Field evaluation of a rota- and adenovirus immunochromatographic assay using stool samples from children with acute diarrhea in Ghana

We evaluated the Rida Quick rotavirus/adenovirus Combi rapid immunochromatographic test (ICT) under field conditions with Ghanaian children with acute diarrhea. Compared to PCR results, sensitivities and specificities were 75% and 95% for rotavirus and 22% and 84% for adenovirus. In resource-poor settings, ICTs may help to overcome difficulties in the diagnosis of rotavirus infection.     

Evaluation of an immunochromatographic dip strip test for simultaneous detection of Cryptosporidium spp, Giardia duodenalis, and Entamoeba histolytica antigens in human faecal samples

Immunochromatographic (IC) tests may play an important role in the future diagnosis of parasitic diseases because of their speed and simplicity of use. A recently developed test to detect Cryptosporidium spp, Giardia duodenalis and Entamoeba histolytica was evaluated. Microscopy and PCR were the "gold standard" reference techniques and the results of this IC test were compared with those obtained with ELISA and IC single test for the three parasites. One hundred sixty stool samples were assayed. Using microscopy, 22 samples were diagnosed as positive for Cryptosporidium spp., 31 for Giardia duodenalis, 41 for Entamoeba histolytica/dispar, and 68 had a negative diagnosis for the three parasites. Results of IC tests show sensitivities of 70-72% for Cryptosporidium, 90-97% for Giardia and 62.5% for Entamoeba histolytica. Specificities were of 93.6-94.9%, >99% and 96.1%, respectively. In all diagnoses, agreement with microscopy and PCR was over 90%, except in the triple test and microscopy in E. histolytica detection that was 76.3%, due to the inability of microscopy to differentiate E. histolytica from nonpathogenic species such as E. dispar or E. moshkovskii. The triple stool immunoassays provide adequate sensitivities and specificities for use in outbreak situations, for screening proposals and for massive assays in endemic areas where a large number of samples must be analysed or as complementary test for individual diagnosis.     

Evaluation of three commercial assays for detection of Giardia and Cryptosporidium organisms in fecal specimens

There is an increasing demand for diagnostic testing for Giardia intestinalis (G. lamblia) and Cryptosporidium parvum, with a priority being placed on obtaining diagnostic results in an efficient and timely manner. Several commercial companies have developed rapid diagnostic tests that are simple to perform and can be completed in less time than traditional methods for detecting Giardia and Cryptosporidium: We compared one of these rapid tests, the ImmunoCard STAT! (Meridian Bioscience, Inc.) lateral-flow immunoassay, with the MERIFLUOR direct fluorescent-antibody (DFA) test, the ProSpecT EZ microplate assay for Giardia and the ProSpecT microplate assay for Cryptosporidium, and modified Kinyoun's acid-fast stained smears for the detection of Cryptosporidium using 246 specimens. The MERIFLUOR DFA (Meridian Bioscience, Inc.) test detected the largest number of cases (32 Giardia and 37 Cryptosporidium) infections and was used to calculate the sensitivity and specificity of the other tests. For Giardia, the sensitivities of the ImmunoCard STAT! and the ProSpecT Giardia EZ microplate assay (Alexon-Trend, Inc.) were 81 and 91%, respectively. For detection of Cryptosporidium, the sensitivities of the ImmunoCard STAT!, the ProSpecT Cryptosporidium microplate assay (Alexon-Trend, Inc.), and modified Kinyoun's acid-fast stained smears were 68, 70, and 78%, respectively. Test specificities were equal to or greater than 99%. Specimens with very small numbers of organisms were not detected by the ImmunoCard STAT!, the ProSpecT microplate assay or modified Kinyoun's acid-fast stained smears.     

Performances of four fourth‐generation human immunodeficiency virus‐1 screening assays

