
• Never add a single drug to a failing regimen — that is functional monotherapy against an already-resistant population, and resistance to the added drug follows within weeks
• Fixed-dose combinations prevent inadvertent monotherapy, which is why they are used in tuberculosis, leprosy and HIV programmes where supervision cannot be guaranteed
• Detecting resistance genes is faster than growing the organism, and CBNAAT reporting rifampicin resistance in 2 hours has changed the management of tuberculosis in India more than any new drug
• A gene may be present without being expressed, so molecular resistance testing complements rather than replaces culture and phenotypic sensitivity

Applied Aspects

• Antibiotics do not create resistance; they select for it — resistant mutants arise spontaneously, and the drug kills the sensitive majority and leaves them to multiply without competition
• Conjugation is why resistance spreads so fast — a plasmid carrying several resistance genes can move between species in the gut of a single patient, so using one antibiotic can select resistance to others
• Combination therapy prevents resistance in tuberculosis, leprosy and HIV because the probability of simultaneous resistance to several drugs is the product of the individual probabilities
• Molecular detection of rifampicin resistance guides treatment within hours instead of weeks, which is the single most useful diagnostic advance in Indian tuberculosis practice
• Infection control limits the spread of resistant organisms, and no prescribing policy compensates for poor hand hygiene; the two must go together
• India has among the highest rates of antimicrobial resistance in the world, driven by over-the-counter sale, incomplete courses and agricultural use; the National Action Plan and Schedule H1 are steps toward control

Normal Flora

• Beneficial roles — colonisation resistance (competing for nutrients and receptors and producing inhibitory substances), synthesis of vitamin K and B group vitamins, stimulation of the immune system and of gut-associated lymphoid tissue, and aid to digestion
• Harmful roles — they act as opportunistic pathogens when displaced from their site (E. Coli in the urinary tract), when a barrier is breached (bowel perforation, a catheter), or when immunity fails; and they are a reservoir of resistance genes

Host-parasite Relationship

Determinants of Bacterial Virulence

• Adhesion — pili and fimbriae, adhesins and surface proteins; without attachment there is no infection, which is why the flushing action of urine and the mucociliary escalator are so protective
• Invasion — invasins and enzymes: hyaluronidase ("spreading factor"), collagenase, streptokinase (fibrinolysin), lecithinase
• Evasion of host defences — the capsule (antiphagocytic); protein A of staphylococci (binds the Fc portion of IgG the wrong way round); M protein of Streptococcus pyogenes; IgA protease; coagulase, which walls the organism off in fibrin; intracellular survival (Mycobacterium, Salmonella, Listeria); and antigenic variation
• Toxin production — exotoxins and endotoxin
• Iron acquisition by siderophores, competing with host transferrin and lactoferrin
• Biofilm formation on devices and damaged tissue

Host Defence Mechanisms

• Mechanical barriers — intact skin, mucous membranes, the mucociliary escalator, coughing and sneezing, the flushing of urine and tears
• Chemical barriers — gastric acid, lysozyme in tears and saliva, fatty acids and low pH of skin, lactoferrin, defensins, the acidic vaginal pH
• Biological — the normal flora itself
• Innate cellular and humoral — phagocytes (neutrophils and macrophages), natural killer cells, the complement system, interferons, acute phase proteins, fever and inflammation
• Adaptive — humoral (antibody) and cell-mediated immunity, with memory

Koch Postulates and Their Limitations

• The postulates — the organism must be found in all cases of the disease and not in healthy individuals; it must be isolated in pure culture; it must reproduce the disease when inoculated into a susceptible animal; and it must be RE-isolated from that animal
• Limitations — some organisms cannot be cultured (M. Leprae, Treponema pallidum); some have no animal model; asymptomatic carriers harbour the organism without disease, breaking the first postulate; disease may require several organisms or a particular host state; and ethical considerations forbid human inoculation
• Modified molecular postulates now relate a gene to virulence — the gene should be present in pathogenic strains, its inactivation should reduce virulence, and restoring it should restore virulence
• They remain a useful discipline of thought — before attributing a disease to an organism, ask whether it is present in all cases, absent in health, and capable of producing the illness

Applied Aspects

• Distinguish colonisation from infection before prescribing — a positive wound swab, sputum culture or catheter urine in a well patient usually needs no antibiotic, and treating it selects resistance
• Every indwelling device breaches a barrier; catheters, cannulae and tubes should be removed as soon as they are not needed, which prevents more infection than any antibiotic
• Immunosuppressed patients are threatened by their own flora and by organisms of low virulence, so the differential diagnosis of fever is entirely different
• Chronic carriers sustain outbreaks — the typhoid carrier with gallbladder colonisation is the classical example, and food handlers must be identified and treated
• Do not suppress gastric acid unnecessarily; it is a genuine defence, and its removal increases enteric infection, C. Difficile and pneumonia
• Handwashing removes transient flora and is the single most effective measure against cross-infection; alcohol rub is faster and better for most organisms, but soap and water is required for C. Difficile spores
• Resident flora cannot be eliminated, only reduced, which is why surgical scrub uses a substantive agent such as chlorhexidine that goes on acting under the glove
• Probiotics aim to restore the flora, and faecal microbiota transplantation for recurrent C. Difficile infection is a striking demonstration that the flora itself can be therapeutic
• The microbiome is increasingly implicated beyond infection — in obesity, immune development and inflammatory disease — which reframes the normal flora as an organ rather than a set of passengers
• Opportunistic infection defines the differential in the immunosuppressed — organisms of low virulence that would never trouble a normal host, so the threshold for investigation must be far lower
• Malnutrition, diabetes, HIV and chronic kidney disease are the common causes of impaired defence in Indian practice, and each shifts the likely pathogens in a predictable direction
• Virulence and host resistance are two sides of one balance; the same organism causes asymptomatic carriage in one person and fatal sepsis in another, which is why treating the host matters as much as killing the organism
• Vaccination works by shifting that balance before exposure, and is the only intervention that prevents infection without selecting for resistance
• Nutrition is an underrated determinant of infection outcome, and in India undernutrition remains a major reason why tuberculosis, measles and diarrhoea kill children who would otherwise recover
• Splenectomised patients are at lifelong risk from capsulated organisms — pneumococcus, meningococcus and Haemophilus — and require vaccination, prophylaxis and a card, because the spleen is what clears them
• Take an occupational, travel and animal contact history; brucellosis, leptospirosis, scrub typhus, anthrax and rabies are all suggested by exposure long before any test is available

Definition and Classification

• Adaptive immunity is further classified as active (the host makes its own response, slow to develop but long-lasting, with memory) or passive (preformed antibody is transferred, giving immediate but temporary protection with no memory)
• Active natural — clinical or subclinical infection; active artificial — vaccination
• Passive natural — maternal IgG across the placenta and IgA in colostrum; passive artificial — immunoglobulin, antivenom, antitoxin

Organs of the Immune System

Cells of the Immune System

Innate Defence Mechanisms in Detail

• Mechanical barriers — intact skin, mucous membranes, the mucociliary escalator, coughing, sneezing, and the flushing action of urine and tears
• Chemical barriers — gastric acid, lysozyme in tears and saliva (which splits peptidoglycan), fatty acids and low pH of skin, lactoferrin sequestering iron, and defensins
• Biological — the normal flora, through colonisation resistance
• Phagocytosis — chemotaxis, adherence (greatly enhanced by opsonins: IgG and C3b), ingestion, and killing by oxygen-dependent (respiratory burst, myeloperoxidase) and oxygen-independent (lysozyme, lactoferrin, defensins) mechanisms
• Complement, interferons, acute phase proteins (C-reactive protein, mannose-binding lectin), fever and inflammation
• Pattern recognition — TOLL-like receptors recognise conserved microbial structures: TLR4 binds LPS, TLR2 peptidoglycan, TLR5 flagellin, TLR3, 7, 8 and 9 nucleic acids

Cytokines

Applied Aspects

• Maternal IgG protects the infant for about 3 to 6 months, which is why the primary immunisation schedule begins at 6 weeks — earlier vaccination is partly neutralised by maternal antibody
• Splenectomised patients are at lifelong risk from capsulated organisms — pneumococcus, meningococcus, Haemophilus — and require vaccination, penicillin prophylaxis and a warning card
• Defects in phagocyte function cause recurrent pyogenic infection, as in chronic granulomatous disease, where the respiratory burst fails and catalase-positive organisms survive inside the phagocyte
• Passive immunisation gives immediate protection and no memory, which is why antivenom, antitoxin and immunoglobulin are used for immediate threats and are given alongside the vaccine in post-exposure prophylaxis
• Adjuvants work by provoking innate signals; alum and newer adjuvants convert a weakly immunogenic protein into an effective vaccine by supplying the danger signal that dendritic cells require
• Undernutrition impairs cell-mediated immunity particularly, and is a major reason why tuberculosis, measles and diarrhoea remain lethal in Indian children who would otherwise recover

Antigen — Definitions

• Determinants of immunogenicity — foreignness (the greater the phylogenetic distance, the stronger the response), large molecular size (above about 10 kDa), chemical complexity (proteins are the best immunogens, then polysaccharides; lipids and nucleic acids are poor unless conjugated), degradability, and the dose, route and genetic constitution of the host

Immunoglobulin Structure

The basic unit is a Y-shaped glycoprotein of four polypeptide chains → two identical heavy chains (about 50 kDa) and two identical light chains (about 25 kDa), joined by disulphide bonds → Light chains are either kappa or lambda — never both in one molecule → Each chain has a variable (V) region at the amino end and a constant (C) region → The variable regions of one heavy and one light chain together form the antigen-binding site, containing three hypervariable regions (complementarity-determining regions) that make the actual contact → The hinge region gives flexibility so the two arms can grip epitopes at varying distances → The heavy chain constant region forms the Fc portion, which determines the class and all the effector functions

Classes of Immunoglobulin

Primary and Secondary Immune Response

• The secondary response is the entire basis of booster doses and of why a vaccinated person is protected: on real exposure the response is fast enough to prevent disease

Monoclonal Antibodies

• Produced by hybridoma technology — a B cell from an immunised animal is fused with a myeloma cell, giving an immortal clone secreting a single antibody of one specificity (Köhler and Milstein)
• Nomenclature indicates origin and immunogenicity — "-omab" murine (most immunogenic, provoking human anti-mouse antibodies), "-ximab" chimeric, "-zumab" humanised, "-umab" fully human (least immunogenic)
• Diagnostic uses — ELISA and rapid tests, immunohistochemistry, blood grouping, flow cytometry, and tumour marker assays
• Therapeutic uses — rituximab (anti-CD20), trastuzumab (anti-HER2), infliximab and adalimumab (anti-TNF), tocilizumab (anti-IL-6R), omalizumab (anti-IgE), eculizumab (anti-C5), and the checkpoint inhibitors in cancer
• Passive immunisation — palivizumab against respiratory syncytial virus, and monoclonal antibodies developed against SARS-CoV-2 and rabies
• Limitations — very high cost, parenteral administration, infusion reactions, immunogenicity, and infection risk with immunosuppressive agents; biosimilars produced in India have substantially improved access

Applied Aspects

• IgM in the newborn indicates intrauterine infection, since maternal IgM cannot cross the placenta — the practical basis of diagnosing congenital rubella, toxoplasmosis, syphilis and cytomegalovirus
• A fourfold rise in antibody titre between acute and convalescent sera is diagnostic; a single titre is hard to interpret where the population has high background seropositivity, as in India
• IgM indicates recent infection and IgG past exposure or immunity, which is why both are measured in dengue, hepatitis and torch screening
• Secretory IgA in colostrum protects the newborn gut, which is a concrete immunological argument for breastfeeding and for feeding through diarrhoeal illness
• Selective IgA deficiency is the commonest primary immunodeficiency, and these patients may develop anaphylaxis to blood products containing IgA
• Monoclonal antibodies are now major therapeutic agents — rituximab, trastuzumab, infliximab and the checkpoint inhibitors
• Hapten-carrier conjugation explains drug allergy — penicillin is far too small to be immunogenic alone, and becomes so only by binding host protein; this is also why cross-reactivity occurs within a drug class
• Superantigens explain toxic shock syndrome — a small amount of TSST-1 activates a huge fraction of T cells at once, and the resulting cytokine storm causes the shock rather than any direct toxicity
• Adjuvants are what make a purified protein vaccine work; without an innate danger signal the adaptive system responds poorly, which is why alum is added to toxoid and recombinant vaccines
• A booster dose exploits the secondary response, producing higher-affinity IgG within days; the interval between doses matters because memory cells need time to develop
• Immunoglobulin levels vary with age — a physiological trough at 3 to 6 months as maternal IgG wanes before the infant’s own production rises, which is when antibody deficiencies first declare themselves
• Serum protein electrophoresis is a useful screen — a monoclonal band suggests myeloma, a diffuse polyclonal rise suggests chronic infection or liver disease, and a reduced gamma region suggests hypogammaglobulinaemia
• Antibody neutralises toxin but cannot reach an intracellular organism, which is why humoral immunity protects against diphtheria and tetanus and cell-mediated immunity is required against tuberculosis
• Passive antibody interferes with live vaccines, so measles vaccination is deferred for some months after immunoglobulin or blood transfusion
• Affinity maturation takes weeks, which is why the antibody of a primary response is less protective than that of a secondary one even at the same titre

