Infectious Diseases

• Specimens are taken with particular cognisance of the site of the infection, and hence the yield;
• Timing of specimen taking should be early enough to coincide with the period of maximal viral shedding;
• For fluids, prompt delivery to the laboratory in sterile containers is crucial, while for swabs, special viral transport media like the BSS (?Bile Salt Sugar) solution enriched with antibiotics to inhibit the growth of bacterial contaminants are required;
• Swabs and NPA should be cell-rich; swabs should ensure fairly vigorous rubbing of the (suspected) infected site to ensure adequacy of cellular contents;
• Transportation to the lab should prompt and specimens are preferably delivered on ice, as against frozen specimens; lab may need to be consulted in advance in case an unusual specimen is to be delivered

Techniques include:

• Electron microscopy (EM);
• Antigen detection, (Indirect) Fluorescent Antibody technique test;
• Antibody detection, i.e. Serological techniques like CFT, HAI etc;
• Virus culture in specific cell-culture media of human or animal sources; examples include HEK Cell line, Human Embryonic lung fibroblasts, monkey kidney cell lines, Hep Cell line for RSV, suckling mice inoculation for Cox Sackie viruses. Arboviruses and rabies

Virus isolation after the above culture and subsequently, identifying the virus, by using the peculiar pathological changes caused by the presence of the suspected virus, i.e. Cytopathic effect, or CPE; e.g. RSV and Herpes Simplex Virus which cause syncytia and giant-cell formation.

Such identification of cellular pathology requires just light microscopy under low magnification

Molecular biology methods for detecting specific nucleic acids peculiar to the virus, after the appropriate amplification – these tests are sometimes referred to as nuclear amplification tests with PCR test (i.e. Polymerase Chain Reaction test) being a classic example.

• Becoming increasingly important because of the corresponding surge in the population of the immunocompromised paediatric hosts, in whom specific antifungal treatment may be needed after empirical broad spectrum antimicrobial therapy! (Remember that empirical antifungal treatment, especially with the less safe amphotericin B CANNOT be justified unless there is a pending tissue or serological samples for diagnostic confirmation;
• In general fungal infections can be categorized as superficial or deep mycoses;
• Skin scrapings from superficial skin lesions like Taenia corporis or T. capitis may be examined under the microscope (at low magnification) for hyphae of the causative fungal agents after pre-treatment of sample with a weak solution (20%) of KOH to dissolve the ‘entangled’ keratin;
• The gold standard fungal staining technique is the Indian ink preparation, while many superficial dermatophytes can be cultured using the Sabouraud’s maltose-peptone agar medium;
• Others: DTM (dermatophyte test medium with nutrient agar with cycloheximide, gentamycin and chlortetracycline to inhibit the growth of non-pathogenic fungi and bacteria.

In contemporary times, the mycotic infections especially the deep mycoses like candidaemia, aspergillosis, coccidioidomycoses, blastomycetes or histoplasma infections are usually identified using one or more of:

• Wood’s light examination, ie examination which uses a lamp that emits UV light, at a wavelength of 365nanometers, is particularly useful for diagnosing superficial fungal lesions by Micrsporon and trichophyton – principle is the characteristic fluourescence by the fungi in a darkened room.
• Small-spore ectothrix + fluourescence of M. audouinii, and large-spore ectothrix + no fluorescence of other spp of Microsporum and certain spp. of Trichophyton;
• Endothrix invasion with no fluorescence of Tricophyton tonsurans with hyphae growth and fragmentation within the hair shaft causing several broken shaft and alopecia. Fluorescence with endothrix invasion is seen in T. schoenleinii infection associated with ‘favus’
• Skin testing, using the specific fungal antigen’ frequently reversible;
• Routine blood culture especially for candidaemia (90% positive e yield), or culture from BAL or bronchoscopic specimens in candida infection of the tracheobronchial infections;
• Serological technique using CFT, or immunodiffusion;
• Elevated eosinophil (from FBC), or specific IgE, as exemplified by Aspergillosis infection, ABPA;
• Enzyme-linked immunosorbent assay (EIA) to detect specific fungal antigen – reputedly most specific for many including blastomycetes;
• Histoplasma capsulatum or Pneumocystis jirovecci can be identified from the sputum, other relevant respiratory specimens or lung biopsy specimens;

Diagnosis Pneumocytis jiroveccii infection

• For P. jirovecci, four stains can be used to identify the cyst forms or trophozoites or sporozoites–
• Giemsa or similar polychrome stains for tropho- or sporozoites ;
• Grocott-Gomori stain and toluidione blue for the cyst form;
• Fluorescein-labelled monoclonal antibody stains for both

For most parasitic infections, laboratory diagnosis is usually made using microscopy of clinical specimens at low magnification, as exemplified by:

• Direct microscopy of unstained (wet) stool or (spun) urine specimens, for helminth eggs, ova, cysts and trophozoites (e.g. Ascaris lumbricoides, Strongyloides stercoralis, Schistosoma mansoni eggs, Entamoeba histolytica and Giardia lamblia cysts and trophozoites in stool, eggs of Schistosoma haematobium in spun urine samples.
• Also, direct microscopy of blood or bone marrow smears is invaluable for:
• Identifying Plasmodium species in malaria (thick and thin film) and Babesia in babesiosis;
• Identifying Leishmania spp. in bone marrow smear (in Leishmaniasis).
• Serology for ‘difficult’ parasites with poor sensitivity of stool sample examination like Strongyloides stercoralis. Other parasites that are preferably detected serologically (because of difficulty with direct microscopy) include Toxoplasma and Trichinella spp.

