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 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.     

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.     

Comparison of conventional stool concentration and preserved-smear methods with Merifluor Cryptosporidium/Giardia Direct Immunofluorescence Assay and ProSpecT Giardia EZ Microplate Assay for detection of Giardia lamblia.

Giardiasis is the most common parasitic infection in the United States. Variation in the numbers of cysts and/or trophozoites that are present along with the need for a skilled microscopist offer challenges in diagnosis. We compared the sediment wet preparation and permanent stained smear results (concentration in formalin-ethyl acetate and preparation of a smear prepared from a polyvinyl alcohol-preserved specimen) from 512 consecutive specimens with the results obtained by using the Merifluor Cryptosporidium/Giardia Direct Immunofluorescence Assay (DFA; Meridian Diagnostics, Inc., Cincinnati, Ohio) and the ProSpecT Giardia EZ Microplate Assay (EIA; Alexon, Inc., Sunnyvale, Calif.). The Merifluor DFA detected 33 of 33 positive specimens, and the ProSpecT EIA detected 32 of 33 positive specimens. The diagnostic sensitivities of the Merifluor DFA and the ProSpecT EIA were 100 and 97%, respectively. The specificities of the assays were 99.8%. The Merifluor DFA and the ProSpecT EIA appear to be equally sensitive, and both are more sensitive than conventional microscopy.     

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 seven commercial antigen detection tests for Giardia and Cryptosporidium in stool samples

Stool samples from patients with abdominal symptoms were used to evaluate different copro-diagnostic assays for the detection of Giardia and Cryptosporidium. Results from microscopical examination following conventional stool concentration and direct fluorescent-antibody methods were compared with various commercially available immunochromatographic and enzyme immunoassays. Of 220 samples, 45 were positive for Giardia and 17 for Cryptosporidium. For Giardia, the sensitivities obtained by Ridascreen Giardia, Rida Quick Giardia, Rida Quick Combi and Giardia-Strip were 82%, 80%, 80% and 44%, respectively. For Cryptosporidium, the sensitivities obtained by Rida Quick Cryptosporidium, Ridascreen Cryptosporidium, Rida Quick Combi and Cryptosporidium-Strip were 88%, 82%, 82% and 75%, respectively. The specificity of all tests was > or = 98%. Other intestinal parasites were present in 68 samples, but cross-reactions with other protozoan or helminthic parasites were not observed. Overall, the copro-antigen assays were less time-consuming and easier to perform, but were less sensitive than conventional microscopical methods. Thus, these tests might be a useful addition to, but not a substitute for microscopical methods in the diagnosis of travel-associated giardiasis and cryptosporidiosis.     

Evaluation of Four Enzyme Immunoassays for the Detection of Giardia lamblia Antigen in Stool Specimens

 Four enzyme immunoassays for the detection of Giardia lamblia antigen in stool specimens were evaluated: the ProSpecT Giardia Microplate Assay (Alexon, USA), the Giardia CELISA (Cellabs, Australia), the DSl-Giardia-ELISA (DSL, Germany), and the Melotest Giardiasis Ag (Melotec, Spain). Microscopic examination and enzyme immunoassays were performed on 168 stool specimens collected from 168 patients suspected to have giardiasis. All assays were easy to perform. The ProSpecT Giardia assay had the highest sensitivity of the assays evaluated (91%), and its interpretation was the easiest. The sensitivity of the three other assays ranged from 63 to 81%. The ProSpecT Giardia assay can be useful to detect Giardia lamblia and may replace microscopic examination in areas of high endemicity.     