Definition and Components

• Inactivated at 56°C for 30 minutes — the basis of "heat inactivation" of serum before certain serological tests
• C3 is the most abundant and the central component, at which all three pathways converge

The Three Pathways

All three pathways generate a C3 convertase → C3 → C3a + C3b → C3b binds the surface (opsonisation) and forms the C5 convertase → C5 → C5a + C5b → C5b binds C6, C7, C8 and multiple C9 molecules → membrane attack complex (MAC, C5b-9) forms a transmembrane pore → Osmotic lysis of the cell

Biological Functions

Complement Deficiencies and Regulation

• Regulatory proteins prevent damage to host cells — C1 inhibitor, factor H and factor I, DAF and CD59. Host cells are protected; microbial surfaces are not, which is what gives the alternative pathway its selectivity

Measurement and Clinical Interpretation

• Falling C3 and C4 indicate active consumption and are used to track disease activity, whereas a persistently low level with no illness suggests an inherited deficiency

Applied Aspects

• Test complement in recurrent neisserial infection; terminal component deficiency is easily missed and the patient benefits from meningococcal vaccination and prophylaxis
• Hereditary angio-oedema is repeatedly mistaken for anaphylaxis and treated with adrenaline, antihistamines and steroids, none of which work; the absence of urticaria and itch is the clue, and a low C4 supports the diagnosis
• Low C3 and C4 indicate active consumption, and are used to monitor disease activity in SLE and post-streptococcal glomerulonephritis
• Eculizumab blocks C5 and prevents MAC formation, transforming paroxysmal nocturnal haemoglobinuria and atypical haemolytic-uraemic syndrome — but it produces exactly the deficiency described above, so patients must be vaccinated against meningococcus before treatment
• Anaphylatoxins explain "pseudo-allergic" reactions to radiocontrast, vancomycin and some drugs, which look like anaphylaxis but involve no IgE and may occur on first exposure
• Heat serum at 56°C for 30 minutes to destroy complement before certain serological tests, or non-specific lysis confuses the result
• C3 deficiency is the most dangerous, because opsonisation fails and both the classical and alternative pathways converge on it; such children present with severe pyogenic infection in infancy
• Complement is consumed in disseminated intravascular coagulation and severe sepsis, and low levels there reflect consumption rather than deficiency
• The alternative pathway is active from the first moment of infection, which is why complement is classed as innate immunity despite its classical arm needing antibody
• Immune complexes are cleared on erythrocytes through CR1, so anaemia and splenectomy both impair clearance and predispose to immune complex deposition
• C1 inhibitor also regulates the contact and kinin systems, which is why its deficiency causes bradykinin-mediated angio-oedema rather than a complement-mediated illness, and why ACE inhibitors precipitate attacks
• Complement is heat-labile at 56°C but the antibody is not, which is the basis of inactivating serum without destroying the specific antibody being measured
• The alternative pathway distinguishes self from non-self by regulation — host surfaces carry factor H and other inhibitors, microbial surfaces do not, so amplification proceeds only on the invader
• Complement therapeutics are expanding — eculizumab and its successors block C5, and their cost and the meningococcal risk they create are both practical considerations
• Vaccinate against meningococcus before starting a C5 inhibitor, and consider continuous penicillin prophylaxis; the drug creates precisely the deficiency that predisposes to neisserial disease
• C-reactive protein and mannose-binding lectin are innate opsonins that activate complement without antibody, which is why CRP rises so early in bacterial infection
• Do not confuse low complement with immunodeficiency in an acutely ill patient; consumption in active SLE or sepsis is far commoner than an inherited defect
• Complement bridges innate and adaptive immunity — C3d binding CR2 lowers the threshold for B-cell activation, so the innate system amplifies the adaptive one it also instructs
• Splenectomy impairs complement-mediated clearance as well as removing the site of response to blood-borne capsulated organisms, which is why these patients need vaccination and prophylaxis for life

Definition and Classification

• Types I, II and III are antibody-mediated and can therefore be transferred by serum; type IV is cell-mediated and can be transferred only by cells. This is the single most useful way to hold the classification
• A type V (stimulatory) is sometimes added for antibodies that stimulate rather than destroy — the TSH-receptor antibody of graves disease

Type I — Immediate Hypersensitivity

First exposure — antigen drives a Th2 response with IL-4 → B cells class-switch to produce IgE → IgE binds high-affinity FcepsilonRI receptors on mast cells and basophils — the person is now sensitised but has no symptoms → second exposure — antigen cross-links adjacent IgE molecules → degranulation within seconds → preformed mediators — histamine, tryptase, heparin, chemotactic factors → newly synthesised mediators — leukotrienes (the old SRS-A), prostaglandins, platelet-activating factor, cytokines → early phase (minutes) — vasodilatation, increased permeability, bronchospasm, itch; then a late phase at 4–12 hours with eosinophil infiltration and sustained inflammation

• Examples — anaphylaxis, atopic asthma, allergic rhinitis, urticaria and angio-oedema, food and drug allergy, insect sting reactions
• Tests — skin prick test, intradermal test, serum specific IgE (RAST), and total IgE
• Treatment — adrenaline intramuscularly in anaphylaxis; antihistamines, corticosteroids and mast cell stabilisers for lesser reactions; allergen avoidance; and desensitisation (immunotherapy) in selected patients

Type II — Cytotoxic Hypersensitivity

• Mechanism — IgG or IgM binds an antigen on a cell surface or basement membrane, and damage follows by (1) complement activation and lysis, (2) opsonisation and phagocytosis, (3) antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells
• Examples — ABO-incompatible transfusion reaction; haemolytic disease OF the newborn (Rh incompatibility); autoimmune haemolytic anaemia and immune thrombocytopenia; goodpasture syndrome (anti-glomerular basement membrane antibody affecting kidney and lung); rheumatic carditis (molecular mimicry with streptococcal M protein); drug-induced cytopenias; and hyperacute graft rejection
• Antibodies that block or stimulate rather than destroy — myasthenia gravis (blocking the acetylcholine receptor) and graves disease (stimulating the TSH receptor)
• Test — the COOMBS (antiglobulin) test: direct detects antibody already on the patient’s red cells; indirect detects free antibody in the serum, and is used in cross-matching and antenatal screening

Type III and Type IV

Diagnostic Tests in Hypersensitivity

Applied Aspects

• In tuberculosis and leprosy, much of the tissue damage is type IV hypersensitivity rather than the organism itself — which is why corticosteroids are given in tuberculous meningitis and pericarditis, and in type 1 lepra reactions to save nerve function
• The Mantoux test measures exposure, not disease, and in a country with universal BCG and high background infection its positive predictive value is limited; a negative test does not exclude tuberculosis, particularly in miliary disease, HIV or malnutrition, where anergy occurs
• Give anti-D immunoglobulin to Rh-negative mothers after delivery of an Rh-positive infant and after any sensitising event; it prevents haemolytic disease of the newborn by removing fetal cells before the mother can respond
• Ask about previous reactions before giving heterologous serum — antivenom, antitoxin — and have adrenaline drawn up; but never withhold antivenom from an envenomated patient for fear of a reaction, since the alternative is death
• Adrenaline is the only drug that reverses every feature of anaphylaxis, and antihistamines and steroids must never delay it
• Contact dermatitis is a common occupational problem — nickel, chromate in cement, rubber accelerators, and topical antibiotics; patch testing identifies the agent and avoidance is the treatment
• Read the Mantoux as induration, not erythema, at 48 to 72 hours; measuring redness is a common and consequential error
• Desensitisation (allergen immunotherapy) works by shifting the response from IgE toward IgG blocking antibody and regulatory T cells; it is effective for venom and pollen allergy but must be done where anaphylaxis can be treated
• Type II and type III are distinguished by where the antigen is — fixed on a cell surface in type II, soluble and forming circulating complexes in type III; the mediators overlap but the pattern of damage differs entirely
• Serum sickness is now seen chiefly with antivenom, antitoxin and some monoclonal antibodies, appearing 7 to 10 days after administration with fever, rash, arthralgia and proteinuria
• Atopy runs in families and tends to march — infantile eczema, then food allergy, allergic rhinitis and asthma; recognising the pattern allows earlier and better management
• Hypersensitivity pneumonitis is occupational and preventable — farmer’s lung, bagassosis, bird fancier’s lung; identifying and removing the exposure prevents irreversible fibrosis
• Type IV reactions to drugs may be severe — Stevens-Johnson syndrome and toxic epidermal necrolysis, associated with particular HLA types and with carbamazepine, allopurinol, sulphonamides and nevirapine
• Transfusion reactions span several types — acute haemolytic (type II, from ABO incompatibility and usually a clerical error), febrile non-haemolytic, allergic and anaphylactic (type I, notably in IgA deficiency)
• Check the identity of patient and unit at the bedside; ABO-incompatible transfusion is a preventable type II reaction and the fault is almost always administrative rather than immunological
• Latex allergy is an occupational hazard for healthcare workers, and cross-reacts with banana, avocado and kiwi; powder-free and synthetic gloves have greatly reduced it
• Anaphylaxis to antivenom must never prevent its use in an envenomated patient; adrenaline is drawn up first, the reaction treated, and the antivenom continued

Antigen Processing and Presentation

The Humoral Immune Response

Antigen is captured by a dendritic cell and carried to the lymph node → A naive B cell binds native antigen through its surface immunoglobulin and internalises it → The B cell presents processed peptide on MHC class II to a CD4 helper T cell → The T cell provides CD40L and cytokines — the "second signal" → The B cell proliferates, forming a germinal centre → class switching (IgM to IgG, IgA or IgE) and affinity maturation by somatic hypermutation → Differentiation into plasma cells (antibody factories) and memory B cells

• T-dependent antigens (proteins) require helper T cells, and therefore produce class switching, affinity maturation and memory
• T-independent antigens (polysaccharides, LPS) activate B cells directly but give IgM only, with NO memory and a poor response in infants under 2 — which is precisely why plain polysaccharide vaccines fail in young children and why conjugation to a protein carrier works
• Effector functions of antibody — neutralisation of toxins and viruses, opsonisation, complement activation, ADCC, agglutination and precipitation, and mucosal exclusion by secretory IgA

The Cell-mediated Immune Response

• Essential against — intracellular organisms (mycobacteria, Listeria, Salmonella, Brucella, Leishmania), viruses, fungi, tumour cells, and grafts; and it mediates type IV hypersensitivity
• CD4 Th1 cells release interferon-gamma, which is the principal macrophage-activating cytokine; activated macrophages then kill organisms they had merely contained
• CD8 cytotoxic T cells kill by releasing perforin (which forms pores) and granzymes (which trigger apoptosis), and by the Fas-Fas ligand pathway
• Granuloma formation is the response to antigen that cannot be eliminated — epithelioid cells, giant cells and lymphocytes wall it off; the characteristic lesion of tuberculosis, leprosy, sarcoidosis and deep mycoses
• Tests of cell-mediated immunity — delayed hypersensitivity skin tests (mantoux, lepromin), lymphocyte transformation, cytokine assays, and interferon-gamma release assays, which are unaffected by prior BCG

Immunological Tolerance and its Failure

• Tolerance is specific unresponsiveness to an antigen — and tolerance to self is what prevents autoimmunity
• Central tolerance — deletion of strongly self-reactive lymphocytes during maturation in the thymus and bone marrow
• Peripheral tolerance — anergy (recognition without the second signal), suppression by T regulatory cells, deletion, and sequestration of antigens in privileged sites (eye, testis, brain)
• Mechanisms of failure, causing autoimmunity — molecular mimicry (streptococcal M protein resembling cardiac myosin, giving rheumatic fever; Campylobacter and Guillain-Barré syndrome); release of sequestered antigen (sympathetic ophthalmia after eye injury); polyclonal activation by superantigens; failure of regulatory cells; and genetic predisposition, strongly linked to particular HLA types

