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.     

Monoclonal antibody-based enzyme immunoassay for Giardia lamblia antigen in human stool

A visually readable monoclonal antibody-based antigen-capture enzyme immunoassay for the detection of Giardia lamblia antigen in human stool specimens was developed and found to be 97% (30 of 31 stool specimens) sensitive for formalinized stools and 82% (49 of 60 stool specimens) sensitive for unfixed stool specimens by visual reading. The storage of specimens in 10% Formalin resulted in increased absorbance in 20 of 26 G. lamblia-positive specimens tested as both formalinized and unfixed specimens; the increase averaged 1,336%. The assay was specific for antigens of this organism and for antigens derived from the cyst, as opposed to the trophozoite, stage. The assay could detect the antigens of five cysts per well, but could not detect antigen in in vitro-cultured trophozoites. A mouse monoclonal antibody of the immunoglobulin G1 (IgG1) subclass, which was prepared against cysts of G. lamblia, was used as the solid-phase capture antibody. The antibody was reactive with the cyst wall, as determined by immunofluorescence. Polyclonal rabbit anti-cyst IgG was used as the secondary antibody, and peroxidase-labeled goat anti-rabbit IgG was used as the tertiary antibody in the assay format. Maximal capture of antigen from stool specimens occurred by 30 min. Optimal dilution of specimens was in the range of 1:60 to 1:600. Preliminary characterization of affinity-purified antigen recognized by the monoclonal antibody showed that it is heat stable (100 degrees C, 12 min) and resistant to sodium periodate treatment and that it may exist in multiple molecular weights from 45,000 to 110,000.     

Comparison of microscopy, real-time PCR and a rapid immunoassay for the detection of Giardia lamblia in human stool specimens

Giardia lamblia is one of the most common intestinal parasites worldwide, with microscopy being the diagnostic reference standard for use with human stools. However, microscopy is time-consuming, labour-intensive and lacks sensitivity when single stools are examined. In the present study, microscopy, real-time PCR and a rapid immunoassay were compared for the detection of G. lamblia in human stools. All three methods were highly sensitive, with values of 99%, 100% and 98%, respectively. Specificity and positive and negative predictive values were >or=97%, except when using real-time PCR, for which the specificity and positive predictive value were 92% and 93%, respectively. The lower specificity of real-time PCR was associated mostly with failure to detect specimens regarded as true positives for G. lamblia DNA, although cross-contamination was suspected in a minority of cases because of the large amount of G. lamblia DNA present in most positive specimens. It was concluded that microscopy should remain the primary diagnostic tool for identifying G. lamblia in human stools, mainly because of its ability to simultaneously detect other gastrointestinal parasites. However, the simple and rapid immunoassay is a valuable tool to decrease turn-around time. Real-time PCR provides additional sensitivity, although there is a risk of cross-contamination. Based on this observation, and the need for other real-time assays to be developed to detect other intestinal parasites, real-time PCR is currently useful only as an additional test supplementary to microscopy.     

Real-time PCR assay for detection and genotype differentiation of Giardia lamblia in stool specimens

Real-time PCR, using dual-labeled fluorescent probes targeting the beta-giardin gene, was used to detect Giardia lamblia in human stool specimens and to discriminate between isolates from the two major genetic assemblages of G. lamblia infective to humans, assemblages A and B.     

Enzyme immunoassay for detection of Giardia lamblia cyst antigens in formalin-fixed and unfixed human stool.

An antigen-capture enzyme-linked immunosorbent assay employing rabbit and mouse antisera to Giardia lamblia cyst antigens was developed for the diagnosis of Giardia infection through detection of G. lamblia-specific stool antigens in cell-free aqueous eluates of human stool. This is the first report of the use of anti-cyst antibodies in an enzyme immunoassay for G. lamblia. The assay gave a positive result with 54 of 59 stools from patients with symptomatic, clinically diagnosed giardiasis, giving the test a sensitivity of 91.5%. A negative reading was obtained with all of 25 stools from G. lamblia-negative control patients. The assay could detect as few as 20 sonicated cysts added to control stool eluate. The assay was more sensitive to cyst-derived antigens than to trophozoite-derived antigens. With two exceptions, the assay gave a negative result with stools from patients infected with Entamoeba histolytica (seven), Cryptosporidium sp. (four), or Blastocystis hominis (seven) and thus appears to be specific for G. lamblia antigens. Storage of stool eluates for more than 6 months at 4 degrees C as unpreserved aqueous eluates or as formalinized eluates did not affect the ability of the assay to detect the giardial antigens. The enzyme-linked immunosorbent assay proved useful for monitoring the levels of G. lamblia-specific stool antigens in the stool of patients undergoing antigiardial chemotherapy.     

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.     

Use of pooled formalin-preserved fecal specimens to detect Giardia lamblia.

