Evaluation of ColorPAC Giardia/Cryptosporidium Rapid Assay and ProSpecT Giardia/Cryptosporidium Microplate Assay for Detection of Giardia and Cryptosporidium in Fecal Specimens

Detection of Giardia and Cryptosporidium in clinical stool specimens using the ColorPAC and ProSpecT enzyme immunoassays revealed 98.7% agreement for Giardia detection and 98.1% agreement for Cryptosporidium detection. Sensitivities were uniformly 100%. The specificities of the ColorPAC immunoassay for Giardia and Cryptosporidium detection were 100 and 99.5%, respectively, and those for the ProSpecT assay were 98.4 and 98.6%, respectively. The false-positive reactions with the ProSpecT assay occurred with specimens that were grossly bloody.     

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.     

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 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.     

Use of an enzyme immunoassay does not eliminate the need to analyze multiple stool specimens for sensitive detection of Giardia lamblia

The relative sensitivities of a commercially available enzyme immunoassay (EIA) (ProSpecT Giardia; Alexon-Trend Inc., Ramsey, Minn.) and conventional ovum-and-parasite (O&P) examination for the detection of Giardia lamblia in preserved stool specimens were determined. Paired stool samples collected independently within a 7-day period from 103 patients were analyzed by both methods. A total of 54 specimens from 30 patients (18 asymptomatically infected with G. lamblia and 12 with symptoms consistent with intestinal giardiasis) were determined to be positive for G. lamblia, of which 48 (88.9%) were positive by microscopy and 52 (96.3%) were positive by EIA. Both specimens submitted were positive for G. lamblia by O&P examination for 66.7% (20 of 30) of the positive patients; for 26.7% (8 of 30) a single specimen was positive by O&P examination, and for 6.7% (2 of 30) of those determined to be infected with G. lamblia, both samples were negative by microscopy. The sensitivity of conventional O&P examination was somewhat higher in symptomatically infected individuals, with 75% (9 of 12) of patients in this category having G. lamblia detected in both samples, compared with 61% (11 of 18) of asymptomatic patients. A total of 24 positive patients (80%) had G. lamblia antigen detected by EIA in both submitted samples, 4 positive patients (13.3%) had one specimen positive by EIA, and the EIA was negative in both specimens from 2 infected individuals (6.5%), the sensitivity of EIA was substantially equivalent in asymptomatic and symptomatic individuals (77 versus 83% of patients with positive results on both specimens). Although the sensitivity of EIA for the detection of G. lamblia on a single stool specimen was somewhat higher than that of conventional O&P examination in symptomatic patients (83 versus 75%), in asymptomatic patients (77 versus 61%), and overall (80 versus 67%), examination of two specimens by either EIA or microscopy was necessary to achieve a diagnostic sensitivity of greater than 90%.     

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.     

Evaluation of rapid commercial enzyme immunoassay for detection of Giardia lamblia in formalin-preserved stool specimens.

Two hundred twenty-three formalin-preserved stool specimens were evaluated by using ProSpecT Giardia Rapid Assay (membrane bound) (Alexon, Inc., Sunnyvale, Calif.). Enzyme immunoassay (EIA) results were compared with those by conventional microscopic examination. Two hundred four specimens were negative by both methods, and 13 (6.3%) were positive. Five specimens were negative by initial microscopic exam and positive by EIA; three of these specimens were found to be positive upon extensive microscopic reexamination. The remaining two specimens were from patients who previously tested positive and who had recurrent symptoms of or responded to therapy for giardia. Therefore, we consider both cases to be true positives. One specimen exhibited a single cyst by microscopic exam and was negative by EIA Resolved results yielded a relative sensitivity of 95%, a relative specificity of 100%, a positive predictive value of 99.6%, and a negative predictive value of 100%, compared with a sensitivity of 74% for conventional microscopy.     