Immune Evasion BY Pathogens

Applied Aspects

• Conjugate vaccines exist because polysaccharides are T-independent; linking the polysaccharide to a protein recruits T-cell help and produces memory, which is what makes Hib, pneumococcal and meningococcal vaccines effective in infants
• HIV destroys CD4 cells and therefore both arms of the response, since the helper cell coordinates antibody production as well as macrophage activation; this is why the opportunistic infections are so diverse
• Interferon-gamma release assays are preferred to the Mantoux test in a BCG-vaccinated population, since they use antigens absent from BCG; but neither distinguishes latent infection from active disease
• Corticosteroids and other immunosuppressants act chiefly on cell-mediated immunity, which is why they reactivate tuberculosis and predispose to fungal and viral infection rather than to simple pyogenic disease
• Rheumatic fever is a disease of molecular mimicry, and secondary penicillin prophylaxis works by preventing the streptococcal infections that provoke it — an important programme in India
• Anergy in an ill patient is itself informative — a negative Mantoux in miliary tuberculosis, or loss of skin test reactivity in HIV, measles or severe malnutrition, indicates depressed cell-mediated immunity rather than absence of infection
• Antigenic shift causes pandemics and drift causes epidemics — which is why the influenza vaccine must be reformulated annually and why a genuinely novel subtype finds a population with no immunity at all
• NK cells and cytotoxic T cells are complementary: a virus that down-regulates MHC to hide from T cells makes itself a target for NK cells, so evading one arm exposes it to the other
• Live vaccines are contraindicated in T-cell deficiency, since the attenuated organism requires cell-mediated immunity to be contained — BCG has caused disseminated disease in infants with undiagnosed severe combined immunodeficiency
• Immunological memory is why vaccination works and why a second attack of measles is rare; the duration of that memory determines whether boosters are needed, and it differs greatly between vaccines
• Sequestered antigen explains sympathetic ophthalmia, in which injury to one eye exposes previously hidden antigen and the other eye is attacked — a rare but devastating illustration of why privileged sites exist
• Regulatory T-cell failure causes severe early autoimmunity, as in the IPEX syndrome, demonstrating that active suppression, and not merely deletion, is required to maintain tolerance
• Oral tolerance is exploited therapeutically in allergen immunotherapy, where controlled exposure by a tolerogenic route shifts the response away from IgE
• Tolerance is antigen-specific, whereas immunosuppression is not — which is why inducing tolerance to a graft remains the goal of transplantation research: it would remove the infection and malignancy risk that lifelong immunosuppression brings
• The Th1/Th2 balance determines the outcome in leprosy — a strong Th1 response gives paucibacillary tuberculoid disease with few organisms and much tissue damage; a Th2-dominant response gives lepromatous disease with abundant bacilli and little cellular reaction
• Cytotoxic T cells require the cell to display antigen on MHC class I, so a tumour that loses MHC escapes them — and it is that loss which checkpoint inhibitors and NK-based therapies attempt to circumvent
• Cell-mediated immunity is what BCG and the interferon-gamma release assays measure, and its impairment by HIV, malnutrition or steroids is why tuberculosis reactivates in exactly those patients
• The second signal prevents autoimmunity — a T cell meeting antigen without co-stimulation becomes anergic rather than activated, which is how the system distinguishes a genuine threat from harmless self
• Mucosal immunity is a separate compartment with its own IgA response, which is why oral polio vaccine gives gut immunity that the injectable form does not, and why that difference mattered in eradication strategy
• Dendritic cells decide what kind of response follows, so the cytokines they release on first encountering an organism determine whether a Th1, Th2 or Th17 response develops — and therefore whether the infection is controlled
• Vaccination is the deliberate creation of memory without disease, which is the single most successful application of immunology and the only intervention that prevents infection without selecting for resistance

Morphology and Classification

• Gram-positive cocci arranged in irregular clusters resembling a bunch of grapes, because division occurs in several planes; non-motile, non-sporing, facultative anaerobes
• Catalase positive — the single test that separates staphylococci from streptococci, which are catalase negative
• Grow readily on ordinary media; produce a golden-yellow pigment on nutrient agar and beta-haemolysis on blood agar; tolerate high salt, which is exploited in mannitol salt agar

Virulence Factors

Toxin-mediated Disease

Diseases and Laboratory Diagnosis

• Skin and soft tissue — folliculitis, furuncle (boil), carbuncle, impetigo, cellulitis, wound and burn infection, breast abscess, styes
• Deep and systemic — osteomyelitis (the commonest cause), septic arthritis, pneumonia (often following influenza, and necrotising with Panton-Valentine leucocidin), empyema, acute endocarditis (right-sided tricuspid disease in intravenous drug users), bacteraemia and metastatic abscesses, and device-related infection
• Toxin-mediated — food poisoning, toxic shock syndrome, scalded skin syndrome
• Laboratory diagnosis — Gram stain of pus showing gram-positive cocci in clusters with pus cells; culture on blood agar and mannitol salt agar; catalase to separate from streptococci, then coagulase (slide test detects bound clumping factor and is a screening test; the tube test detects free coagulase and is confirmatory); DNase and mannitol fermentation; and antibiotic susceptibility including a cefoxitin disc for MRSA
• In food poisoning the organism or toxin is sought in the food rather than the patient, since the illness is an intoxication

Differentiation Within the Genus

• A coagulase-negative staphylococcus in one blood culture bottle is usually contamination; growth in several bottles from separate sites, or in a patient with a prosthesis or line, is far more likely to be real

Treatment and Applied Aspects

• Drainage is the treatment for an abscess; antibiotics penetrate pus poorly and source control outranks drug choice
• Antibiotic choice — cloxacillin or flucloxacillin for methicillin-sensitive strains (plain penicillin is useless, since over 90% produce penicillinase); vancomycin, linezolid, daptomycin, clindamycin or co-trimoxazole for MRSA
• Nasal carriage is the reservoir; decolonisation with mupirocin to the anterior nares and chlorhexidine washes reduces surgical site infection in carriers
• Staphylococcal bacteraemia is never trivial — it demands a search for endocarditis and metastatic foci, removal of any line, repeat blood cultures, and a prolonged course; treating it as a simple bacteraemia leads to relapse
• Food poisoning is prevented by food handling — excluding staff with skin lesions, refrigeration, and avoiding prolonged holding of cooked food at room temperature, which is a common practice at Indian social gatherings
• Hand hygiene is what limits spread in hospital; staphylococci survive well on skin, clothing and surfaces, and the hands of staff are the principal vehicle
• Recurrent boils suggest carriage or an underlying problem — check for diabetes, and consider decolonisation of the patient and close contacts, since the reservoir is usually the patient's own nose
• Suspect toxic shock in an unexplained febrile illness with rash and hypotension, and look for a retained tampon, nasal packing or an occult wound; the focus may be trivial while the illness is life-threatening
• Separate scalded skin syndrome from toxic epidermal necrolysis; the split is at the granular layer in the first and at the dermo-epidermal junction in the second, which biopsy resolves and which changes both treatment and prognosis
• Never dismiss S. Aureus in urine; unlike coliforms it rarely ascends, so its presence often indicates haematogenous seeding from an unsuspected bacteraemia
• Osteomyelitis and septic arthritis need prolonged treatment and often surgical washout; short courses guided by clinical improvement alone lead to chronic infection and sequestrum formation
• Echocardiography is indicated in staphylococcal bacteraemia, and a transoesophageal study where the transthoracic view is inadequate or suspicion remains high
• Food poisoning needs no antibiotic — the toxin is already formed and the organism may not even be present; treatment is rehydration and the investigation is of the food
• Exclude food handlers with boils or septic lesions; a staphylococcal skin lesion on the hands of someone preparing food is the classical route to an outbreak
• Coagulase-negative staphylococci are not harmless in a patient with a prosthetic valve, joint or shunt, where they are the leading pathogen and the device usually has to be removed

Classification of Streptococci

• Gram-positive cocci in chains or pairs, non-motile, non-sporing, catalase negative

Virulence Factors of Streptococcus Pyogenes

Diseases

• Suppurative (invasive) — pharyngitis and tonsillitis (the commonest); impetigo and pyoderma; erysipelas (a sharply demarcated superficial cellulitis) and cellulitis; necrotising fasciitis; puerperal sepsis; lymphangitis; otitis media and sinusitis; and bacteraemia
• Toxin-mediated — scarlet fever (a punctate erythematous rash sparing the perioral area, with a strawberry tongue and later desquamation) and streptococcal toxic shock syndrome
• Non-suppurative sequelae — the immunologically mediated complications, which are the reason streptococcal sore throat is treated at all

Laboratory Diagnosis

• Throat swab — Gram stain is of little value (the throat carries many organisms); culture on blood agar shows small beta-haemolytic colonies
• Identification — catalase negative; bacitracin sensitivity and PYR positive; Lancefield grouping by latex agglutination
• Rapid antigen detection tests are specific but less sensitive, so a negative result in a child is confirmed by culture
• Serology for the non-suppurative sequelae, where the organism has usually gone by the time the illness appears — ASO titre rises after throat infection, but poorly after skin infection because skin lipids inhibit streptolysin O; anti-DNase B is therefore the better test after pyoderma
• Streptozyme detects several antibodies together

Other Medically Important Streptococci

Treatment and Applied Aspects

• Penicillin remains the drug of choice — remarkably, S. Pyogenes has never developed penicillin resistance after eighty years of use, which is almost unique among bacteria. Erythromycin or a macrolide in penicillin allergy
• Secondary prophylaxis after rheumatic fever — benzathine penicillin every 3 to 4 weeks, continued for years or for life depending on carditis; this prevents recurrent attacks and progressive valve damage, and is the single most important streptococcal intervention in India
• Add clindamycin in necrotising fasciitis and toxic shock — it inhibits toxin production and is unaffected by the high organism density that renders penicillin less effective
• Necrotising fasciitis is a surgical emergency; pain out of proportion to the visible signs is the early clue, and survival depends on early and radical debridement rather than on antibiotics
• Group B streptococcus screening in pregnancy and intrapartum penicillin prevent neonatal sepsis and meningitis
• Viridans streptococci cause subacute endocarditis after dental procedures; prophylaxis is now restricted to the highest-risk cardiac lesions, and good dental hygiene matters more than antibiotic cover
• Use a clinical score to decide who needs a throat swab; most sore throats are viral, and treating all of them wastes antibiotics while treating none permits rheumatic fever
• Ask specifically about a sore throat weeks earlier in a child with arthritis, chorea or heart failure; the infection is often forgotten by the time the sequel appears
• Isolation of S. Bovis from blood mandates colonoscopy, because of its strong association with colonic carcinoma — a microbiological result that directly triggers a surgical investigation
• Erysipelas and cellulitis differ in their margins; erysipelas is sharply demarcated and raised, involving the upper dermis and lymphatics, whereas cellulitis has an indistinct edge
• Penicillin sensitivity in S. Pyogenes has never been lost, so routine sensitivity testing is unnecessary for penicillin, though macrolide resistance does occur and matters in allergic patients
• Rheumatic fever is diagnosed by the JONES criteria with evidence of preceding streptococcal infection; the microbiology supports a clinical diagnosis rather than making one
• Anti-DNase B is the better serological test after skin infection, since skin lipids inhibit streptolysin O and the ASO titre may not rise at all

Morphology and Cultural Characteristics

• Gram-positive, non-motile, non-sporing, non-capsulated bacilli that are club-shaped (swollen at one end) and markedly pleomorphic
• Arranged at angles in "chinese letter" or cuneiform patterns, because the cells snap after division rather than separating cleanly
• Metachromatic granules (volutin, Babes-Ernst granules) of polymetaphosphate are demonstrated by ALBERT stain, appearing bluish-black against a green bacillus, and by Neisser and Ponder stains

Diphtheria Toxin

The tox gene is carried by a bacteriophage, not by the bacterium — lysogenic conversion by beta-phage → Toxin production requires a low iron concentration in the medium and in tissue → The toxin has two fragments — B binds the cell surface receptor and A is the active enzyme → Fragment A catalyses ADP-ribosylation of elongation factor 2 (EF-2) → protein synthesis stops in the affected cell → A single molecule can inactivate all the EF-2 in a cell, so the toxin is extraordinarily potent → Local necrosis produces the pseudomembrane; absorbed toxin damages heart, nerves and kidney