实时荧光PCR检测蓝氏贾第鞭毛虫和隐孢子虫方法的建立

目的建立贾第鞭毛虫和隐孢子虫的基因检测方法。方法根据GenBank中贾第鞭毛虫和隐孢子虫相对保守序列,设计引物及其相应的Taqman探针。通过对引物和探针浓度、Taq酶以及反应条件等优化筛选后,建立检测贾第鞭毛虫和隐孢子虫的荧光PCR方法。结果贾第鞭毛虫和隐孢子虫的检测结果为阳性,对其它DNA样本如日本血吸虫、刚地弓形虫、溶组织内阿米巴、旋毛虫和阴道毛滴虫的检测均为阴性。本法的敏感性高,可检测到10～102copies/μl的DNA浓度。结论建立的基因检测方法,对贾第鞭毛虫和隐孢子虫的检测具有高度的特异性和敏感性,可用于饮用水和临床样本等的快速检测。     

Commercial assay for detection of Giardia lamblia and Cryptosporidium parvum antigens in human fecal specimens by rapid solid-phase qualitative immunochromatography

The ImmunoCard STAT! Cryptosporidium/Giardia rapid assay (Meridian Bioscience, Inc.) is a solid-phase qualitative immunochromatographic assay that detects and distinguishes between Giardia lamblia and Cryptosporidium parvum in aqueous extracts of human fecal specimens (fresh, frozen, unfixed, or fixed in 5 or 10% formalin or sodium acetate-acetic acid-formalin). By using specific antibodies, antigens specific for these organisms are isolated and immobilized on a substrate. After the addition of appropriate reagents, a positive test is detected visually by the presence of a gray-black color bar (regardless of the intensity) next to the organism name printed on the test device. A control is included in the device. Steps include tube preparation (buffer, patient specimen, conjugates A and B), testing (addition of sample onto the test device), and visual reading (total time, 12 min). Test performance was evaluated with known positive and negative stool specimens (170 specimens positive for Giardia and 231 specimens negative for Giardia) (85 specimens positive for Cryptosporidium and 316 specimens negative for Cryptosporidium); they were tested with trichrome, iron-hematoxylin, or modified acid-fast stains or the Meridian Bioscience, Inc., Giardia/Cryptosporidium Merifluor combination reagent; specimens with discrepant results were retested by using the Merifluor combination reagent. On the basis of the results of the reference methods, the sensitivities, specificities, and positive and negative predictive values were as follows: for G. lamblia, 93.5, 100, 100, and 95.5%, respectively; for C. parvum, 98.8, 100, 100, and 99.7%, respectively. False-negative results for G. lamblia were obtained with specimens with low parasite numbers (n = 7) or specimens containing trophozoites only (n = 3); one specimen with a false-negative result contained numerous cysts. The one specimen false negative for C. parvum was confirmed to be positive by immunofluorescence. No cross-reactivity was seen with 10 different protozoa (152 challenges), nine different helminths (35 challenges), or human cells (4 challenges) found in fecal specimens. This rapid test system may be very beneficial in the absence of trained microscopists; however, for patients who remain symptomatic after a negative result, the ova and parasite examination and special stains for other coccidia and the microsporidia should always remain options.     

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.     

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.     

Multisite performance evaluation of an enzyme-linked immunosorbent assay for detection of Giardia, Cryptosporidium, and Entamoeba histolytica antigens in human stool

A novel fecal antigen detection assay for fresh and frozen human samples that detects but does not differentiate Giardia spp, Cryptosporidium spp, and Entamoeba histolytica, the Tri-Combo parasite screen, was compared to three established enzyme-linked immunosorbent assays (ELISAs) at three international sites. It exhibited 97.9% sensitivity and 97.0% specificity, with positive and negative predictive values of 93.4% and 99.1%, respectively. The Tri-Combo test proved a reliable means to limit the use of individual parasite ELISAs to positive samples.     

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.     

Immunochromatographic Strip-Based Detection of Entamoeba histolytica-E. dispar and Giardia lamblia Coproantigen

BIOSITE Triage was 68.3% sensitive and 100% specific for the detection of Entamoeba histolytica-E. dispar (n = 71) compared to Alexon-Trend's ProSpecT test (reference standard) using fresh-frozen stool. Neither test is able to distinguish E. histolytica from E. dispar. Triage was 83.3% sensitive and 100% specific compared to microscopy (formalin-ether concentrates and permanent stains) for the detection of Giardia lamblia.     