ImmunoCard STAT! cartridge antigen detection assay compared to microplate enzyme immunoassay and modified Kinyoun's acid-fast staining technique for detection of Cryptosporidium in fecal specimens

Cryptosporidium species infect humans and a wide range of animals worldwide; outbreaks of cryptosporidiosis have been reported in several countries. Routine diagnostic methods may be insufficient to demonstrate the presence of these organisms. The study assessed the diagnostic accuracy of the antigen detection immuno-cartridge test, ImmunoCard STAT! (Meridian Bioscience Inc., Cincinnati, OH, USA), compared to the combined gold standard: modified Kinyoun's acid-fast technique confirmed with the microplate enzyme immunoassay (EIA) for the detection of Cryptosporidium in fecal specimens. Three hundred fifteen formalin-fixed stool specimens were submitted for testing. The Kinyoun's acid-fast-stained smear revealed 24 positive samples for Cryptosporidium (of which 23 specimens were confirmed by the EIA) and 291 negative samples (of which 289 were negative by EIA). Agreement between the three used tests was shown in 22 positive and 288 negative samples for Cryptosporidium. Kappa score of agreement between the immuno-cartridge test and EIA was 0.957, p?=?0.000. The sensitivity of the immuno-cartridge test was 96% (95% confidence interval (CI), 87% to 104%) and the total accuracy of the test was 97% (95% CI, 93-103). The ImmunoCard STAT! Cryptosporidium cartridge assay is easy to use and does not require specialized training or equipment and is useful in routine diagnosis and screening for Cryptosporidium especially where rapid, point of care testing is needed or where other reliable tests are unfeasible with a performance comparable to the EIA and acid-fast technique.     

Evaluation of the Alexon-trend ProSpecT Campylobacter microplate assay

We evaluated stool specimens known to contain or be free of Campylobacter by traditional culture, using the ProSpecT Campylobacter microplate assay (Alexon-Trend, Ramsey, Minn.). This rapid enzyme immunoassay for the detection of Campylobacter-specific antigens demonstrated 96% sensitivity and 99% specificity and is an acceptable alternative method of Campylobacter detection.     

Rapid detection of Campylobacter jejuni in stool specimens by an enzyme immunoassay and surveillance for Campylobacter upsaliensis in the greater Salt Lake City area

The Alexon-Trend, Inc. (Ramsey, Minn.), ProSpecT Campylobacter microplate assay was compared with culture on a Campy-CVA plate (Remel, Lenexa, Kans.) and blood-free campylobacter agar with cefoperazone (20 microg/ml), amphotericin B (10 microg/ml), and teicoplanin (4 microg/ml) (CAT medium; Oxoid Limited, Hampshire, England) with 631 patient stool samples. The CAT medium was used to isolate Campylobacter upsaliensis. The enzyme immunoassay (EIA) had a sensitivity and a specificity of 89 and 99%, respectively, and the positive and negative predictive values were 80 and 99%, respectively. Even though we extensively looked for C. upsaliensis in stool samples from patients from the greater Salt Lake City area, we did not isolate this species during the study period. The overall excellent specificity of the EIA allows rapid detection and treatment of positive patients; however, a negative result should be confirmed by culture when clinical suspicion is high.     

Physician use of parasite tests in the United States from 1997 to 2006 and in a Utah Cryptosporidium outbreak in 2007