Clinical Features

• Faucial diphtheria is commonest — a tough, adherent, greyish-white pseudomembrane over the tonsils and pharynx, which bleeds when removed (unlike a candidal or streptococcal exudate). It consists of fibrin, necrotic epithelium, leucocytes and organisms
• "bull neck" from marked cervical lymphadenopathy and oedema, with severe toxaemia disproportionate to the modest fever
• Laryngeal diphtheria — hoarseness, stridor and respiratory obstruction from membrane extension, which may require tracheostomy; a particular danger in young children
• Other sites — nasal (often mild and a source of carriage), cutaneous (common in tropical countries and an important reservoir), conjunctival, genital
• Complications from absorbed toxin — myocarditis in the second week (arrhythmia, heart block, cardiac failure — the commonest cause of death); palatal palsy with nasal regurgitation and a nasal voice around the third week; and later polyneuritis including ocular and diaphragmatic paralysis; acute tubular necrosis

Laboratory Diagnosis and Tests for Toxigenicity

• Swabs from the edge of and beneath the membrane, and from the nose
• Direct smear — Albert stain for granules and Gram stain; suggestive but not confirmatory, since diphtheroids are normal throat flora
• Culture — Loeffler slope (rapid), tellurite (selective) and blood agar, examined at 6 to 8 hours and again at 24 to 48 hours
• ELEK test — an in-vitro immunoprecipitation test: a filter paper strip soaked in antitoxin is laid on the agar and the strain streaked at right angles; arrowhead lines of precipitation where toxin meets antitoxin indicate toxigenicity
• PCR for the tox gene — rapid, though it detects the gene rather than its expression
• In-vivo tests (guinea-pig inoculation, intradermal test) are of historical interest only
• SCHICK test — a historical intradermal test of susceptibility, measuring circulating antitoxin; now replaced by serology and no longer performed

Other Corynebacteria and Differential Diagnosis

Treatment, Prevention and Applied Aspects

• Antitoxin is given immediately on clinical suspicion, without waiting for laboratory confirmation, because it neutralises only circulating toxin — once the toxin has bound to tissue the damage cannot be reversed. Delay of even a day increases mortality substantially. A test dose is given, since it is horse serum
• Antibiotics (penicillin or erythromycin) eliminate the organism and stop further toxin production and transmission, but they do not substitute for antitoxin
• Supportive care — strict bed rest for weeks because of myocarditis, cardiac monitoring, airway management with tracheostomy if needed, and isolation until two negative cultures
• Prevention by toxoid — formalin-treated toxin that is immunogenic but non-toxic, given as DPT or pentavalent vaccine in India's Universal Immunisation Programme, with boosters. Immunity wanes, so adult boosters matter — the resurgence of diphtheria in the former Soviet Union followed a fall in adult immunity
• Contacts — culture, antibiotic prophylaxis, and a booster dose
• Diphtheria persists in parts of India in older children and adults with waning immunity and in under-vaccinated pockets, so it must remain in the differential of membranous pharyngitis; the diagnosis is clinical and treatment cannot wait
• Do not attempt to remove the membrane; it bleeds and increases toxin absorption, and may obstruct the airway
• Nurse the patient with strict bed rest for 2 to 3 weeks and monitor the electrocardiogram; myocarditis appears in the second week, when the throat is improving and the patient feels well enough to move about
• Cutaneous diphtheria is an important and neglected reservoir in tropical countries, maintaining transmission silently in communities where faucial disease is rare
• Immunisation does not prevent carriage, only disease — so a vaccinated population can still transmit, which is why outbreak control requires contact tracing and prophylaxis rather than vaccination alone
• Send swabs before starting antibiotics, but never delay antitoxin for the result; the laboratory confirms the diagnosis, it does not make it
• Antitoxin is horse serum, so a test dose is given and adrenaline kept ready; serum sickness may follow a week later, and neither risk justifies withholding it in suspected diphtheria
• Notify the case and investigate the contacts; diphtheria is a notifiable disease and a single case indicates a gap in immunisation coverage in that community
• Check and complete immunisation in every child presenting with any illness; opportunistic vaccination catches those missed by the routine schedule, and diphtheria is a disease of the unimmunised
• Isolate the patient until two consecutive negative cultures taken at least 24 hours apart and after antibiotics have finished
• Give a booster to close contacts as well as prophylaxis, since their immunity is likely to have waned in the same way as the index case's
• Toxoid is one of the oldest and most effective vaccines in use, and the near-disappearance of diphtheria from well-vaccinated populations is among the clearest demonstrations of what immunisation achieves
• Low iron promotes toxin production, which is a curious detail with a practical corollary: the organism is most dangerous in exactly the iron-poor conditions found at a mucosal surface
• Diphtheria antitoxin supply is limited worldwide, and delays in obtaining it contribute to mortality; hospitals in endemic areas should know where their nearest supply is held before they need it
• The Elek test remains the standard toxigenicity test in many laboratories, and its result determines whether contacts need prophylaxis and whether the case is notifiable as diphtheria
• Consider diphtheria in an unimmunised adult with a sore throat and disproportionate toxaemia, particularly with cervical swelling; the low fever is deceptive and delays the diagnosis
• Laryngeal obstruction may develop rapidly in a small child, so the airway must be assessed repeatedly and tracheostomy facilities kept available

Clostridium Tetani — Morphology and Toxin

• Gram-positive, motile, obligate anaerobic bacillus with a terminal, spherical, bulging spore giving the classical drumstick appearance
• Swarms over the surface of blood agar; grows in Robertson cooked meat medium; spores are widespread in soil, dust and animal and human faeces, and survive for years
• Non-invasive — it remains at the site of inoculation, and the entire illness is caused by the toxin

Spores enter a wound and germinate where the oxidation-reduction potential is low — devitalised tissue, foreign body, deep puncture, or co-infection with aerobes → The vegetative organism releases tetanospasmin → The toxin travels by retrograde axonal transport up peripheral motor nerves, and by blood to distant nerve endings → It reaches the spinal cord and brainstem → It cleaves synaptobrevin, blocking release of the inhibitory transmitters glycine and GABA from Renshaw cells → Loss of inhibition — not stimulation → Unopposed motor neuron activity gives sustained rigidity and spasms, with sympathetic overactivity

Clinical Features and Management of Tetanus

• Incubation 4 to 21 days; the shorter the incubation and the shorter the period of onset (the interval between the first symptom and the first spasm), the worse the prognosis
• Features — trismus (lockjaw), often the first sign; risus sardonicus from facial muscle spasm; neck stiffness; dysphagia; opisthotonus; board-like abdominal rigidity; and generalised reflex spasms triggered by light, sound or touch. The patient remains fully conscious throughout
• Autonomic instability — labile blood pressure, tachycardia, arrhythmia and sweating, which is now a leading cause of death in ventilated patients
• Death from respiratory failure, laryngeal spasm, aspiration or autonomic dysfunction
• Forms — generalised, local, cephalic (after head wounds, with cranial nerve palsies), and neonatal tetanus from unclean cord practices
• Management — nurse in a quiet, darkened room; human tetanus immunoglobulin to neutralise unbound toxin; metronidazole (preferred to penicillin, which is a GABA antagonist); surgical debridement of the wound; diazepam or magnesium for spasms; neuromuscular blockade and mechanical ventilation in severe cases; and active immunisation during convalescence, since the disease itself does not confer immunity — the lethal dose of toxin is smaller than the immunising dose

Gas Gangrene — Clostridium Perfringens

• Gram-positive, non-motile, capsulated bacillus with a subterminal spore that is rarely seen in clinical material; an anaerobe found in soil and in the large intestine
• Alpha toxin is a lecithinase (phospholipase C) that splits lecithin in cell membranes, causing haemolysis, tissue necrosis and myocardial toxicity — the principal virulence factor
• NAGLER reaction — on egg yolk agar, lecithinase produces a zone of opalescence which is inhibited on the half of the plate spread with antitoxin; a rapid and specific identification test
• Stormy clot (stormy fermentation) reaction in litmus milk — acid clot torn apart by vigorous gas production
• Grows rapidly with a "target" or double zone of haemolysis on blood agar
• Clinical features — a rapidly progressive myonecrosis with severe pain, tense oedema, crepitus from gas in tissues, a thin foul brownish discharge, discoloured skin and profound toxaemia
• Also causes food poisoning — type A enterotoxin, an incubation of 8 to 24 hours, watery diarrhoea and cramps without much vomiting, from meat dishes cooked and held warm; and necrotising enteritis (pigbel) from type C
• Treatment — urgent and radical surgical debridement is the definitive treatment, sometimes requiring amputation; penicillin with clindamycin (clindamycin suppresses toxin production); and hyperbaric oxygen where available

Prevention of Tetanus and Gas Gangrene

Comparison of the Pathogenic Clostridia

• All are gram-positive, spore-forming, obligate anaerobes whose spores persist in soil and dust for years, and all cause disease chiefly through exotoxins rather than by invasion

Applied Aspects

• Tetanus and gas gangrene are clinical diagnoses; treatment must never await laboratory confirmation, which is often negative and always late
• Assess every wound for tetanus risk and immunisation status, and give toxoid, immunoglobulin or both accordingly; this is among the commonest omissions in a busy casualty department
• Do not rely on antibiotics in gas gangrene — they cannot reach ischaemic necrotic muscle, and delay in taking the patient to theatre costs limbs and lives
• Immunise during convalescence from tetanus, since the disease confers no immunity — a point frequently and dangerously assumed the other way
• Neonatal tetanus was a major cause of newborn death in India and has fallen dramatically with maternal immunisation and clean cord care; the residual cases follow home deliveries with traditional cord applications
• Avoid applying dung, ash or oil to the umbilical stump or to wounds, a practice that persists in some communities and directly inoculates spores
• Penicillin is a GABA antagonist and may aggravate spasms, which is why metronidazole is now preferred in tetanus — a small pharmacological point with direct bedside consequence
• Crepitus is a late sign in gas gangrene; severe pain with tense oedema and toxaemia out of proportion to the wound should prompt surgical exploration before gas is palpable
• C. Perfringens food poisoning is a disease of bulk catering — meat cooked in large quantities, cooled slowly and held warm, which describes many institutional and wedding kitchens
• Anaerobic culture is often negative in clostridial disease, and a negative report must never delay surgery or antitoxin; the organisms are fastidious and the diagnosis is clinical
• The period of onset predicts severity in tetanus — the shorter the interval between the first symptom and the first spasm, the worse the outlook, and this guides early transfer to intensive care
• Autonomic instability now kills more ventilated tetanus patients than the spasms do, and magnesium sulphate together with sedation has improved outcomes
• Gas in the tissues is not always gas gangrene; it also occurs with other organisms and after trauma or surgery, so the toxaemia and the appearance of the muscle at operation decide the diagnosis
• Clindamycin is added to penicillin in clostridial myonecrosis because it suppresses toxin synthesis and works even at high organism density, when penicillin's effect falls away
• Ask about the injury and its management; a wound closed primarily over contaminated or devitalised tissue creates exactly the anaerobic conditions these organisms require
• Booster immunity to tetanus wanes over a decade, so adults presenting with wounds frequently need a dose even if they were fully immunised as children
• Neonatal tetanus elimination in India rests on two measures — maternal toxoid and clean cord care — neither of which requires a hospital, which is why it succeeded
• Give tetanus immunoglobulin at a different site from the toxoid and with a separate syringe, or the antibody neutralises the vaccine antigen
• Cephalic tetanus follows head and facial wounds and presents with cranial nerve palsies rather than the classical picture, so it is easily missed until trismus appears

Bacillus Anthracis — Morphology

• Large, gram-positive, aerobic, spore-bearing bacillus with square-cut (truncated) ends, arranged in long chains giving a "bamboo stick" appearance
• Non-motile — which distinguishes it from almost all other Bacillus species, which are motile
• The only bacterium with a polypeptide capsule, made of poly-D-glutamic acid; being D-amino acid it resists proteolysis and is poorly immunogenic. The capsule is formed IN tissue (and in culture with bicarbonate and carbon dioxide), not on ordinary media
• Spores are central, oval and non-bulging, formed only in the presence of oxygen — so they are not formed in the living host, but appear once the carcass is opened. This is why anthrax carcasses must not be opened or skinned, and are burned or buried deeply

Anthrax Toxin and Pathogenesis

• Three components, each harmless alone — protective antigen (PA), which binds the cell and forms the channel; oedema factor (EF), a calmodulin-dependent adenylate cyclase that raises cAMP and causes massive oedema; and lethal factor (LF), a protease that cleaves map kinase kinases, killing macrophages and releasing cytokines
• PA + EF = oedema toxin; PA + LF = lethal toxin. Protective antigen is essential to both, which is why it is the component used in the vaccine — antibody to it blocks delivery of the other two
• Both toxin and capsule are plasmid-encoded (pXO1 and pXO2); loss of either plasmid attenuates the organism, which is the basis of the Sterne vaccine strain

