SN7 SHORT NOTE 5M

Describe the collection of specimens and the methods of antimicrobial susceptibility testing.

Microbiology · 2nd Professional — Para-Clinical

Principles of Specimen Collection

Collect before antibiotics are started wherever possible — a single dose can render cultures sterile and leave the patient without a microbiological diagnosis for the remainder of the illness
Collect from the actual site of infection, avoiding contamination with normal flora; the deeper and more specific the specimen, the more interpretable the result
Adequate quantity — a swab is the least satisfactory specimen; pus, tissue and fluid are far better
Correct container and transport medium, correctly labelled, with the clinical details and antibiotic history on the request form
Prompt transport; where delay is unavoidable, refrigerate urine but never refrigerate cerebrospinal fluid (meningococci and pneumococci die) or specimens for anaerobic culture

Specimen	Key points
Blood culture	The most important microbiological investigation in a septic patient. Take at least 2 sets from separate sites, with strict skin antisepsis; volume is the single greatest determinant of yield (8–10 mL per bottle in adults); take before antibiotics
Urine	Clean-catch midstream; significant bacteriuria is conventionally ≥105 colony-forming units/mL of a single organism, though lower counts matter in symptomatic young women and in suprapubic samples. Growth of three or more organisms indicates contamination
Sputum	Deep cough, preferably early morning; assess quality first — many squamous cells means saliva. Two specimens for tuberculosis
Cerebrospinal fluid	Process immediately; do not refrigerate; divide for cell count, biochemistry and microbiology; Gram stain guides immediate treatment
Pus and wound	Aspirate with a syringe rather than swab; send tissue where possible
Stool	Fresh sample in a clean container; Cary-Blair for Vibrio and Campylobacter; three samples for parasites

CLINICAL PEARL

Volume matters more than technique in blood culture, and this is consistently underestimated. Bacteraemia is often low-grade, with only a few organisms per millilitre, so every extra millilitre raises the yield measurably. Two properly filled sets from separate sites, taken before antibiotics, will detect the great majority of bloodstream infections — and a single under-filled bottle taken after the first dose will frequently detect nothing at all.

Antimicrobial Susceptibility Testing

Method	Principle	Comments
Disc diffusion (Kirby-Bauer)	Antibiotic discs on a lawn of the organism on Mueller-Hinton agar; the antibiotic diffuses outward, creating a concentration gradient, and the zone OF inhibition is measured	Simple, cheap and the standard method in most Indian laboratories. Interpreted against CLSI or EUCAST breakpoints as sensitive, intermediate or resistant. Inoculum density, agar depth and disc potency must be standardised
Broth or agar dilution	Doubling dilutions determine the minimum inhibitory concentration (MIC)	The reference method; gives a quantitative result needed for endocarditis and for reduced-susceptibility organisms
E-test	A strip with a gradient of antibiotic; the MIC is read where the elliptical zone meets the strip	Combines the simplicity of diffusion with a quantitative MIC; more expensive
Automated systems	Growth measured turbidimetrically in wells	Rapid identification and MIC together; costly
Molecular detection	Detects the resistance gene rather than the phenotype	MecA for MRSA, rpoB for rifampicin resistance (CBNAAT), carbapenemase genes; fast, but a gene may be present without being expressed

"Sensitive" means the organism is likely to respond to the usual dose at the usual site — it is a clinical prediction, not a chemical measurement, which is why breakpoints differ between sites (a urinary breakpoint differs from a meningeal one)
Special tests — the D-test for inducible clindamycin resistance; double-disc synergy for ESBL; modified Hodge and combined disc tests for carbapenemases; cefoxitin disc for MRSA

Serological and Molecular Diagnosis

Antibody detection — a fourfold rise in titre between acute and convalescent samples is diagnostic; a single titre is difficult to interpret in an endemic area where background seropositivity is high. IgM indicates recent infection
Common tests — WIDAL (typhoid; poor specificity and much over-used in India), ELISA, agglutination, complement fixation, and immunofluorescence
Antigen detection — faster than antibody and positive early: rapid tests for malaria, dengue NS1, cryptococcal antigen, streptococcal antigen, and lateral flow assays generally
Molecular methods — PCR, real-time PCR for viral load, CBNAAT for tuberculosis and rifampicin resistance, and sequencing; they detect nucleic acid rather than viability and are prone to contamination
MALDI-TOF mass spectrometry identifies an isolate within minutes from a colony by its protein fingerprint, and has transformed turnaround time where available

Applied Aspects

Take cultures before the first antibiotic dose, and do not delay treatment of a septic patient to do so — the two take a few minutes together
Give the laboratory clinical information; a request marked only "culture and sensitivity" cannot prompt the special media and prolonged incubation that tuberculosis, anaerobes, fungi or fastidious organisms require
Interpret a result in context — ask whether the organism fits the site and the illness, whether it could be contamination or colonisation, and whether the clinical course matches
De-escalate when the sensitivity returns; the purpose of the test is to narrow therapy, and failing to act on it wastes both the specimen and the opportunity
The Widal test is over-used and over-interpreted in India; a single raised titre in an endemic population is weak evidence, and blood culture remains the definitive test for typhoid
Local antibiograms should guide empirical therapy rather than textbook spectra, since resistance patterns differ markedly between hospitals and regions

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