Parasitic infection is uncommon in the United States, but surveys suggest that physicians test when the presence of parasites is unlikely and fail to order appropriate testing when suspicion is high. Numerous studies confirm that immunoassays are more sensitive for Giardia and Cryptosporidium detection, but our experience was that physicians preferentially used ovum and parasite examination (O&P). We conducted a retrospective study of fecal parasite testing at a referral laboratory nationally (1997 to 2006) and during a Cryptosporidium outbreak (Utah, 2007) to correlate physician use of O&P and enzyme immunoassays (EIAs) with the yield of parasites detected. Nationally, of 170,671 episodes, 76.0% (n = 129,732) included O&P, 27.9% (n = 47,666) included Giardia EIA, and 5.7% (n = 9,754) included Cryptosporidium EIA. Most pathogens were Giardia or Cryptosporidium. More episodes were positive when EIA was performed (n = 1,860/54,483 [3.4%]) than when O&P only was performed (n = 1,667/116,188 [1.4%]; P < 0.001), and EIA was more sensitive than O&P. However, more O&P results were positive among patients with both O&P and EIA performed (2.5%) than among those with O&P only performed (1.4%; P < 0.001), suggesting that patients tested by O&P only may have been at lower risk. During the first 10 weeks of the outbreak, physicians also preferentially used O&P over EIA, but no Cryptosporidium cases were detected by O&P. We conclude that clinicians frequently use O&P testing when test performance and epidemiology support the use of immunoassays or no testing. We recommend that stool O&P be limited to patients with negative immunoassay results and persistent symptoms or individuals at increased risk for non-Giardia, non-Cryptosporidium infection. An evidence-based algorithm for the evaluation of patients with suspected intestinal parasitic infection is proposed.     

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 performance and potential clinical impact of ProSpecT Shiga toxin Escherichia coli microplate assay for detection of Shiga Toxin-producing E. coli in stool samples

Shiga toxin-producing Escherichia coli bacteria (STEC) are emerging pathogens capable of producing sporadic and epidemic diarrhea, hemorrhagic colitis, and potentially life-threatening hemolytic-uremic syndrome. Although the presence of E. coli O157 can be readily detected in stool by sorbitol-MacConkey agar culture (SMAC), STEC non-O157 serotypes cannot. In contrast to culture, testing for the presence of Shiga toxins 1 and 2 in stool detects both O157 and non-O157 STEC serotypes capable of causing disease. Over two consecutive summers, we evaluated the performance of the ProSpecT Shiga toxin E. coli Microplate assay (Alexon-Trend, Ramsey, Minn.), an enzyme immunoassay for the detection of Shiga toxins 1 and 2, on all stools submitted for culture of enteric pathogens, and the potential clinical impact of Shiga toxin detection. Twenty-nine stool specimens were STEC positive by ProSpecT assay. Twenty-seven of 29 STEC-positive isolates were confirmed by SMAC and serotyping or by a second enzyme immunoassay and PCR (positive predictive value, 93%). Thirteen of 27 confirmed Shiga toxin-producing strains were serotype O157. The remaining 14 strains represented 8 other serotypes. The ProSpecT assay was 100% sensitive and specific for detection of E. coli O157 in stool (7 of 7) compared to SMAC. In addition, the ProSpecT assay detected twice as many STEC as SMAC. Fifty-two percent of confirmed STEC-positive stools were nonbloody. Thus, in our population, screening strategies that test only visibly bloody stools for STEC would miss a majority of cases. Eleven (41%) STEC-positive patients were hospitalized, and eight (30%) developed severe disease (two developed hemolytic-uremic syndrome, and six developed hemorrhagic colitis). Prior to detection of STEC infection, seven (26%) and eight patients (30%) underwent unnecessary diagnostic procedures or received potentially deleterious empirical treatment, respectively. We propose that establishing a specific diagnosis of STEC may have prevented these potentially harmful interventions. We conclude that the ProSpecT assay is sensitive and specific for the detection of Shiga toxins 1 and 2 in stool and has potentially significant clinical impact for the individual patient and public health. Shiga toxin assays should be considered for routine use in settings where prevalence of STEC disease warrants testing.     