Clinical Forms of Anthrax

• Treatment — ciprofloxacin or doxycycline (penicillin is effective against sensitive strains but resistance has been engineered), with combination therapy and antitoxin (raxibacumab) in systemic disease; the cutaneous lesion is not excised, since surgery may disseminate it
• Anthrax is a recognised bioterrorism agent, because spores are stable, easily disseminated and cause high mortality by inhalation; this is why inhalational anthrax in an unexplained setting is treated as a public health emergency

Other Aerobic Spore-bearers

Laboratory Diagnosis and Prevention of Anthrax

• Specimens — vesicle fluid or material from the eschar edge, blood, sputum, cerebrospinal fluid, and tissue or animal material
• Handle at BSL-3 where inhalational anthrax is suspected, and warn the laboratory, since anthrax is a recognised cause of laboratory-acquired infection
• Direct examination — Gram stain showing large square-ended bacilli in chains, and the McFADYEAN capsule reaction
• Culture on blood and nutrient agar, with Medusa head colonies, non-motility, non-haemolysis, gamma-phage susceptibility and the string of pearls test
• Serology and PCR for toxin and capsule genes; the ASCOLI test where material is decomposed
• Prevention — animal vaccination (the Sterne live attenuated non-capsulated strain) is the key control measure; safe carcass disposal by burning or deep burial with lime; protective equipment for those handling hides and wool; and human vaccination with protective antigen for high-risk occupational groups and military personnel. Post-exposure prophylaxis with ciprofloxacin for 60 days, because inhaled spores may germinate late

Applied Aspects

• Do not open or skin a carcass suspected of anthrax; spores form only on exposure to oxygen, and opening the animal contaminates the soil for decades. Carcasses are burned or buried deeply with lime
• Anthrax is a notifiable and occupational disease in India, occurring in agricultural and animal-handling communities in several states; control depends on animal vaccination and safe carcass disposal rather than on human treatment
• Suspect anthrax in a painless black eschar with disproportionate oedema in someone with animal contact — the painlessness distinguishes it from staphylococcal and streptococcal lesions
• Reheated rice is the classic vehicle for Bacillus cereus emetic disease; the spores survive boiling, germinate as the rice cools slowly, and the toxin is not destroyed by reheating — so cooked rice must be cooled rapidly and refrigerated
• Bacillus species in a blood culture are usually contaminants, but should not be dismissed automatically in a neutropenic patient or one with an intravascular device
• Spore-formers define sterilisation standards — the biological indicators for every sterilising process are Bacillus spores, because they are the most resistant form of life to that process
• Distinguish B. Anthracis from the anthracoid bacilli carefully; the latter are common contaminants, and the practical differences are motility, haemolysis, capsule and gamma-phage susceptibility
• Cutaneous anthrax is not excised, since surgery risks dissemination; it is treated with antibiotics and the eschar allowed to separate
• Post-exposure prophylaxis after inhalational exposure runs for 60 days, because spores may lie dormant in the mediastinal nodes and germinate long after exposure — a far longer course than intuition suggests
• Occupational health matters more than clinical medicine here; vaccinating livestock, disposing of carcasses safely and protecting hide and wool workers prevents almost all human anthrax
• Warn the laboratory when anthrax is suspected; several documented laboratory-acquired infections have followed routine handling of an unlabelled specimen on an open bench
• Inhalational anthrax presents as a widened mediastinum without pneumonia, which is an unusual radiological combination and should raise the possibility immediately
• An unexplained case of inhalational anthrax is a public health emergency and must be notified at once, since the natural form is now vanishingly rare
• Cook rice in the quantity needed and refrigerate promptly; the Bacillus cereus emetic toxin is not destroyed by reheating, so the prevention lies entirely in rapid cooling rather than in the final heating
• Anthrax spores survive in soil for decades, so land where an infected animal died remains hazardous long after any living memory of the event
• Bacillus cereus also causes severe eye infection after penetrating trauma, progressing to loss of the eye within a day, so a contaminated ocular injury must be treated urgently rather than observed

General Characters of the Enterobacteriaceae

• Gram-negative, non-sporing bacilli, motile by peritrichous flagella or non-motile; facultative anaerobes growing readily on ordinary media
• All ferment glucose, all reduce nitrate to nitrite, all are oxidase negative and catalase positive
• The oxidase test is the crucial first discrimination — a gram-negative bacillus that is oxidase positive is not an enterobacterium, and is more likely Pseudomonas, Vibrio, Aeromonas or Campylobacter

Escherichia Coli — General Features

• The predominant facultative anaerobe of the normal large bowel, and therefore an indicator OF faecal contamination of water and food
• IMViC reactions ++–– — indole positive, methyl red positive, Voges-Proskauer negative, citrate negative; this distinguishes it from Klebsiella, which is ––++
• Ferments lactose with acid and gas; on EMB agar produces a characteristic green metallic sheen
• Extra-intestinal disease — the commonest cause of urinary tract infection (uropathogenic strains adhering by P fimbriae); neonatal meningitis (K1 capsule); intra-abdominal sepsis and peritonitis; wound infection; and gram-negative bacteraemia

Diarrhoeagenic Escherichia Coli

• Antibiotics are contraindicated IN EHEC — they increase Shiga toxin release by inducing the prophage, and raise the risk of haemolytic uraemic syndrome. Antimotility drugs are likewise avoided

Laboratory Diagnosis and Treatment

• Urine — clean-catch midstream sample; significant bacteriuria is conventionally 105 colony-forming units per mL, though lower counts matter in a symptomatic young woman or a suprapubic specimen
• Stool — culture on MacConkey and selective media; serotyping and toxin or gene detection are needed to identify a diarrhoeagenic type, since ordinary culture cannot distinguish them from commensal E. Coli
• Blood, pus and cerebrospinal fluid as appropriate; identification by IMViC, urease, and automated systems or MALDI-TOF
• Treatment — guided by sensitivity, since resistance is now extensive; nitrofurantoin or fosfomycin for uncomplicated cystitis; aminoglycosides, carbapenems or beta-lactam with inhibitor combinations for serious infection. ESBL and carbapenemase production are widespread in India
• Most diarrhoea needs only oral rehydration, with zinc in children; antibiotics are reserved for dysentery, severe illness and specific organisms

Biochemical Identification of the Enterobacteriaceae

Applied Aspects

• E. Coli in water indicates faecal contamination, and coliform counts are the standard of water safety; this remains a central public health measurement in India
• Do not give antibiotics for bloody diarrhoea until EHEC is excluded in a child with an appropriate history, since they worsen the outcome
• Uncomplicated cystitis in a young woman needs no culture in most settings; culture is reserved for treatment failure, pregnancy, men, children and recurrent or complicated infection
• Asymptomatic bacteriuria is treated only in pregnancy and before urological instrumentation; treating it otherwise selects resistance without benefit
• Hand hygiene, safe water and sanitation prevent far more E. Coli disease than any antibiotic, and the burden of childhood diarrhoea in India reflects this directly
• Take a travel and food history in acute diarrhoea; the pattern of illness, incubation and the presence of blood narrow the likely organism before any laboratory result
• Watery diarrhoea points to a toxin acting on the small bowel (ETEC, cholera), whereas blood and pus point to invasion of the colon (Shigella, EIEC, Campylobacter) — a distinction that guides both investigation and whether antibiotics are needed at all
• Suspect haemolytic uraemic syndrome in a child with bloody diarrhoea who becomes pale or oliguric, and check the platelet count, film and creatinine rather than simply treating the diarrhoea
• Serotyping is needed to identify diarrhoeagenic E. Coli, so routine stool culture reporting "E. Coli grown" from a diarrhoeal specimen means nothing — it is normal flora
• Neonatal meningitis caused by E. Coli carries the K1 capsule, and the organism should be sought in blood and cerebrospinal fluid in any septic neonate
• Zinc for 14 days with oral rehydration is standard in childhood diarrhoea in India, reducing both the current episode and further episodes over the following months
• Give ORS by spoon or cup, not by bottle, and continue after every loose stool; the commonest reason oral rehydration "fails" is that too little is given, too slowly
• Do not use antibiotics for watery diarrhoea in the absence of cholera, severe illness or a specific indication; the great majority are self-limiting and antibiotics select resistance and prolong carriage
• Recurrent urinary infection in a child warrants imaging for reflux or structural abnormality, since repeated pyelonephritis causes permanent scarring

The Organism

• Gram-negative bacilli, motile by peritrichous flagella, non-capsulated (except the Vi antigen), non-lactose fermenting
• Nomenclature — almost all human isolates belong to Salmonella enterica subspecies enterica, named by serotype: S. Typhi, S. Paratyphi A, B and C, S. Typhimurium, S. Enteritidis
• Antigenic structure and the kauffmann-white scheme — classifies over 2,500 serotypes by their O (somatic) and H (flagellar) antigens; S. Typhi additionally carries the Vi (capsular) antigen, which is antiphagocytic, masks the O antigen, and is the basis of both a vaccine and carrier detection
• Biochemical features — S. Typhi produces acid but NO gas from glucose (other salmonellae produce both), and produces only a trace of H2S, whereas other salmonellae produce it abundantly

Pathogenesis of Enteric Fever

Ingestion of a large inoculum in contaminated water or food; gastric acid is a barrier, so achlorhydria and antacids lower the infecting dose → The organism invades the ileal mucosa and PEYER patches → It multiplies within macrophages — an intracellular pathogen, which is why cell-mediated immunity matters and why intracellular antibiotics are needed → Spread to mesenteric lymph nodes and by the thoracic duct to blood — primary bacteraemia, which is asymptomatic → Seeding of liver, spleen, bone marrow and gallbladder, where further multiplication occurs during the incubation period of 7 to 14 days → secondary bacteraemia — and it is now that the illness begins → Organisms excreted in bile re-infect the Peyer patches, which undergo hyperplasia, necrosis and ulceration along the long axis of the ileum → Hence perforation and haemorrhage in the third week

Clinical Features and Complications

• Week 1 — step-ladder fever rising over days, headache, malaise, dry cough, constipation more often than diarrhoea in adults; relative bradycardia (Faget sign)
• Week 2 — sustained high fever, toxaemia, rose spots (sparse blanching pink macules on the trunk, difficult to see on pigmented skin), splenomegaly, abdominal distension, and apathy; leucopenia with an absent eosinophil count is characteristic
• Week 3 — the dangerous week: intestinal perforation and haemorrhage from the ulcerated terminal ileum; also myocarditis, encephalopathy ("typhoid state"), and shock
• Other complications — cholecystitis, osteomyelitis, arthritis, meningitis, nephritis, abortion; and relapse in up to 10%, usually milder
• Carriers — convalescent (up to 3 months) and chronic (over 1 year), in whom the organism persists in the gallbladder, particularly with gallstones; commoner in older women. "Typhoid Mary" is the historical example, and food handlers are the public health concern

Laboratory Diagnosis

• Interpreting the Widal test — O antibody is IgM and indicates recent or active infection; H antibody is IgG, persists for years and rises after vaccination, so a raised H titre alone means little. False positives occur in other infections and in autoimmune disease; false negatives occur early, after antibiotics, and in the immunosuppressed

Differential Diagnosis of Prolonged Fever in India

• Take blood cultures before antibiotics in any prolonged fever; a single prior dose is the commonest reason enteric fever is never confirmed, and it then becomes an untreatable diagnostic problem for weeks

