Detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum antigens in human fecal specimens using the triage parasite panel enzyme immunoassay.

The Triage parasite panel (BIOSITE Diagnostics, San Diego, Calif.) is a new qualitative enzyme immunoassay (EIA) panel for the detection of Giardia lamblia, Entamoeba histolytica/E. dispar, and Cryptosporidium parvum in fresh or fresh, frozen, unfixed human fecal specimens. By using specific antibodies, antigens specific for these organisms are captured and immobilized on a membrane. Panel performance was evaluated with known positive and negative stool specimens (a total of 444 specimens) that were tested by the standard ova and parasite (O&P) examination as the "gold standard," including staining with both trichrome and modified acid-fast stains. Specimens with discrepant results between the reference and Triage methods were retested by a different method, either EIA or immunofluorescence. A number of samples with discrepant results with the Triage device were confirmed to be true positives. After resolution of discrepant results, the number of positive specimens and the sensitivity and specificity results were as follows: for G. lamblia, 170, 95.9%, and 97.4%, respectively; for E. histolytica/E. dispar, 99, 96.0%, and 99.1%, respectively; and for C. parvum, 60, 98.3%, and 99.7%, respectively. There was no cross-reactivity with other parasites found in stool specimens, including eight different protozoa (128 challenges) and three different helminths (83 challenges). The ability to perform the complete O&P examination should remain an option for those patients with negative parasite panel results but who are still symptomatic.

Detection of Giardia lamblia and Cryptosporidium parvum antigens in human fecal specimens using the ColorPAC combination rapid solid-phase qualitative immunochromatographic assay.

The ColorPAC Giardia/Cryptosporidium (Becton Dickinson) is a solid-phase qualitative immunochromatographic assay that detects and distinguishes between Giardia lamblia and Cryptosporidium parvum in human stool. Agreement between the Alexon-Trend ProSpecT Giardia Rapid EIA and the ColorPAC assay was 166 of 172 (96.5%). Agreement between the Alexon-Trend ProSpecT Cryptosporidium Rapid EIA and the ColorPAC assay was 169 of 171 (98.8%). No cross-reactions were seen with other parasites or human cells.

Evaluation of nine immunoassay kits (enzyme immunoassay and direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvum in human fecal specimens.

It is well known that Giardia lamblia and Cryptosporidium parvum can cause severe symptoms in humans, particularly those who are immunologically compromised. Immunoassay procedures offer both increased sensitivity and specificity compared to conventional staining methods. These reagents are also helpful when screening large numbers of patients, particularly in an outbreak situation or when screening patients with minimal symptoms. The data obtained by using 9 diagnostic kits were compared: direct fluorescent-antibody assay (DFA) kits (TechLab Giardia/Crypto IF kit, TechLab Crypto IF kit, and Meridian Merifluor Cryptosporidium/Giardia) and enzyme immunoassay (EIA) kits (Alexon ProSpecT Giardia EZ Microplate Assay, Alexon ProSpecT Cryptosporidium Microplate Assay, Cambridge Giardia lamblia Antigen Microwell ELISA, Meridian Premier Giardia lamblia, Meridian Premier Cryptosporidium, TechLab Giardia CELISA, Trend Giardia lamblia EIA). The test with the Meridian Merifluor Cryptosporidium/Giardia kit was used as the reference method. In various combinations, 60 specimens positive for Giardia, 60 specimens positive for Cryptosporidium, 40 specimens positive for a Giardia-Cryptosporidium mix, and 50 negative fecal specimens were tested. Different species (nine protozoa, three coccidia, one microsporidium, five nematodes, three cestodes, and one trematode) were included in the negative specimens. The sensitivity of EIA for Giardia ranged from 94% (Alexon) to 99% (Trend and Cambridge); the specificity was 100% with all EIA kits tested. The sensitivity of EIA for Cryptosporidium ranged from 98% (Alexon) to 99% (Meridian Premier); specificities were 100%. All DFA results were in agreement, with 100% sensitivity and specificity; however, the TechLab reagents resulted in fluorescence intensity that was generally one level below that seen with the reagents used in the reference method. In addition to sensitivity and specificity, factors such as cost, simplicity, ease of interpretation of results (color, intensity of fluorescence), equipment, available personnel, and number of tests ordered are also important considerations prior to kit selection.

Evaluation of intestinal protozoan morphology in polyvinyl alcohol preservative: comparison of zinc sulfate- and mercuric chloride-based compounds for use in Schaudinn's fixative.