Three formalin-preserved fecal specimens from the same child attending a child-care center were pooled and compared with the three separate individual specimens by a single microscopic examination of concentration sediment for Giardia lamblia. The sensitivity of the pooled system was 100% when two or more individual specimens were positive and 88% when only one individual specimen was positive. The organism density in a single specimen was not a factor of whether the pool of specimens was positive or negative. Nearly half of the pools that contained positive specimens had only one of three specimens with positive results, reinforcing the need for multiple stool examinations when diagnosing G. lamblia infections.     

Evaluation of an antigencapture enzyme immunoassay for detection ofEntamoeba histolytica in stool samples

In order to identify the prevalence ofEntamoeba histolytica in tourists with diarrhoea returning from countries of the developing world, sensitivity and specificity of a commercially available enzyme immunoassay (EIA) kit for the detection ofEntamoeba histolytica coproantigen in stool were evaluated. Five hundred seventy-seven specimens from 469 patients were examined by microscopy and EIA. Sixty-two specimens from 49 patients were considered positive forEntamoeba histolytica. Compared with microscopic examination of stool samples, the EIA was found to be slightly more sensitive (90.3% vs. 87.1%) and was 97.7% specific forEntamoeba histolytica.     

Evaluation of new rapid commercial enzyme immunoassay for detection of Cryptosporidium oocysts in untreated stool specimens.

Sixty-six stool specimens were evaluated by the ProSpecTR Cryptosporidium rapid enzyme immunoassay (EIA) (Alexon, Sunnyvale, Calif.). Approximately 2 g of untreated stool suspended in buffer was filtered through membranes labelled with anti-Cryptosporidium-specific antigen antibody. Anti-Cryptosporidium-specific antigen antibody was labelled with biotin, horseradish peroxidase conjugated to streptavidin, and tetramethylbenzidine, and each labelled antibody was added in sequence to the membranes. Each membrane had a positive control and test area. EIA results were compared with those of the modified acid-fast procedure. Twenty-three specimens were positive by the initial acid-fast procedure and the EIA. Forty-two specimens were negative by the initial acid-fast test and the EIA. One specimen was negative by the initial acid-fast test and positive by the EIA (sensitivity, 100%; specificity, 98.5%). This technique is easy to use by comparison with the cumbersome, labor-intensive, and more subjective microscopic methods currently available, and its sensitivity equals that of current microscopic methods.     

Evaluation of an immunoassay for direct detection of Escherichia coli O157 in stool specimens.

An enzyme-linked immunosorbent assay (ELISA) produced by LMD Laboratories, Inc., Carlsbad, Calif., was compared with culture for the detection of Escherichia coli O157. Nine of 185 stool specimens evaluated had positive results by the LMD E. coli O157 ELISA and grew E. coli O157 on culture; 174 had negative by LMD E. coli O157 ELISA results and did not grow E. coli O157 on culture. Of 174 specimens negative by LMD E. coli O157 ELISA, 117 specimens grew other enteric pathogens: Campylobacter spp. (46 isolates), Salmonella spp. (43 isolates), Yersinia spp. (20 isolates), and Shigella spp. (8 isolates). There were two indeterminant results by the LMD E. coli O157 ELISA. One stool specimen did not grow other enteric pathogens on culture, and one grew a Campylobacter sp. on culture. Both had negative LMD E. coli O157 ELISA results upon repeat testing. The LMD E. coli O157 ELISA is an accurate, easy-to-read screening method for the detection of E. coli O157 in fecal specimens.     

Evaluation of reliability of pooling stool specimens from different patients and detection of Giardia lamblia antigen by microtiter enzyme-linked immunosorbent assay.

We have shown that stool samples from different patients can be pooled at a 1:2 dilution and reliably assayed for Giardia lamblia antigen by a commercial microtiter enzyme-linked immunosorbent assay (ELISA) system (LMD Laboratories, Inc., Carlsbad, Calif.). Laboratories can reduce reagent costs by pooling specimens submitted for the detection of Giardia antigen by ELISA.     

An immunoenzymatic dot-ELISA for the detection of Giardia lamblia antigen in stool eluates of clinical cases of giardiasis

A dot-ELISA technique for the detection of G. lamblia specific antigen in stool eluates of clinical cases of giardiasis was developed and evaluated employing monospecific antibodies to a G. lamblia specific coproantigen with a molecular mass of 66 kDa. The assay detected 22 (91.7%) of the 24 microscopically confirmed cases of giardiasis while none of the stool eluates from 20 patients with gastrointestinal parasites other than G. lamblia and 20 apparently healthy subjects had any detectable levels of G. lamblia-specific coproantigen. Of the 25 stool eluates from clinically suspected cases of giardiasis, 13 (52%) were found to contain G. lamblia-specific coproantigen. A 3-month-follow up of five of such cases where stool eluates has antigen detected by dot-ELISA assay, revealed the presence of G. lamblia cysts on repeated stool examinations. All the clinically suspected cases with detectable levels of G. lamblia-specific coproantigen by dot-ELISA were relieved of their clinical symptoms following metronidazole therapy. Single stool eluate examination by dot-ELISA was found to be sufficient to confirm the diagnosis. The dot-ELISA is an easy, rapid, sensitive and specific procedure for confirming the diagnosis of suspected cases of giardiasis. It should be a valuable diagnostic aid under field conditions as well as in the laboratory.     