Fourth‐generation human immunodeficiency virus‐1 (HIV‐1) screening assays have improved sensitivity, but vary in performance characteristics. The purpose of this study was to evaluate four different fourth‐generation HIV‐1 assays. These assays included the AxSYM HIV Ag/Ab Combo (Abbott diagnostics, Delkenheim, Germany), ARCHITECT HIV Ag/Ab Combo (Abbott), Elecsys 2010 HIV Combi (Roche Diagnostics GmbH, Mannheim, Germany), and Elecsys HIV Combi PT (Roche). A total of 1,306 samples that included 1,225 clinical samples and 81 samples consisting of seroconversion panels, an HIV‐1 p24 antigen sensitivity panel, and dilution series of HIV‐1 lysates and HIV‐1 antibodies were tested. All of the assays had sensitivities of 100% on clinical samples. The specificities of the AxSYM, ARCHITECT, Elecsys 2010 HIV Combi, and Elecsys HIV Combi PT were 99.6, 99.6, 99.0, and 99.5%, respectively. Of the 81 samples with different levels of HIV antigen or antibody and/or subtypes, Elecsys HIV Combi PT and ARCHITECT HIV Ag/Ab Combo showed better analytical sensitivities than the other two assays. In summary, the performance characteristics of AxSYM, ARCHITECT, and Elecsys HIV Combi PT were comparable and satisfactory for clinical samples. ARCHITECT HIV Ag/Ab Combo and Elecsys HIV Combi PT have the higher analytical sensitivities, and would be preferable for reducing the window period. J. Med. Virol. 84:1884–1888, 2012. © 2012 Wiley Periodicals, Inc.     

Evaluation of two commercial enzyme immunoassays for the detection of norovirus in faecal samples from hospitalised children with sporadic acute gastroenteritis

Two commercially available enzyme immunoassays (EIAs), IDEIA and Ridascreen, for norovirus antigen detection were evaluated with 117 faecal samples from hospitalised children with acute gastroenteritis. Eighteen of 39 samples positive by RT-PCR were characterised by sequence analysis, and 17 of these were related to norovirus genogroup II. When compared with RT-PCR, the sensitivity and specificity values were 76.9% and 85.9%, respectively, for the IDEIA assay, and 59.0% and 73.1%, respectively, for the Ridascreen assay. The sensitivity and specificity of both EIA tests require improvement, but they could both eventually be of use in the diagnosis of norovirus diarrhoea in clinical laboratories.     

Evaluation of multiplex tandem real-time PCR for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical stool samples

The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites.     

Evaluation of rapid antigen point-of-care tests for detection of Giardia and Cryptosporidium species in human fecal specimens

In Bangladesh, a new parasite rapid antigen test was investigated demonstrating accuracy and feasibility. For Giardia species, it had a sensitivity and specificity of 94% and 100%, respectively. For Cryptosporidium species, it had a sensitivity and specificity of 100% and 100%, respectively. These are higher than or equal to the sensitivities and specificities of other tests on the market.     

Evaluation of an enzyme-linked immunosorbent assay for detection of Cryptosporidium spp. in stool specimens.

We evaluated a commercially produced enzyme-linked immunosorbent assay (ELISA; LMD Laboratories, Inc.) for the detection of Cryptosporidium spp. in 296 stool specimens submitted to the Mayo Clinic parasitology laboratory for routine examination. The specimens examined were fresh (4 specimens), were stored frozen at -65 degrees C (49 specimens), or were preserved in 10% formalin (243 specimens). Results were compared with those obtained by indirect immunofluorescent antibody detection (Merifluor Cryptosporidium/Giardia; Meridian Diagnostics, Inc.). One hundred of the specimens were positive by indirect immunofluorescent antibody and ELISA, while 187 were negative by both methods; 91 of these negative stool samples contained 121 parasites of 17 different species. Eight ELISA false negatives and one false positive were observed. The ELISA sensitivity was 93%, specificity was 99%, and the positive predictive value was 99%. Storage of specimens preserved in 10% formalin or frozen fresh at -65 degrees C for up to 18 months did not appear to affect the results. There was no cross-reactivity with Giardia lamblia (54 negative specimens) or with the 16 other parasites present in the ELISA-negative stool samples. The ELISA is a fast, easy-to-read, and accurate method for the detection of Cryptosporidium spp. in stool specimens.     

Evaluation of Two Enzyme Immunoassays for Detection of Human Rotaviruses in Fecal Specimens

The two assays evaluated in this study (the Ridascreen rotavirus and the Pathfinder rotavirus) exhibited comparable sensitivities (100%) but highly divergent positive predictive values (93.74 and 57.7%, respectively) when compared on 393 specimens. This difference should be considered when using these tests on collectives with an unknown or low prevalence.     

Evaluation of a combination rapid immunoassay for detection of Giardia and Cryptosporidium antigens

A combination cassette format nonenzymatic rapid immunoassay for detection of Giardia and Cryptosporidium antigens was evaluated by using 556 patient stool specimens from three clinical laboratories. This assay (Genzyme Diagnostics Contrast Giardia/Cryptosporidium), which can be used with fresh or formalin-fixed specimens, had unadjusted sensitivities and specificities of 96.1 and 98.5% for Giardia and 100 and 98.7% for Cryptosporidium, respectively, in this study.     

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.     