As a result of disposal problems related to the use of mercury compounds, many laboratories have considered switching from mercuric chloride-based Schaudinn's and polyvinyl alcohol (PVA) stool preservatives to other non-mercury-based preservatives. The primary use for PVA-preserved specimens is the permanent stained smear, the most important technique in the routine ova and parasite examination for the identification and confirmation of intestinal protozoa. A comparison of organism recovery and morphology of the intestinal protozoa was undertaken with PVA containing either a zinc sulfate base or the "gold standard" mercuric chloride base. Paired positive fecal specimens (106 from 64 patients) were collected and examined microscopically by the trichrome stain technique. There were 161 instances in which organism trophozoite and/or cyst stages were identified and 3 in which human cells were identified. Morphology, clarity of nuclear and cytoplasmic detail, overall color differences, and the ease or difficulty in detecting intestinal protozoa in fecal debris, as well as the number of patients with a missed diagnosis, were assessed from the permanent stained smear. Overall organism morphology of the intestinal protozoa preserved in zinc sulfate-PVA was not always equal in nuclear and cytoplasmic detail or range of color after permanent staining to that seen with mercuric chloride-PVA. However, the same organisms were usually identified in both specimens, with the exception of situations in which organism numbers were characterized as rare (no organisms per 10 oil immersion fields at x1,000 magnification but at least one organism in the smear) [9 of 161 (5.6%)] or the organism was missed because of poor morphologic detail [12 of 161 (7.5%)]. In only six of these cases [6 of 161 (3.7%)] did the results involve pathogens. The patient diagnosis was missed in four cases of amebiasis and two cases of giardiasis; in both situations the organism numbers were rare. There were no discrepant results with Dientamoeba fragilis. Overall agreement between the two PVA-based results was 87.0% (140 of 161); when the instances of rare organisms were disregarded, the overall agreement was 92.5% (149 of 161). On the basis of these findings, zinc-PVA is viable substitute for mercuric chloride-PVA used for trichrome permanent stained smears.

Sinus tract extension of a liver hydatid cyst and recovery of diagnostic hooklets in sputum.

A 67-year-old man, born in Turkey but living within the United States since 1975, presented with a four-month history of right lower chest pain. Chest x-ray revealed a right lower lobe infiltrate. Liver scan revealed multiple calcified cysts consistent with unilocular hydatid disease. The patient was taken to surgery for liver cysts removal. Although there was no specific evidence of lung cysts, it was recommended that sputum specimens be submitted for evidence of hydatid sand, i.e., hooklets and scolices. Hooklets were found, thus confirming the sinus tract connection between lung and liver. This case emphasizes the point that hooklets can be recovered in sputum and identified, even when there are few present. This approach also represents a noninvasive procedure that, along with serology, could be used as an alternative to biopsy technics under certain conditions.

Babesiosis: problems in diagnosis using autoanalyzers.

A 76-year-old white man previous diagnosed as having Waldenstrom's macroglobulinemia continued with persistent fevers and sweats for two and a half years. Recently, repeated automated differentials during 11 days of hospitalization failed to note any intracellular inclusions in the RBCs. Blood sent to the Microbiology Laboratory was noted to contain Babesia species. A review of the hematology slides revealed that Babesia species was present on all the slides the analyzer had screened. This failure to note infected RBCs may pose serious diagnostic problems.

Techniques for the recovery and identification of Cryptosporidium oocysts from stool specimens.

Due to increasing numbers of patients with documented infections with Cryptosporidium and other coccidia, it is important for the physician and clinical laboratory to be aware of the appropriate diagnostic techniques necessary for organism recovery and identification. Although Cryptosporidium is found in the gastrointestinal tract, tissue biopsies may be insufficient for organism recovery; the examination of stool specimens is a noninvasive procedure and will provide better overall opportunities for organism recovery. Human clinical specimens were examined from 45 patients with confirmed cryptosporidiosis or suspected of having the infection. Tissue biopsy sections, fecal wet preparations, and permanent stained smears were examined. Stool specimens were submitted in 10% Formalin, 2.5% potassium dichromate, and polyvinyl alcohol and were examined for oocysts by using 15 different methods: phase-contrast and light microscopy; Sheather's sugar flotation; Formalin concentration techniques; 10% potassium hydroxide; Giemsa; trichrome; periodic acid-Schiff; modified periodic acid-Schiff; silver methenamine; acridine orange; auramine-rhodamine; Kinyoun acid-fast; Ziehl-Neelsen carbolfuchsin; and a modified acid-fast procedure. Each technique or combination of techniques was assessed by organism quantitation, organism morphology, and ease of visual recognition. Based on these comparative studies, the modified Ziehl-Neelsen carbolfuchsin stain on 10% Formalin-preserved stool is recommended for the recovery and identification of Cryptosporidium.

Comparison of indirect fluorescent-antibody amoebic serology with counterimmunoelectrophoresis and indirect hemagglutination amoebic serologies.

Patients ranged from those with no prior diagnosis of or suspected exposure to Entamoeba histolytica to those with proven amoebic liver abscesses (extraintestinal disease). A comparison of serologies from patients with proven and suspected amoebiasis or possible past exposure revealed good correlation between the indirect fluorescent antibody (IFA) procedure and the other methods used, counterimmunoelectrophoresis and indirect hemagglutination. Titers from patients with proven extraintestinal amoebiasis were in the expected high range previously reported by other authors. Patients with clinical histories suggestive of exposure to E. histolytica but no proven disease had lower titers which indicated possible background exposure. The IFA procedure provides a rapid method of antibody detection; results obtained on an emergency basis provide essential information in making the diagnosis of amoebic abscess, pyogenic abscess, or tumor. The IFA procedure is rapid, reliable reproducible, and relatively inexpensive to perform, provided a good source of antigen is consistently available.