Treatment, Prevention and Applied Aspects

• Ceftriaxone or azithromycin are now first-line in India; fluoroquinolone resistance is widespread, and nalidixic acid resistance predicts it. Chloramphenicol, ampicillin and co-trimoxazole retain activity against some strains, and multidrug-resistant and XDR strains are an increasing problem
• Fever settles slowly, over 3 to 5 days, and persistent fever is not by itself evidence of treatment failure — changing antibiotics prematurely is a common error
• Dexamethasone reduces mortality in severe typhoid with shock or altered consciousness
• Chronic carriers — prolonged ciprofloxacin or amoxicillin, and cholecystectomy where there are gallstones and treatment fails
• Prevention — safe water, sanitation, food hygiene, exclusion and treatment of carriers among food handlers; and vaccination with Vi polysaccharide, oral live Ty21a, or the newer Vi conjugate vaccine (TCV), which unlike plain polysaccharide works in infants and gives longer protection
• Enteric fever remains common in India and must be considered in any prolonged fever; blood cultures should be taken before antibiotics, which in practice is the commonest reason the diagnosis is never confirmed
• Watch for perforation in the third week — sudden abdominal pain with a falling temperature and rising pulse in a patient who was improving; it is a surgical emergency and easily attributed to the illness itself
• Do not treat non-typhoidal Salmonella gastroenteritis with antibiotics in an otherwise healthy patient; they prolong carriage without shortening the illness
• Screen and treat carriers among food handlers; a single chronic carrier can sustain an outbreak for years, and gallstones make eradication difficult
• The typhoid conjugate vaccine works in infants, unlike the plain Vi polysaccharide, and its introduction is a significant addition to control in endemic areas
• Bone marrow culture remains positive after antibiotics and is the most sensitive test; it is worth doing where the diagnosis matters and blood cultures were taken too late
• Interpret a single Widal titre against local baseline values, which differ between regions; many laboratories in India report against a fixed cut-off that has never been validated locally
• Relapse occurs in up to 10%, typically 1 to 3 weeks after treatment ends and usually milder; it is not necessarily evidence of resistance
• Vaccination does not remove the need for safe water; typhoid is a disease of sanitation, and the durable solution is environmental rather than immunological
• Extensively drug-resistant typhoid has emerged in the subcontinent, resistant to fluoroquinolones and third-generation cephalosporins, leaving azithromycin and carbapenems — a serious narrowing of options for a common disease
• Do not stop treatment when the fever settles; the full course is required to prevent relapse and to clear the organism from the gallbladder

The Organism

• Vibrio cholerae — a gram-negative, comma-shaped (curved) bacillus with a single polar flagellum giving rapid darting or "shooting star" motility, seen in a hanging drop and abolished by specific antiserum
• Oxidase positive — which immediately separates it from the Enterobacteriaceae
• Alkaliphilic — it tolerates pH up to 9.2 but is killed rapidly by acid, which is exploited in both enrichment and epidemiology
• Classification — over 200 O serogroups, but only O1 and O139 cause epidemic cholera. O1 has two biotypes, classical and EL TOR (the current pandemic strain, which is hardier, produces more asymptomatic carriers and survives longer in the environment), and three serotypes: OGAWA, INABA and Hikojima

Cholera Toxin

The toxin has one A subunit and five B subunits ("A-5B") → The B subunits bind the GM1 ganglioside receptor on the enterocyte → The A subunit enters the cell → It catalyses ADP-ribosylation of the Gs alpha subunit of the regulatory G protein → This locks Gs in its active state — the GTPase that would switch it off is disabled → adenylate cyclase is permanently ON and intracellular cAMP rises enormously → Active secretion of chloride from crypt cells and blocked absorption of sodium → Water follows osmotically — massive isotonic fluid loss into the lumen

Clinical Features

• Incubation a few hours to 5 days; most infections are asymptomatic or mild, particularly with El Tor, and these people spread it
• Abrupt painless profuse watery diarrhoea with effortless vomiting; the stool becomes "rice-water" — colourless with flecks of mucus, with a characteristic inoffensive fishy odour
• NO fever, NO abdominal pain, NO blood or pus in the stool; the patient remains afebrile and mentally clear until shock supervenes
• Fluid loss may reach 1 litre per hour, giving severe dehydration within hours — sunken eyes, "washerwoman's hands", loss of skin turgor, feeble pulse, hypotension
• Consequences — hypovolaemic shock, metabolic acidosis from bicarbonate loss, hypokalaemia (paralytic ileus, arrhythmia), acute kidney injury, and in children hypoglycaemia
• Death may occur within hours, and untreated mortality reaches 50%; with adequate rehydration it falls below 1%

Treatment and Prevention

• Rehydration IS the treatment — everything else is secondary. Ringer lactate intravenously for severe dehydration, replacing the estimated deficit within 3 to 4 hours and then matching ongoing losses; oral rehydration solution for mild and moderate cases and to continue after resuscitation
• ORS composition depends on glucose-coupled sodium transport; the reduced-osmolarity formulation is now standard and lessens vomiting and stool output
• Monitor and replace potassium, which is lost heavily and is often overlooked while attention is on volume
• Antibiotics are adjunctive — a single dose of doxycycline, or azithromycin in children and pregnancy; they shorten the illness, reduce stool volume by about half and shorten excretion, but they do not save the life — fluid does
• Zinc in children reduces duration and severity
• Prevention — safe water and sanitation above all; food hygiene; chlorination; case isolation and disinfection of excreta; and oral killed vaccines (Shanchol, Dukoral), used for outbreak control and in endemic areas, though they do not replace clean water

Assessment of Dehydration and Fluid Replacement

Applied Aspects

• Assess dehydration and start fluid immediately; no investigation should delay resuscitation, and the diagnosis in an outbreak is clinical
• A single case of cholera constitutes an outbreak and must be notified; it indicates faecal contamination of a water supply serving many people
• Cholera remains endemic in parts of India with seasonal outbreaks during and after the monsoon, and in flooded and displaced populations where sanitation has failed
• Oral rehydration therapy was developed largely in the Indian subcontinent and is among the most important therapeutic advances of the twentieth century, having saved tens of millions of lives at negligible cost
• Send stool in Cary-Blair and tell the laboratory cholera is suspected, so that alkaline peptone water and TCBS are set up; ordinary stool culture will miss it
• Treat the water supply, not just the patient; chlorination, safe storage and sanitation end an outbreak, whereas treating cases one by one does not
• Weigh the patient on admission and again after rehydration; the change in weight is the most reliable measure of the deficit replaced, particularly in children
• Do not use plain glucose saline or dextrose alone; the losses are isotonic with bicarbonate and potassium, and Ringer lactate replaces them far better
• Continue feeding and breastfeeding throughout; withholding food prolongs recovery and worsens the nutritional consequences, particularly in children
• Set up a cholera treatment centre with cholera cots in an outbreak, allowing continuous measurement of stool losses and matched replacement — simple organisation that reduces mortality more than any drug
• Give a single dose of doxycycline to household contacts in an outbreak, alongside the measures directed at water and sanitation
• El Tor produces many mild and asymptomatic infections, so the cases presenting to hospital represent a small fraction of those infected and spreading it
• Distinguish cholera from other watery diarrhoeas by its sheer volume and by the absence of fever and abdominal pain; the clinical picture is characteristic once seen
• Vibrio dies rapidly in ordinary transport, so use Cary-Blair or alkaline peptone water and inform the laboratory, or the culture will be falsely negative
• Hypoglycaemia is a cause of death in children with cholera and must be anticipated and treated, since it is easily missed behind the dehydration
• Oral cholera vaccine is a supplement, not a substitute, and is most useful in outbreak control and in displaced populations where sanitation cannot be provided quickly
• Cholera follows the breakdown of infrastructure — floods, displacement, conflict — which is why its appearance is as much a signal about water supply as about microbiology
• Chlorinate the water and disinfect excreta around a case; the organism is killed readily by chlorine, drying and heat, so control measures are simple where they can be applied
• O139 emerged as a genuinely new epidemic strain in 1992 from the Bay of Bengal, infecting people already immune to O1 — a reminder that immunity to cholera is serogroup-specific

General Features of the Neisseriae

• Gram-negative diplococci with adjacent flattened sides, giving a kidney or coffee-bean shape
• Oxidase positive and catalase positive — the oxidase test is the rapid presumptive identification
• Fastidious and delicate — they require enriched media (chocolate agar), a capnophilic atmosphere of 5 to 10% carbon dioxide, and are killed rapidly by drying, cold and sunlight. Specimens must therefore be plated immediately or sent in transport medium; they must never be refrigerated
• THAYER-MARTIN (or modified New York City) medium is chocolate agar made selective with vancomycin, colistin and nystatin, for isolation from sites with normal flora
• Sugar utilisation — meningococcus ferments glucose and maltose; gonococcus ferments glucose only

Neisseria Meningitidis

• Capsulated; serogroups A, B, C, W, X and Y cause nearly all disease. Group A caused the African "meningitis belt" epidemics; B and C predominate in Europe; A, B and C occur in India
• Man is the only reservoir; carriage in the nasopharynx is common (5 to 10%, higher in crowded conditions) and is usually harmless — disease is the exception, not the rule
• Transmission by respiratory droplets, favoured by crowding — hostels, barracks, pilgrimages and mass gatherings
• Clinical forms — meningitis (fever, headache, neck stiffness, photophobia, altered consciousness); meningococcaemia with a petechial and purpuric rash that does not blanch; and fulminant waterhouse-friderichsen syndrome — shock with bilateral adrenal haemorrhage and disseminated intravascular coagulation, which may kill within hours
• Complement deficiency of the terminal components (C5–C9) and asplenia predispose to recurrent meningococcal disease and should be sought after a second episode
• Diagnosis — lumbar puncture with Gram stain (gram-negative diplococci, often intracellular), culture of cerebrospinal fluid and blood, latex antigen detection and PCR (which remains positive after antibiotics)
• Treatment — ceftriaxone or benzylpenicillin, given immediately; in suspected meningococcal sepsis with a rash, the first dose should be given before transfer and before lumbar puncture
• Chemoprophylaxis of close contacts — rifampicin, ciprofloxacin or ceftriaxone, given promptly; and conjugate vaccines (ACWY and protein-based MenB), with vaccination required for Haj pilgrims

Neisseria Gonorrhoeae

• Non-capsulated; virulence depends on pili (adhesion, and antigenically highly variable), Opa proteins, IgA protease, and endotoxin. Antigenic variation of the pili is why infection confers NO lasting immunity and reinfection is common
• Transmission is almost exclusively sexual, and vertically at delivery; the organism does not survive outside the body
• In men — acute urethritis 2 to 5 days after exposure with purulent discharge and dysuria; complications are epididymo-orchitis, prostatitis and urethral stricture
• In women — frequently asymptomatic or mild (cervicitis, discharge), which is why it goes untreated and progresses to pelvic inflammatory disease with infertility, ectopic pregnancy and chronic pelvic pain. Perihepatitis (Fitz-Hugh-Curtis syndrome) may occur
• Ophthalmia neonatorum — acquired during passage through an infected birth canal; a purulent conjunctivitis within 2 to 5 days that rapidly causes corneal ulceration and blindness if untreated
• Disseminated gonococcal infection — fever, pustular skin lesions, tenosynovitis and migratory arthritis; associated with terminal complement deficiency
• Diagnosis — a Gram stain of urethral pus showing intracellular gram-negative diplococci within polymorphs is diagnostic in symptomatic men but insufficient in women; culture on Thayer-Martin with immediate plating; and nucleic acid amplification tests, which are now the most sensitive and can be done on urine
• Treatment — ceftriaxone intramuscularly, usually with azithromycin; resistance has developed to sulphonamides, penicillin, tetracyclines and fluoroquinolones in turn, and reduced cephalosporin susceptibility is now reported, so surveillance is essential

Comparison of the Two Pathogenic Neisseriae

Prevention and Applied Aspects

• Treat the sexual partners and screen for other sexually transmitted infections, particularly chlamydia, syphilis and HIV; co-infection with chlamydia is common, which is why dual therapy is given
• Syndromic management is used in India where laboratory facilities are limited, treating urethral or vaginal discharge for both gonococcus and chlamydia at the first visit
• Prevent ophthalmia neonatorum with prophylactic eye care at birth and by treating maternal infection antenatally; blindness from this cause is entirely preventable
• Any purulent conjunctivitis in the first week of life is gonococcal until proved otherwise, and requires systemic treatment, not merely drops
• Give the first antibiotic dose in suspected meningococcal disease before transfer; this single practice has measurably reduced mortality
• Investigate recurrent neisserial infection for terminal complement deficiency, and vaccinate such patients — a small group in whom a simple test changes lifelong management
• Plate gonococcal and meningococcal specimens immediately and never refrigerate them; the organisms are delicate, and a negative culture from a properly infected patient is usually a transport failure
• Vaccinate Haj and Umrah pilgrims against meningococcus, which is a requirement for entry and has prevented outbreaks associated with mass gatherings
• Gonorrhoea is often asymptomatic in women, so partner notification and treatment matter more than symptoms; the sequelae are borne almost entirely by women
• Gonococcal resistance has advanced through every drug class in turn, and there is now real concern about untreatable gonorrhoea; culture with sensitivities should be preserved wherever possible rather than relying only on molecular tests
• Do not delay the lumbar puncture beyond the antibiotic in suspected meningococcal disease — but equally, do not delay the antibiotic for the lumbar puncture; PCR and blood culture will usually still yield the diagnosis
• Look for the rash with the patient undressed and in good light, and use the tumbler test; early petechiae are sparse and easily missed on pigmented skin
• Meningococcal carriage is common and usually harmless, so throat swabbing of contacts is not useful; prophylaxis is given on the basis of exposure, not of carriage
• Give prophylaxis to close contacts within 24 hours — household members, those sharing a dormitory, and anyone with direct exposure to respiratory secretions including staff who intubated the patient
• Ceftriaxone is the preferred prophylaxis in pregnancy, since ciprofloxacin and rifampicin are avoided; and rifampicin reduces the effectiveness of oral contraceptives, which must be mentioned
• Screen for other sexually transmitted infections whenever one is found, and offer HIV testing with counselling; single-infection thinking misses the commonest pattern
• Ophthalmia neonatorum requires systemic treatment, not merely topical; and both parents must be examined and treated
• Chlamydial ophthalmia appears later than gonococcal — at 5 to 14 days rather than 2 to 5 — which is a useful clinical discriminator while awaiting laboratory confirmation
• Disseminated gonococcal infection presents as arthritis and skin lesions rather than as urethritis, and the genital infection may be silent, so it is regularly mistaken for a reactive or septic arthritis
• Meningococcal vaccines do not cover group B well in most formulations, because its capsule resembles human neural tissue; protein-based vaccines were developed specifically to solve that problem

