16.3 Microscopy, Antigen/Molecular Detection, Insects and Arthropods

16.3 Microscopy, Antigen/Molecular Detection, Insects and Arthropods

Quick Answer: A complete O&P is a direct wet mount (motility), a concentration (sedimentation or flotation), and a permanent stain (Wheatley trichrome for protozoan nuclei). Add modified acid-fast for Cryptosporidium oocysts, Giemsa thick and thin films for malaria and trypanosomes, Knott concentration for microfilariae, and cellulose tape for pinworm. Direct EIA and NAAT, including multiplex GI panels, detect Giardia and Cryptosporidium with higher sensitivity than a single smear. Ticks are disease vectors; lice, mites, bed bugs, and scabies are primarily infestations. Parasite culture is SM-only and is not M-tested.

Outline III.C.3 is microscopic and macroscopic identification. III.C.4 is direct antigen and molecular detection. III.C.2.e is insects and arthropods (ticks, mites, lice, bed bugs, scabies). III.C.5 Culture remains SM-only. Blood and brain morphology stay in 16.1; intestinal forms stay in 16.2. This section is how the bench actually demonstrates those organisms.

Microscopic and macroscopic identification

The classic ova and parasite (O&P) examination is a three-part procedure, not a single wet mount.

1. Direct wet mount (saline, sometimes iodine) of fresh stool. This is the only step that shows motility: falling-leaf Giardia, directional E. histolytica, jerky Trichomonas on a genital mount. Delayed, refrigerated, or PVA-fixed stool has already killed trophozoites. Iodine kills motility but highlights cysts. A wet mount alone is insensitive; never report no parasites from a wet mount without concentration and a permanent stain when a full O&P was ordered.

2. Concentration increases recovery of eggs, larvae, and cysts. Formalin–ethyl acetate sedimentation is the workhorse: formalin fixes, ethyl acetate extracts debris and fat, and centrifugation pellets parasites. It recovers operculated eggs, schistosome eggs, and Giardia cysts that flotation can miss. Zinc sulfate flotation (specific gravity about 1.18–1.20) floats many protozoan cysts and some eggs but can collapse operculated eggs and miss heavy schistosome eggs. Use sedimentation when trematodes, cestode operculated eggs, or infertile Ascaris are possible. Concentrate formalin-fixed stool, not a random unfixed cup that sat on the counter.

3. Permanent stained smear. Wheatley trichrome (or iron-hematoxylin) is the nuclear stain for intestinal protozoa after PVA or similar fixation. Cytoplasm stains blue-green; chromatin and chromatoid bodies stain red-purple. This is how you call E. histolytica versus E. coli versus Giardia. Trichrome is the wrong primary stain for Cryptosporidium.

Modified acid-fast (cold Kinyoun or hot Ziehl–Neelsen with a weaker decolorizer, often 1% sulfuric acid) is the oocyst stain: Cryptosporidium 4–6 µm, Cyclospora 8–10 µm, Cystoisospora large oval. Report size; a modified-AF oocyst without a diameter is an incomplete identification. Auramine-rhodamine fluorescent screens exist; confirm morphology.

Blood films. Giemsa thick films screen and quantitate malaria and trypanosomes; thin films speciate Plasmodium (16.1). Make both promptly. Scan adequate fields under oil; a five-minute glance is not a negative malaria smear.

Knott concentration (formalin plus centrifugation, or membrane filtration) concentrates microfilariae from blood. Collect W. bancrofti at night (nocturnal periodicity) and Loa loa by day. Knott is not a stool method and not a malaria method.

Cellulose-tape preparations recover Enterobius eggs from perianal skin. Skin scrapings in mineral oil recover Sarcoptes mites. Skin snips (not blood) recover Onchocerca microfilariae. Sputum may show Paragonimus eggs or Strongyloides filariform larvae in hyperinfection. Macroscopic identification includes adult Ascaris, tapeworm proglottids (count uterine branches), and hydatid sand — submit these in saline, not formalin, if motility or scolex examination is still needed, following the SOP.

Poor fixation is the most common reason a trichrome is unreadable. If the laboratory cannot see nuclei, do not guess species; request a re-collect in the correct kit.

Direct antigen and molecular detection

Microscopy remains required for malaria speciation and for helminth eggs. For the two most common intestinal protozoa, antigen and NAAT outperform a single O&P.

Giardia and Cryptosporidium EIA (and DFA) detect stool antigen or fluorescent oocysts and cysts and are widely used as screens, especially in outbreak and pediatric settings. Sensitivity is higher than a single wet mount. A positive crypto EIA in an AIDS patient with watery diarrhea is diagnostic without waiting for modified AF, though many labs still stain to confirm and to size-split Cyclospora.

NAAT and multiplex GI panels amplify Giardia, Cryptosporidium, and often E. histolytica (molecular assays that distinguish E. histolytica from E. dispar), plus bacterial and viral targets. They do not replace O&P when the question is helminths, Cyclospora (unless the panel includes it), or an unusual parasite. They also do not speciate malaria. Use the panel when the syndrome is community gastroenteritis; add a full O&P plus modified AF when travel, eosinophilia, worm exposure, or immunosuppression suggests something the panel does not cover.

Trichomonas NAAT (or antigen) on genital specimens is more sensitive than a cooled wet mount. Malaria rapid diagnostic tests detect HRP2 (P. falciparum) and pan-species pLDH or aldolase; they are adjuncts, not replacements for films, and HRP2-deletion strains can false-negative falciparum antigen tests. Toxoplasma PCR on amniotic fluid or CSF supports congenital and CNS disease; serology remains first-line in immunocompetent hosts (serology chapter).

Do not answer an M item with parasite culture. In vitro culture of T. vaginalis and agar-plate culture of Strongyloides exist in specialist practice and are listed as III.C.5 (SM ONLY). The M-tested tools are microscopy, antigen, and molecular detection.

Insects and arthropods: vectors versus infestation

The outline examples are ticks, mites, lice, bed bugs, and scabies. The exam distinction is whether the arthropod transmits a pathogen (vector) or is the disease (infestation).

Ticks are the medically important vectors on this list. Ixodes scapularis vectors Lyme borreliosis, Babesia, and Anaplasma. Dermacentor is linked to Rocky Mountain spotted fever and tularemia. Amblyomma (lone star) is linked to ehrlichiosis. The parasitology bench may be asked to identify the tick (capitulum, scutum, festoons, mouthparts) when a patient submits one. That identification is entomology, not a Giemsa species call. Do not report a tick as Plasmodium.

Scabies, lice, and bed bugs produce local infestation. Sarcoptes mites tunnel in stratum corneum; crusted (Norwegian) scabies in immunocompromised hosts sheds enormous mite burdens and is a contact-control emergency. Head lice stay on hair; body lice live in clothing. Bed bugs hide in mattress seams, bite at night, and are not competent vectors of malaria, Chagas disease, or filaria in routine clinical teaching.

Other vectors belong with their parasites but are not the named insect list: Anopheles mosquitoes (Plasmodium), reduviid bugs (T. cruzi), tsetse flies (T. brucei), sand flies (Leishmania), blackflies (Onchocerca), and freshwater snails (schistosomes — mollusks, not insects). If a stem asks about ticks versus bed bugs, the tick is the vector and the bed bug is the infestation.

Close the loop: match the method to the organism. Wet mount and trichrome for Giardia and Entamoeba, modified AF or EIA/NAAT for Cryptosporidium, films for malaria, tape for pinworm, Knott for lymphatic filaria, scrapings for scabies. Do not reach for SM-only culture to answer an M question.