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 a Screening Test for Detection of Giardia and Cryptosporidium Parasites

The Giardia/Cryptosporidium Chek test (TechLab, Inc.), a screening test for Giardia and Cryptosporidium, was evaluated with 136 fecal samples. Using the results of the Giardia II test and Cryptosporidium II test as gold standards, it was 98.4% sensitive and 100% specific and had positive and negative predictive values of 98.7% and 99.3%.     

Prospective comparison of direct immunofluorescence and conventional staining methods for detection of Giardia and Cryptosporidium spp. in human fecal specimens.

In a prospective comparative study, 2,696 consecutive fresh stool specimens over the course of 1 year were examined for Giardia lamblia and Cryptosporidium parvum by using a direct immunofluorescent-monoclonal antibody stain (for unspun specimens) and conventional staining methods (chlorazol black E for Giardia cysts and modified Kinyoun acid-fast for Cryptosporidium oocysts). The direct immunofluorescent-monoclonal antibody method resulted in a significantly increased detection rate for both giardia (118 versus 79 specimens, 49.4%; P = 0.006) and cryptosporidia (39 versus 23 specimens, 69.6%; P = 0.055).     

Comparison of techniques for detecting antigens of Giardia lamblia and Cryptosporidium parvum in faeces.

AIM--To compare the use of commercial monoclonal antibody test systems--the Giardia CEL IF test and the Crypto CEL IF test--for the detection of Giardia lamblia and Cryptosporidium parvum antigens in faeces with conventional techniques. METHODS--Sensitivity and specificity were evaluated using preparations of cysts of G lamblia and purified oocysts of C parvum. Evaluation of 59 random faecal samples passing through the Department of Clinical Parasitology, Hospital for Tropical Diseases, London, was carried out for both organisms. RESULTS--The fluorescence staining techniques proved more sensitive than other tests routinely used for diagnosis.     

Performance of three enzyme immunoassays and two direct fluorescence assays for detection of Giardia lamblia in stool specimens preserved in ECOFIX

ECOFIX is a single-vial stool preservative that is both formalin- and mercury-free. We evaluated the abilities of three commercial Giardia lamblia-specific enzyme immunoassays (EIAs) (ProSpecT Giardia Microplate Assay [Alexon-Trend Inc.], Giardia Test [Techlab], and Premier Giardia lamblia [Meridian Diagnostics, Inc.]) and two commercial direct fluorescent-antibody (FA) assays for G. lamblia (Crypto/Giardia IF Test [Techlab] and Merifluor Cryptosporidium/Giardia [Meridian Diagnostics, Inc.]) to detect G. lamblia in 34 G. lamblia-positive and 44 G. lamblia-negative stool specimens (determined by traditional examination for ova and parasites) preserved in ECOFIX compared to their abilities to detect G. lamblia in the same specimens preserved in formalin as the "gold standard" for each assay. Of the 34 formalin-fixed positive specimens, the number detected by each assay was as follows:, Alexon EIA, 34; Meridian EIA, 27; Techlab EIA, 29; Meridian FA assay, 31; and Techlab FA assay, 28. Both FA tests and the Alexon EIA performed well with ECOFIX, but the other two EIAs detected fewer positive specimens (the difference was statistically significant with the Techlab EIA) when ECOFIX was the preservative. Use of G. lamblia cyst antigen from cultured organisms preserved in formalin and ECOFIX demonstrated that the Alexon EIA could detect smaller amounts of antigen in ECOFIX than the other two EIAs could and suggested that cyst antigen is more stable in formalin. We recommend that laboratories use an FA assay or the Alexon EIA if they plan to use ECOFIX as their stool preservative.     

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.