Evaluation of the ProSpecT Microplate Assay for detection of Campylobacter: a routine laboratory perspective

Objectives  To evaluate the use of the new enzyme-linked immunosorbent assay, the ProSpecT Campylobacter Microplate Assay (Alexon-Trend, Minneapolis, MN, USA), which allows 2-h detection of both Campylobacter jejuni and Campylobacter coli antigen directly in stool specimens.
Methods  Over 4 months, all stool samples preserved in Cary–Blair medium, or fresh specimens, from non-hospitalized children and HIV-infected patients (adults and children), submitted to our laboratory were evaluated with the ProSpecT Campylobacter Microplate Assay. Results were compared with those obtained by routine culture methods using both a specific medium and a filtration method for the recovery of Campylobacter spp.
Results  Of the 1205 stool specimens cultured, 101 were found to be positive for either C. jejuni or C. coli , giving an overall recovery rate of 8.38%. Ninety samples were positive by both culture and ProSpecT Campylobacter Microplate Assay, and 11 were positive by culture only, giving a sensitivity of 89.1%. In addition, of 1104 samples negative by culture, 25 were initially positive by ProSpecT Campylobacter Microplate Assay. We found no cross-reaction with other bacterial enteropathogens isolated from stool specimens. These results thus confirm a high specificity (97.7%) for both C. jejuni and C. coli. The positive and negative predictive values found were 78.3% and 99%, respectively. There was no statistically significant difference in sensitivity and specificity if the stool was fresh or preserved with Cary–Blair medium.
Conclusion  These data suggest that the ProSpecT Campylobacter Microplate Assay is a rapid and easy-to-use test for the detection of both C. jejuni and C. coli in stool specimens. It could be used for patients for whom early antibiotic therapy is needed or for epidemiologic studies.     

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.     

Stool diagnosis of giardiasis using a commercially available enzyme immunoassay to detect Giardia-specific antigen 65 (GSA 65).

A commercially available enzyme immunoassay for the diagnosis of giardiasis was evaluated in a clinical trial. The ProSpecT/Giardia diagnostic test (Alexon, Inc., Mountain View, Calif.) was compared with the standard ova and parasite (O&P) microscopic examination. Additionally, several widely used stool fixatives and a commonly used transport medium were assessed for compatibility with the immunoassay. A total of 325 stool specimens were collected and used to evaluate assay performance. Of those, 93 specimens were collected from symptomatic Giardia O&P-positive patients and 232 specimens were randomly collected from patients as part of a routine health screening procedure. All 93 Giardia O&P-positive stool specimens were strongly positive by visual and spectrophotometric examination using the immunoassay. Of the 232 randomly collected specimens, 16 were positive by O&P examination and immunoassay, 6 were negative by O&P examination but positive by immunoassay, and 1 was positive by O&P examination and negative by immunoassay. There was substantial supportive evidence that indicated that the six immunoassay-positive, O&P-negative specimens were true-positives. When these six specimens were accepted as true-positives, the immunoassay detected almost 30% more cases of Giardia infection than did O&P examination. Its sensitivity and specificity were 96 and 100%, respectively, while the sensitivity and specificity of O&P examination were 74 and 100%, respectively. The immunoassay also performed well on specimens treated with 10% neutral Formalin, sodium acetate-Formalin fixative, and Cary-Blair transport medium. However, the test was not compatible with polyvinyl alcohol-treated specimens. Overall, the ProSpecT/Giardia test was a sensitive, specific immunoassay which was easy to run and interpret. It offers a simple solution to traditional difficulties encountered in diagnosing Giardia infection.     

Cryptosporidiosis: comparison of three diagnostic methods and effects of storage temperature on detectability of cryptosporidia in cattle faeces