Laboratory Options and Precautions

Laboratory examination of stool samples may be:

• Wet mount of fresh stool– for motile organisms;
• Concentrated stool sample – to improve yield;
• Stained sample, using permanent stains like trichrome before microscopic examination;
• Modified staining technique for certain parasites which may be otherwise missed by regular staining technique. These include modified AFB staining for detecting Cyclospora and Cryptosporidium, modified trichrome staining for Microsporidia;
• EIA detection of antigen is reputedly sensitive for detecting Giardia and Cryptosporidium from stool samples

Parasite Detection in Stool: Precautions

• Avoid stool contamination with water or urine as the former may contain confusable free-living organisms, while urine may kill some otherwise motile agents in the stool;
• Stool sample collection and examination should be preferably delayed for approximately one to one-and-a-half weeks after patient’s ingestion of mineral oil, barium or bismuth, all of which are known to adversely affect the detection of stool parasites;
• For Giardia lamblia with intermittent stool shedding of its eggs, three or more samples collected on alternate days are required for a possibly successful examination;
• Stool preservatives to sustain the organisms therein may be required if delay in lab delivery is anticipated;

• Essentially for pathogens that are difficult to detect by direct microscopy or to culture in vitro; These include:
• viruses like HIV, EBV, Hepatitis viruses;
• Rickettsia;
• Spirochaetes like Treponema pallidum, Borrelia burgdoferi
• Bacterial agents like Legionella pneumophilia, Bartonella spp;
• Mycoplasma pneumoniae and other Mycoplasma agents like Ureaplasma
• Essentially aimed at detecting one or both of specific IgM or IgG of the infectious agent
• IgM peaks faster (7-10days) and disappears after a few weeks or months in hepatitis, while IgG peaks in 4-6 weeks and is usually detectable throughout the life of the individual;
• IgM suggests recent infections and a single sample is all that is needed to make a diagnosis, whereas IgG may indicate a recent or past infection;
• To make a serological diagnosis of a recent infectious disease using IgG, two samples are needed to demonstrate one of either seroconversion (from negative to positive), or a four-fold rise in the specific IgG titre between the acute (phase) titre and the convalescent titre; the acute and convalescent titres are preferably sought 2-3weeks apart and it is this inevitable time frame that limits its usefulness in making a prompt diagnosis;
• Single high IgG titre may however indicate infection in certain clinical settings like Bartonella, Legionella and rickettsia infections

Again, indicated when culture and serology are either difficult to accomplish, too slow or unavailable;

Molecular diagnostic modalities can be categorized into two:

• Direct detection using DNA probes;
• Nucleic acid amplification, using polymerase chain reaction (PCR)

Direct detection with DNA probes

• Identify the pathogen via hybridization of the probe to complementary sequences in DNA or ribosomal RNA;
• Requires a large population of the target pathogen for a successful detection;
• Principal usefulness is in further identifying pathogens that have been isolated in culture but requires complex or difficult logistics for confirmation;
• Example include Mycobacterium tuberculosis that causes human TB or M. avium-intracellulare, which causes atypical mycobacterial infections.

PCR amplification

• Method of choice for direct detection of the nucleic acids of target pathogens from clinical specimens;
• Specimens are obtained into sterile containers and sent as promptly as possible for lab analyses;
• Using a thermal cycler, the basis of the PCR is the ability of thermostable DNA or RNA polymerase to copy targeted gene sequences, using complementary nucleotides to serve as primers, and hence amplify a conserved region of the organism;
• Each cycle in the thermal cycler doubles the amount of the target nuceic acids and produce million-fold amplification in 30 cycles of PCR;
• Highly sensitive with a wide spectrum of potentially identifiable pathogens including several viruses (including the dreaded Hepatitis C and HIV, HSV,CMV, EBV, etc.), Chlamydia species, M. tuberculosis, B. pertussis, M. pneumoniae, Legionella, Bartonella etc.;
• Main drawback is the propensity for yielding false-positives which can come from contaminant organisms, whose conserved gene sequence might have been similarly amplified;
• Also, PCR is expensive, complex as well as personnel and labor intensive;
• Cost, and need for the appropriate expertise have restricted the use of PCR to a few reference laboratories, especially in the developing world.