Haemophilus Influenzae

• A small, pleomorphic, gram-negative coccobacillus, non-motile, non-sporing; fastidious
• Requires X and V factors — X is haemin and V is NAD. Both are released when blood is heated, which is why it grows on chocolate agar but not on plain blood agar
• Satellitism — on blood agar it grows only as tiny colonies close to a streak of Staphylococcus aureus, which supplies V factor by lysing the red cells; a simple and characteristic identification test
• Capsulated strains are typed a to f; type b (Hib), with a polyribosyl ribitol phosphate capsule, causes almost all invasive disease. Non-capsulated (non-typeable) strains cause mucosal infection
• Diseases — meningitis in unvaccinated children aged 3 months to 3 years; acute epiglottitis, a paediatric emergency in which examination of the throat may precipitate complete obstruction; pneumonia; septic arthritis; cellulitis; and, from non-typeable strains, otitis media, sinusitis and exacerbations of chronic obstructive pulmonary disease
• Prevention — the Hib conjugate vaccine, included in the pentavalent vaccine in India, has almost eliminated invasive disease where coverage is good. Rifampicin prophylaxis for household contacts

Bordetella Pertussis

• A small gram-negative coccobacillus, strictly aerobic and highly fastidious
• BORDET-GENGOU medium (potato, glycerol and blood, with penicillin to suppress other flora) gives characteristic "mercury drop" colonies; charcoal-cephalexin agar is now preferred
• Virulence factors — pertussis toxin (which ADP-ribosylates the inhibitory Gi protein, so adenylate cyclase is disinhibited and cAMP rises — the mirror image of cholera toxin); filamentous haemagglutinin and pertactin (adhesion); tracheal cytotoxin, which destroys ciliated epithelium and is responsible for the cough; and adenylate cyclase toxin
• Clinical course of whooping cough — a catarrhal stage of 1 to 2 weeks resembling a coryza, which is the most infectious period and the one in which the diagnosis is not made; then the paroxysmal stage of 2 to 6 weeks with bouts of coughing ending in an inspiratory whoop and vomiting, and a marked absolute lymphocytosis; then a convalescent stage over weeks to months (the "hundred-day cough")
• Infants under 6 months may have apnoea and cyanosis without a whoop, and account for nearly all deaths
• Diagnosis — a per-nasal swab (not a throat swab) for culture or PCR, which is now the test of choice; serology late in the illness
• Treatment and prevention — a macrolide (azithromycin), which reduces transmission but alters the illness little once paroxysms have begun; prophylaxis for household contacts; and vaccination with whole-cell (in DPT and pentavalent vaccine in India) or acellular vaccine. Maternal immunisation in pregnancy protects the newborn through transplacental IgG before the infant's own course begins

Brucella

• Small, non-motile, gram-negative coccobacilli; strict aerobes, some requiring extra carbon dioxide; slow-growing, requiring prolonged incubation of up to 4 weeks — so the laboratory must be warned or cultures will be discarded as negative
• An intracellular pathogen surviving within macrophages, which explains the chronicity, the granulomatous response and the need for prolonged intracellular antibiotics
• Species and hosts — B. Melitensis (goats and sheep; the most virulent for man), B. Abortus (cattle), B. Suis (pigs), B. Canis (dogs)
• A zoonosis acquired from unpasteurised milk and milk products, or by direct contact with infected animals, placentas and aborted material — an occupational disease of farmers, veterinarians, abattoir workers and laboratory staff
• Clinical features — undulant fever with drenching sweats having a characteristic odour, malaise, weight loss, arthralgia and back pain; hepatosplenomegaly and lymphadenopathy; sacroiliitis and spondylitis; orchitis; endocarditis (the chief cause of death); and neurobrucellosis
• Diagnosis — blood and bone marrow culture with prolonged incubation; standard agglutination test with a fourfold rise; 2-mercaptoethanol test to detect IgG in chronic infection; and PCR. Brucella is a leading cause of laboratory-acquired infection, so it must be handled at BSL-3
• Treatment — doxycycline with rifampicin for 6 weeks, or doxycycline with streptomycin or gentamicin; combination therapy is essential and monotherapy leads to relapse

Comparison of the Three Genera

Applied Aspects

• Do not examine the throat in suspected epiglottitis; a child sitting forward, drooling and unable to swallow needs a controlled airway in theatre, and inspection may precipitate fatal obstruction
• Suspect pertussis in a prolonged cough, including in adolescents and adults whose immunity has waned; they are the reservoir from which unimmunised infants are infected, and the diagnosis is regularly missed
• Immunise pregnant women against pertussis, which protects the infant during the vulnerable weeks before the primary course takes effect
• Ask about unpasteurised milk and animal contact in prolonged fever; brucellosis is under-diagnosed in India, where raw milk consumption is common, and it is frequently treated as tuberculosis or as a fever of unknown origin
• Warn the laboratory when brucellosis is suspected — both to ensure prolonged incubation and to protect staff, since it is among the commonest laboratory-acquired infections
• Treat brucellosis with a combination for at least 6 weeks; incomplete or single-drug treatment produces relapse and chronic disease that is far harder to cure
• Hib disease has become uncommon where vaccination coverage is good, and epiglottitis is now rare in vaccinated populations — but it persists where coverage is patchy, so it must remain in mind
• A per-nasal swab, not a throat swab, is required for pertussis; the wrong specimen is a common reason for a negative result
• Pertussis is most infectious in the catarrhal stage, before anyone suspects it, which is why household prophylaxis is offered as soon as a case is identified
• Brucella endocarditis is the chief cause of death and usually requires valve replacement in addition to prolonged antibiotics
• Pasteurisation of milk prevents brucellosis, tuberculosis and Q fever together, which is why raw milk consumption remains a genuine public health issue in India
• Consider pertussis in an infant with apnoea or cyanosis and no whoop; the classical picture is a feature of older children, and infants under 6 months account for nearly all the deaths
• Ask about occupation in unexplained fever — abattoir work, veterinary practice, dairy farming and laboratory work each point toward specific organisms that routine investigation will not find
• Haemophilus grows only on chocolate agar, so a request for cerebrospinal fluid or respiratory culture should say what is suspected if the organism is to be isolated at all
• Brucellosis is frequently mistaken for tuberculosis in India, since both cause prolonged fever, weight loss and spinal disease; the treatments differ entirely, and the milk and animal contact history is what separates them
• Haemophilus ducreyi causes chancroid, a painful genital ulcer with suppurating inguinal nodes, distinguishing it from the painless chancre of syphilis
• Whole-cell pertussis vaccine is more reactogenic but gives longer protection than the acellular form; India uses the whole-cell vaccine, and the resurgence of pertussis in countries using acellular vaccine is relevant to that choice
• Consider Brucella in culture-negative endocarditis, along with Coxiella, Bartonella and the HACEK organisms; all require the laboratory to be told and to incubate for longer
• Vaccinate before splenectomy against Haemophilus, pneumococcus and meningococcus, all three being capsulated organisms cleared by the spleen
• Brucella agglutinin titres remain raised long after cure, so a single positive result does not prove active disease and follow-up is judged clinically rather than serologically
• Bordetella parapertussis causes a milder similar illness not prevented by the pertussis vaccine, which explains some apparent vaccine failures
• Immunity to pertussis wanes after about a decade from both vaccination and natural infection, so adolescents and adults sustain transmission and boosters are increasingly recommended

Morphology and the Cell Wall

• Slender, slightly curved, beaded bacilli, non-motile, non-sporing and non-capsulated, often occurring in parallel bundles
• Acid-fast — they resist decolourisation by 20% sulphuric acid and 3% alcohol, and are demonstrated by the ZIEHL-neelsen stain as bright red bacilli against a blue background
• The cell wall is up to 60% lipid, which is the source of nearly every distinctive property of the genus

Cultural Characteristics

• An obligate aerobe — which is why disease favours the lung apices, where the oxygen tension is highest, and why cavities teem with organisms while closed caseous lesions do not
• Extremely slow growth — a generation time of 15 to 20 hours (compared with 20 minutes for E. Coli); visible colonies take 2 to 8 weeks
• Lowenstein-JENSEN medium — the standard solid medium: coagulated egg with malachite green (which inhibits other organisms) and glycerol (which favours M. Tuberculosis); colonies are rough, tough and BUFF-coloured
• Middlebrook 7H10 and 7H11 (agar-based) and liquid MGIT systems, which detect growth automatically in about 2 weeks and are now preferred
• Resistance — survives in dried sputum for weeks, and resists acids and alkalis (used for decontamination of sputum before culture) and many disinfectants; but is killed by direct sunlight, ultraviolet light, heat and pasteurisation. Ventilation and daylight are therefore genuine control measures

Pathogenesis — Primary Tuberculosis

Inhalation of droplet nuclei containing very few bacilli — the infecting dose is small → Bacilli reach the alveoli and are ingested by alveolar macrophages → They inhibit phagosome-lysosome fusion and multiply inside the macrophage, unchecked for the first weeks → A subpleural lesion forms in the lower part of the upper lobe or upper part of the lower lobe — the GHON focus → Spread to draining hilar lymph nodes; the focus with the nodes is the primary (GHON) complex → At 4 to 6 weeks cell-mediated immunity develops — the tuberculin test converts to positive → Activated macrophages contain the infection; a caseating granuloma forms and the lesion usually heals with fibrosis and calcification → A few bacilli survive in a dormant state — latent infection

• The granuloma consists of central caseous necrosis surrounded by epithelioid cells, langhans giant cells (with peripherally arranged nuclei), lymphocytes and fibroblasts — the histological signature of the disease
• Most primary infections heal; about 5% progress directly, and a further 5% reactivate later — a lifetime risk of about 10%. In HIV this becomes about 10% per year

Post-primary Tuberculosis

• Dissemination — miliary tuberculosis from haematogenous spread, with innumerable small lesions in lung, liver, spleen and marrow; and metastatic foci that may reactivate years later, including the SIMON focus in the lung apex

Classification of the Mycobacteria

Applied Aspects

• Tuberculosis is an airborne infection, so ventilation, natural light and respiratory hygiene reduce transmission; India's burden makes these measures important in every waiting room and ward
• Sputum-positive cavitary disease is what spreads the infection; identifying and treating these patients promptly does more for control than any other measure, and is the basis of the national programme
• The apical distribution follows the oxygen tension, which is why the upper zones are examined first on a chest radiograph and why apical cavitation is so characteristic
• HIV transforms the natural history — reactivation risk rises from 10% lifetime to about 10% yearly, the presentation is often atypical and sputum-negative, and every tuberculosis patient should be offered HIV testing
• Pasteurisation and control of bovine tuberculosis prevent M. Bovis disease, which still occurs where raw milk is consumed
• Cell-mediated immunity is what contains the organism, so anything impairing it — HIV, corticosteroids, anti-TNF agents, diabetes, malnutrition, chronic kidney disease — predisposes to reactivation and should prompt screening before immunosuppression
• Diabetes roughly triples the risk of tuberculosis and worsens treatment outcomes; the two diseases are both common in India and each should prompt testing for the other
• Smoking and indoor air pollution increase both infection and mortality, and biomass cooking fuel is a substantial and under-recognised contributor in rural India
• Nutritional support is part of treatment, not a luxury; undernutrition is the largest single attributable risk factor for tuberculosis in India, which is why the national programme provides it
• Suspect tuberculosis in any chronic illness with weight loss and fever in an Indian setting; it remains the great mimic, and delayed diagnosis both harms the patient and sustains transmission
• Ventilation and daylight are genuine control measures, since the bacillus is killed by ultraviolet light and diluted by air exchange; this matters in crowded outpatient departments and wards