Three diagnostic methods (a modified Ziehl-Neelsen staining technique (MZN), a negative staining with carbol fuchsine (CF) and a commercial enzyme immunoassay (EIA) kit, ProSpecT? Cryptosporidium Microplate Assay (Remel, Lenexa, KS, USA)) for detection of Cryptosporidium oocysts in cattle faeces were compared regarding sensitivity and suitability under routine laboratory conditions, with particular emphasis on sample storage. In the 103 faecal samples examined, cryptosporidia infections were detected significantly more often by EIA (p<0.05; n=76) than by MZN (n=65) if ten random fields were evaluated microscopically, but not if the whole coverslip was scanned. In contrast, sensitivities of EIA and CF (n=69) did not differ significantly. Results were obtained very rapidly by CF. However, the hands-on time of CF is comparable to EIA, while MZN is more time consuming. EIA is more expensive than CF and MZN but easy to perform and to evaluate and does not need considerably experienced staff in contrast to CF and MZN. Moreover, 45 faecal samples stored for up to 27 days at different temperatures (+6°C, +16°C, +30°C, +40°C) were examined. The sensitivity of microscopic detection of oocysts in stained smears (CF, MZN) decreased in a temperature and time-dependent manner, while EIA results were not influenced by sample storage at any temperature.     

Prospective evaluation of a dot-blot enzyme immunoassay (Directigen RSV) for the antigenic detection of respiratory syncytial virus from nasopharyngeal aspirates of paediatric patients

This study investigated the efficacy of a commercial enzyme immunoassay (Directigen RSV, ColorPAC) in comparison with the shell vial culture method (using Hep-2 cells) for the detection of respiratory syncytial virus (RSV) in nasopharyngeal aspirates from children with bronchiolitis. During the period 1995-2002, 4950 samples were examined. RSV was detected in 1660 (33.5%) samples, with a sensitivity of 80.9%, a specificity of 97.5%, a positive predictive value of 93.8%, a negative predictive value of 91.6%, and a testing efficiency value of 92.2% compared with shell vial culture. In 83 (5%) samples, the ColorPAC was positive and the shell vial assay was negative. Of these, 71 (85.6%) were false-negative by cell culture. The true false-positive results obtained by ColorPAC represented only 0.7% of all RSV-positive samples. In general, no statistically significant differences were detected between the different months and epidemic periods studied. Compared with ColorPAC, the shell vial culture method displayed a sensitivity of 95.8% and a specificity of 100%. Overall, the ColorPAC assay was an acceptable, simple and rapid method for the antigenic detection of RSV in paediatric respiratory samples.     

Evaluation of an enzyme immunoassay kit for detecting cryptosporidium in faeces and environmental samples.

AIMS: To evaluate a commercially available enzyme immunoassay based on a monoclonal antibody to a genus specific Cryptosporidium (IDEIA Cryptosporidium; Dako) antigen for detecting Cryptosporidium oocysts in faecal and environmental samples. METHODS: 435 human faecal samples and post-filtration deposits from 10 reservoir samples, and from six tap water samples seeded with Cryptosporidium oocysts, were examined by EIA according to the manufacturer's instructions, and by microscopic examination of phenolauramine stained smears. Samples giving discrepant results were examined by specific immunofluorescence, before and after concentration of oocysts. RESULTS: Sixteen (3.6%) faecal samples were positive by both microscopy and EIA; five (1.1%) were positive by microscopy of auramine-phenol stained smears (but were not confirmed by specific immunofluorescence) and negative by EIA; one (0.2%) was positive by EIA alone, but confirmed by specific immunofluorescence; and 362 (83.2%) were negative by both microscopy and EIA. Compared with immunofluorescence positive faecal samples, the sensitivity of conventional microscopy and EIA were 94% and 100%, and specificity 76.4% and 100%, respectively. Fifty one (11.7%) were not examined by microscopy due to detection of other pathogens in a previous sample from that patient, but were found to be negative by EIA. Ten reservoir water samples (not suspected of being linked to cases of cryptosporidiosis) were negative by both microscopy and EIA. Of six samples of tap water seeded with varying concentrations of Cryptosporidium oocysts, two (10(2) and 10(3) oocysts/l) were positive by both microscopy and EIA, two (10 and 1/l) by EIA alone, and two (0.1/l and unseeded water) were negative by both microscopy and EIA. CONCLUSIONS: The kit is simple and rapid to use and offers a less subjective method than microscopy for detecting Cryptosporidium in faecal samples submitted to a busy diagnostic laboratory.