Quantitation of Giardia cysts and Cryptosporidium oocysts in fecal samples by direct immunofluorescence assay.

The lack of quick, simple, and sensitive quantitative tests has impeded studies on infection patterns and treatment of Giardia spp. and Cryptosporidium spp. A quantitative direct immunofluorescence assay (FA) using a commercial FA kit was developed and evaluated. Recovery rates of the FA for Cryptosporidium oocysts in calf feces seeded with 1,000, 10,000, 100,000, and 1,000,000 oocysts per g were 14.8, 40.8, 84.2, and 78.2%, respectively. Interassay coefficients of variation were 10.6 to 47.1%. Recovery rates of the FA for Giardia cysts in feces seeded with 1,000, 10,000, and 100,000 cysts per g were 76.4, 96.9, and 89.6%, respectively. Interassay coefficients of variation were 7.4 to 22.1%. By comparison, recovery rates of Giardia cyst by sucrose gradient flotation were only 20.5, 51.2, and 42.9%, respectively. Counts of cysts-per-gram obtained by sucrose gradient flotation with samples from calves, lambs, and ewes were only 49.1 to 54.8% of those obtained by the FA. Zinc sulfate flotation detected only 36.4% of infections when there were < or = 1,000 cysts per g. The quantitative FA offers a useful technique for epidemiological and control studies of these two parasites.     

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 a new monoclonal antibody combination reagent for direct fluorescence detection of Giardia cysts and Cryptosporidium oocysts in human fecal specimens.

Giardia lamblia and Cryptosporidium parvum can cause severe symptoms in humans, particularly in the immunologically compromised. Monoclonal antibody reagents offer increased sensitivity and an excellent alternative to conventional staining methods. These reagents are helpful when screening large numbers of patients or those with minimal symptoms. Problems of false-positive and false-negative results with routine staining methods for stool parasites can be eliminated with monoclonal antibody reagents. Known positive formalinized specimens [Giardia sp. (n = 60), Cryptosporidium sp. (n = 55), and mixed Giardia-Cryptosporidium spp. (n = 10)] and negative formalinized specimens (n = 105), of which 46 contained other yeast or human cells or protozoa), were tested by the MERIFLUOR Cryptosporidium-Giardia direct immunofluorescence detection procedure. The MERIFLUOR reagent exhibited +/- to 4+ (majority, 2+ to 3+) on all Giardia cysts and 2+ to 4+ (majority, 3+ to 4+) on all Cryptosporidium oocysts. The cysts were generally oval (11 to 15 microns), while the oocysts were round (4 to 6 microns); both showed apple-green fluorescence against a background free of nonspecific fluorescence. All specimens positive for Giardia sp. and/or Cryptosporidium sp. showed fluorescence, and all specimens negative for the two organisms showed no fluorescence. There were eight specimens previously negative by the ova and parasite examination which were positive by the direct fluorescence method; four contained Giardia sp., and four contained Cryptosporidium sp. These positive results were confirmed after the examination of additional trichrome and modified acid-fast smears. The MERIFLUOR reagent was very easy to use, and even with a lower fluorescence intensity for Giardia sp. cysts, no false-negative or false-positive results among the specimens tested for either organism were found.     

Intestinal parasitosis: our series from 1992 to 1995

From 1992 to 1995 in our Department 2236 researches of intestinal parasites (2194 on stool specimens, 34 on scotch tests, 8 on enterotests) were carried out on 1200 patients (703 HIV-Ab negative and 497 HIV-Ab positive). On the whole 387 samples (17.34%) of 203 subjects (16.92%) were found parasitized; 92 (13.08%) patients of them were HIV-Ab negative and 111 (22.33%) were HIV-Ab positive. We found more frequently Blastocystis hominis and Giardia intestinalis among HIV-Ab negative subjects and Cryptosporidium sp. and Blastocystis hominis among HIV-Ab positive patients. Isospora belli was found exclusively among HIV-Ab positive people, Cryptosporidium sp. in 54 HIV-Ab positive subjects and 3 HIV-Ab negative children. Strongyloides stercoralis was found only in HIV-Ab positive non-European people.     

Zoonotic Parasites of Bobcats around Human Landscapes

We analyzed Lynx rufus fecal parasites from California and Colorado, hypothesizing that bobcats shed zoonotic parasites around human landscapes. Giardia duodenalis, Cryptosporidium, Ancylostoma, Uncinaria, and Toxocara cati were shed. Toxoplasma gondii serology demonstrated exposure. Giardia and Cryptosporidium shedding increased near large human populations. Genotyped Giardia may indicate indirect transmission with humans.     