Specimen Collection

• Pulmonary — two sputum samples, ideally one spot and one early-morning specimen, which has the highest yield; gastric lavage in young children who swallow sputum; induced sputum or bronchoalveolar lavage where the patient cannot expectorate
• Extrapulmonary — cerebrospinal fluid, pleural, ascitic, pericardial or synovial fluid, lymph node aspirate or biopsy, pus, urine (three early-morning samples), menstrual blood, endometrial curettings
• Collect in a well-ventilated area or a safety cabinet; sputum collection generates aerosols and is a recognised route of transmission to staff
• Specimens from non-sterile sites are decontaminated with sodium hydroxide or the modified Petroff method before culture, exploiting the organism's resistance to alkali while other bacteria are killed

Microscopy

Culture and Molecular Methods

• Molecular tests detect nucleic acid, not viability, so they may remain positive after successful treatment and cannot be used to monitor response
• Culture remains essential for full sensitivity testing, for confirming cure, and for identifying species

Immunological and Other Tests

• Tuberculin (mantoux) test — 0.1 mL of 5 tuberculin units of PPD injected intradermally, read at 48 to 72 hours as the diameter of induration, not erythema. It indicates infection, not disease, is confounded by BCG and by non-tuberculous mycobacteria, and is falsely negative in miliary tuberculosis, HIV, malnutrition, measles and steroid therapy
• Interferon-gamma release assays (IGRA) — measure interferon-gamma released by sensitised T cells in response to ESAT-6 and CFP-10, antigens absent from BCG and from most environmental mycobacteria; therefore not confounded by vaccination. Like the tuberculin test, they cannot distinguish latent infection from active disease
• Histopathology — caseating granulomas with epithelioid cells and Langhans giant cells on biopsy or fine needle aspirate; acid-fast bacilli may be demonstrable
• Adenosine deaminase (ADA) — raised in tuberculous pleural, ascitic and cerebrospinal fluid; a useful and cheap supportive test in paucibacillary extrapulmonary disease
• Chest radiography — sensitive but not specific; useful for screening and for assessing extent

Choosing the Right Test

Applied Aspects

• Send two sputum samples including an early-morning one; a single spot specimen substantially reduces the yield and is a common practical shortcoming
• CBNAAT should be used as the initial test in children, in people living with HIV, in extrapulmonary specimens, in smear-negative patients with suggestive radiology, and in anyone at risk of drug resistance
• Rifampicin resistance detected by CBNAAT is a proxy for multidrug resistance and must prompt immediate referral for a full drug-resistant regimen and second-line testing
• Never diagnose tuberculosis on a tuberculin test alone; in India, where BCG is universal and background infection is high, a positive result is of very limited value
• Ask the laboratory for a mycobacterial culture explicitly; it will not be set up on a routine request, and the specimen will be reported as sterile
• Test every tuberculosis patient for HIV and for diabetes, both of which alter presentation, treatment and outcome, and both of which are common in India
• A positive smear does not distinguish M. Tuberculosis from other mycobacteria, which matters where atypical infection is possible; CBNAAT or culture is needed to identify the species
• Follow-up smears at the end of the intensive phase and at completion assess response; molecular tests cannot be used for this because they detect dead organisms
• Collect sputum safely — outdoors or in a ventilated area, never in a crowded waiting room or a closed cubicle, since the procedure generates infectious aerosols
• Transport specimens promptly and protect them from heat and sunlight, which reduce viability and lower the culture yield
• Decontamination kills some mycobacteria as well as the contaminants, so it lowers the culture yield slightly — a necessary trade-off for specimens from non-sterile sites, but unnecessary and harmful for cerebrospinal fluid or pus from a closed lesion
• Report the smear grade, not merely "positive"; the bacillary load guides infection control and predicts the speed of sputum conversion
• A normal chest radiograph does not exclude tuberculosis in HIV, where disease is frequently disseminated or extrapulmonary with little pulmonary change
• Nikshay, the national notification system, requires every case to be notified, including from the private sector, and this now carries statutory force in India
• Serological tests for tuberculosis are banned in India, having been shown to be inaccurate and misleading; anyone offering one is not following national guidance
• Do not treat on radiology alone where bacteriological confirmation is possible; old fibrotic change is frequently mistaken for active disease and leads to repeated unnecessary courses
• TrueNat allows molecular diagnosis at primary health centre level, being battery-operated and portable, which addresses the geography that has always limited tuberculosis diagnosis in rural India
• Culture remains necessary despite molecular advances — for full sensitivity testing, for species identification, and to confirm cure
• Two negative smears do not exclude the diagnosis in a patient with suggestive symptoms and radiology; proceed to CBNAAT and culture rather than treating with a broad-spectrum antibiotic and reviewing later

The Organism

• Mycobacterium leprae — an acid-fast bacillus, straight or slightly curved, occurring in parallel bundles ("cigar bundles") and in rounded masses called globi within foamy macrophages (virchow or lepra cells)
• Acid-fast to only 5% sulphuric acid (M. Tuberculosis resists 20%), so a modified Ziehl-Neelsen stain is used
• It has never been cultivated in artificial media — a defining practical fact. It is grown in the mouse footpad and in the nine-banded armadillo, which has a low body temperature and permits heavy multiplication
• The slowest-growing bacterium known — a generation time of 12 to 14 days, which explains the very long incubation period of 2 to 5 years or more, and the prolonged treatment
• Prefers cooler parts of the body (about 30°C) — skin, superficial nerves, nose, testis, eye and the extremities; it spares the warm axilla, groin and scalp, which is why the distribution of lesions is characteristic
• An obligate intracellular parasite of macrophages and schwann cells; invasion of Schwann cells is what makes it uniquely neurotropic among bacteria

Transmission and Immunology

• Transmission is chiefly by nasal droplets from untreated multibacillary patients, who may shed millions of bacilli daily; prolonged close contact is usually required
• Most exposed people never develop disease — the outcome depends almost entirely on the host's cell-mediated immunity
• A strong Th1 response gives tuberculoid disease — few organisms, few lesions, and much nerve damage from the vigorous granulomatous reaction
• A weak or Th2-dominated response gives lepromatous disease — abundant organisms, widespread lesions, and less intense but more extensive damage

Classification

• The RIDLEY-jopling spectrum places disease as TT – BT – BB – BL – LL, with the borderline forms being immunologically unstable and liable to shift, which is what produces reactions
• The WHO operational classification, used for treatment, is simpler: paucibacillary (up to 5 skin lesions, no bacilli on smear) and multibacillary (more than 5 lesions, or any positive smear)
• Indeterminate leprosy — the earliest form, a single ill-defined hypopigmented macule which may heal spontaneously or progress

Diagnosis and Treatment

• The WHO cardinal signs — any one establishes the diagnosis: (1) a hypopigmented or reddish skin lesion with definite loss OF sensation; (2) a thickened or tender peripheral nerve with loss of sensation or weakness in its distribution; (3) demonstration of acid-fast bacilli in a slit-skin smear
• Slit-skin smear — taken from the ear lobe and active lesions; reported as the bacteriological index (density of bacilli, on a logarithmic 0 to 6+ scale) and the morphological index (percentage of solidly staining, and therefore viable, bacilli), which falls rapidly with effective treatment
• Nerves to examine — ulnar at the elbow, median, radial cutaneous, common peroneal at the neck of the fibula, posterior tibial, great auricular and supraorbital
• Lepromin test — an intradermal injection of killed bacilli; the Fernandez reaction is read at 48 hours and the mitsuda reaction at 3 to 4 weeks. It is not a diagnostic test — it assesses cell-mediated immunity and is therefore of prognostic and classificatory value
• Multidrug therapy (MDT) — supplied free through the national programme: paucibacillary, rifampicin monthly with daily dapsone for 6 months; multibacillary, rifampicin and clofazimine monthly with daily dapsone and clofazimine for 12 months
• Patients become non-infectious within days of starting rifampicin, so isolation is unnecessary and stigma is unjustified

Deformities and Complications

Applied Aspects

• Test sensation in every hypopigmented patch; this single bedside step distinguishes leprosy from the far commoner pityriasis alba, vitiligo and tinea versicolor, and costs nothing
• Palpate the peripheral nerves routinely, particularly the ulnar and common peroneal; a thickened nerve in a patient with a skin lesion is diagnostic
• Disability is caused by nerve damage, not by the bacillus, and most of it is preventable by early diagnosis and prompt treatment of reactions; deformity appearing during treatment usually means an unrecognised reaction
• Teach self-care of anaesthetic hands and feet — daily inspection, protective footwear, soaking and oiling; ulcers and their consequences arise from unnoticed injury, not from continuing infection
• India accounts for a large share of new cases worldwide despite elimination as a public health problem having been declared; case detection, contact tracing and single-dose rifampicin prophylaxis for contacts remain important
• Stigma remains the greatest obstacle; explaining that treated patients are not infectious, and that the disease is curable and free to treat, changes presentation times more than any laboratory advance
• Examine the eyes at every visit; lagophthalmos and corneal anaesthesia cause blindness silently, and simple measures such as spectacles, lubricants and lid exercises prevent it
• Deformity is not a sign of active infection, and a patient who has completed treatment needs rehabilitation and reconstructive surgery rather than more antibiotics
• Absorption of digits results from repeated unnoticed trauma, not from the organism destroying bone; explaining this makes self-care instructions comprehensible rather than arbitrary
• Single-dose rifampicin prophylaxis for contacts reduces the risk of developing leprosy and is now part of national policy in several countries including India
• Examine all household contacts at diagnosis and annually; early detection in a contact prevents both disability and further transmission
• Clofazimine causes reversible skin darkening, which is distressing and a common reason for stopping treatment; warning patients in advance markedly improves completion
• Leprosy is curable and treatment is free, and saying so plainly at the first consultation does more for adherence and for early presentation of contacts than any other single intervention

The Organism

• Treponema pallidum — a slender spirochaete with 6 to 14 regular, sharp, spiral coils, motile by axial filaments (endoflagella) lying within the periplasm, giving a characteristic rotating, flexing and to-and-fro movement
• Too slender to be seen by ordinary light microscopy or Gram stain; demonstrated by dark-ground (dark-field) microscopy of fresh exudate, by silver impregnation (Fontana), or by immunofluorescence
• It has never been cultivated in artificial media; it is maintained by rabbit testicular inoculation
• Extremely fragile — killed rapidly by drying, heat, soap, antiseptics and oxygen, which is why transmission requires direct, usually sexual, contact and why fomite spread does not occur
• Transmission — sexual contact (the great majority), transplacental passage causing congenital syphilis, blood transfusion, and rarely direct inoculation

Stages of Acquired Syphilis

• Congenital syphilis — early features include "snuffles" (a highly infectious nasal discharge), a bullous rash on palms and soles, hepatosplenomegaly, osteochondritis and pseudoparalysis; late features include hutchinson teeth (notched incisors), interstitial keratitis and eighth nerve deafness (together the Hutchinson triad), saddle nose, sabre tibia and frontal bossing

Serological Diagnosis

Treatment

• Penicillin remains the drug of choice — T. Pallidum has never developed penicillin resistance. Benzathine penicillin as a single intramuscular dose for early syphilis, and weekly for three weeks in late disease
• Neurosyphilis requires intravenous aqueous benzylpenicillin, since benzathine penicillin does not achieve adequate concentrations in cerebrospinal fluid — a distinction that matters
• Doxycycline in penicillin allergy, though penicillin desensitisation is preferred in pregnancy and neurosyphilis, since nothing else is reliably effective
• Jarisch-herxheimer reaction — fever, chills, myalgia and worsening of the rash within hours of the first dose, from the sudden release of treponemal antigen as organisms are killed. It is not an allergic reaction and does not contraindicate further treatment; patients must be warned, and it may precipitate preterm labour in pregnancy
• Follow up with quantitative VDRL or RPR at 3, 6 and 12 months; a fourfold fall in titre indicates successful treatment
• Treat all sexual partners and screen for other sexually transmitted infections, particularly HIV

Interpretation of Serological Patterns