Parasitological diagnosis combining an internally controlled real-time PCR assay for the detection of four protozoa in stool samples with a testing algorithm for microscopy

Molecular detection of gastrointestinal protozoa is more sensitive and more specific than microscopy but, to date, has not routinely replaced time-consuming microscopic analysis. Two internally controlled real-time PCR assays for the combined detection of Entamoeba histolytica , Giardia lamblia , Cryptosporidium spp. and Dientamoeba fragilis in single faecal samples were compared with Triple Faeces Test (TFT) microscopy results from 397 patient samples. Additionally, an algorithm for complete parasitological diagnosis was created. Real-time PCR revealed 152 (38.3%) positive cases, 18 of which were double infections: one (0.3%) sample was positive for E. histolytica , 44 (11.1%) samples were positive for G. lamblia , 122 (30.7%) samples were positive for D. fragilis , and three (0.8%) samples were positive for Cryptosporidium . TFT microscopy yielded 96 (24.2%) positive cases, including five double infections: one sample was positive for E. histolytica / Entamoeba dispar , 29 (7.3%) samples were positive for G. lamblia , 69 (17.4%) samples were positive for D. fragilis , and two (0.5%) samples were positive for Cryptosporidium hominis / Cryptosporidium parvum . Retrospective analysis of the clinical patient information of 2887 TFT sets showed that eosinophilia, elevated IgE levels, adoption and travelling to (sub)tropical areas are predisposing factors for infection with non-protozoal gastrointestinal parasites. The proposed diagnostic algorithm includes application of real-time PCR to all samples, with the addition of microscopy on an unpreserved faecal sample in cases of a predisposing factor, or a repeat request for parasitological examination. Application of real-time PCR improved the diagnostic yield by 18%. A single stool sample is sufficient for complete parasitological diagnosis when an algorithm based on clinical information is applied.     

Rapid detection of giardia antigen in stool with the use of enzyme immunoassays

Enzyme-linked immunosorbent assays (ELISAs) using sera from rabbits and goats immunized with Giardia trophozoites were compared for detection of Giardia antigen in 300 stool specimens, 80 of which had positive results for Giardia by microscopic examination. The diagnostic accuracy of the two assays was similar, with sensitivities of 92% and 87% and specificities of 87% and 91% with the use of rabbit and goat antisera, respectively. Both ELISAs detected Giardia antigen in stool samples from asymptomatic as well as symptomatic excretors and from treated patients after organisms were no longer visualized by microscopic examination. The specificity of the assays was confirmed by consistently negative results on stool specimens from 10 neonates and 27 patients with enteric parasitic infections other than Giardia. Negative results also occurred when stool specimens containing 21 bacterial enteropathogens were tested. Potential confounding variables including multiple freezing and thawing episodes, prolonged storage, and stool filtration did not affect test results from clinical specimens. Antidiarrheal compounds did not interfere with assay results. Preservation of specimens in formalin did interfere with the assay, even if formalinized stool specimens were dialyzed before testing. For diagnosis of giardiasis, the ELISA is a sensitive and specific test that is not influenced by many environmental factors or by other enteropathogens. This test provides a practical and reliable method for evaluating large numbers of specimens in a variety of clinical and epidemiologic settings.     

Diagnosis of Norovirus outbreaks by commercial ELISA or RT-PCR

The IDEIA Norwalk-like virus (Dakocytomation Ltd., Ely, United Kingdom) and the Ridascreen Norwalk-like virus enzyme immunoassay (R-Biopharm AG, Darmstadt, Germany), were evaluated for the diagnosis of outbreaks of acute gastroenteritis. A panel of 158 fecal samples from 23 outbreaks, including confirmed rotavirus and astrovirus outbreaks, was used to determine the sensitivity and specificity of both ELISA kits relative to an RT-PCR protocol that was followed by Southern blot hybridization. Another panel consisted of 6 different genogroup I strains, 12 genogroup II strains and 1 genogroup IV strain and was used to determine the scope of the tests. Compared to the RT-PCR, sensitivities of 38% and 36% and specificities of 96% and 88% were found for the Dako kit and the Ridascreen kit, respectively. Two genogroup I strains, and one genogroup II strain were not detected by the Dako kit, while five genogroup I and five genogroup II strains were not detected by the Ridascreen kit. The sensitivity of both ELISA kits, and the scope of the Ridascreen are considered disappointing. However, the ELISA kits can be useful for a preliminary screening, provided that ELISA negative outbreaks will be re-tested by RT-PCR methods.