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.     

Evaluation of the Triage Micro Parasite Panel for detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum in patient stool specimens

A study comparing the Triage Micro Parasite Panel (Biosite Diagnostics, Inc., San Diego, Calif.) to conventional O&P examination (O&P) was performed using patient fecal specimens. Five hundred twenty-three stool samples were compared. Nineteen specimens were found to be positive by Triage, and 29 were found to be positive by O&P. Seven specimens were positive for Giardia lamblia, four were positive for Entamoeba histolytica/E. dispar, and three were positive for Cryptosporidium parvum as determined by both methods. There was one false positive by Triage (C. parvum) and four false negatives by O&P (two G. lamblia, one E. histolytica/E. dispar, and one C. parvum). The Triage test accurately detected all 18 specimens that contained one of the three organisms that it was designed to detect. The Triage test is a rapid, easy-to-use enzyme immunoassay for the detection of G. lamblia, E. histolytica/E. dispar, and C. parvum in fresh or fresh-frozen fecal specimens. These data suggest that the Triage test can be used as a screen for the immediate testing of stool specimens for these three pathogenic parasites. If Triage test results are negative, O&P can be performed if parasitic infections other than G. lamblia, E. histolytica/E. dispar, or C. parvum are suspected.     

Evaluation of a commercial monoclonal antibody-based enzyme immunoassay for detection of adenovirus types 40 and 41 in stool specimens.

A commercial monoclonal antibody-based enzyme immunoassay (Adenoscreen; Mercia Diagnostics Ltd., Guildford, United Kingdom) for the detection of adenovirus types 40 and 41 in stool specimens was evaluated. Two assay modes were tested. In the first, 177 stool samples were screened for the presence of adenovirus type 40 or 41 (assay mode 1). Virus was detected in 79 of 82 specimens positive for adenovirus type 40 or 41 by a polyclonal antibody-based immune electron microscope test, giving a sensitivity of 96.3%. The enzyme immunoassay was negative in 91 of 95 stool samples which contained either other adenovirus serotypes or other viruses or were virus negative. The specificity was thus 95.8%. The positive and negative predictive values of this assay against immune electron microscopy were 95.2 and 96.8%, respectively, and the diagnostic accuracy was 96.0%. Viruses from the three false-negative enzyme immunoassay stool samples were verified as adenovirus type 40 or 41 by restriction enzyme analysis, monoclonal antibody-based immune electron microscopy, or both. Two of the three false-negative stool samples were subsequently concentrated by ultracentrifugation, and one of the two stool samples was then positive by enzyme immunoassay. The third false-negative virus was typed as adenovirus type 41 in the second (serotyping) enzyme immunoassay mode. The four enzyme immunoassay false-positive stool samples all contained other adenovirus serotypes (two were type 2, and two were type 5), but no cross-reactivity was seen with other strains of these serotypes and the results probably reflected simultaneous excretion of adenovirus type 40 or 41 with other adenovirus serotypes. In the second assay mode viruses from 15 stool samples were serotyped. The results by enzyme immunoassay (4 were type 40 and 11 were type 41) correlated completely with previous results from restriction endonuclease analyses. The commercial enzyme immunoassay system showed excellent sensitivity and specificity for the detection of adenovirus types 40 and 41 in stool specimens and will make an important contribution to the accurate diagnosis of adenovirus gastroenteritis.     

A new enzyme immunoassay for the detection of enteroviruses in faecal specimens

A new enzyme immunoassay (EIA) for direct detection of enteroviruses based on a group-specific monoclonal antibody was evaluated using stool samples from patients with suspected enteroviral infection. The EIA was compared with polymerase chain reaction (PCR) and virus isolation in cell culture. Of 204 samples tested, 20 were positive by EIA, 34 by PCR, and 18 by cell culture. Compared with PCR, the most sensitive method, the sensitivity of EIA was 58% (20/34); the sensitivity of cell culture isolation was 52% (18/34). The results of both assays correlated in only 60% of cases. The combination of EIA and cell culture isolation detected 76% of PCR-positive stool samples. Enterovirus EIA provides results within 3-4 hr and requires only standard EIA equipment. It represents a rapid, reliable, and cost-effective diagnostic tool for enterovirus diagnosis from faecal samples. Negative results must be confirmed by other techniques, such as PCR or virus isolation in cell culture.     

Comparison of an enzyme immunoassay to an indirect fluorescent immunoassay for the detection of antinuclear antibodies

The standard method for detecting antinuclear antibodies (ANAs) is by immunofluorescence assay (IFA), a method that is labor intensive and subjective. In an attempt to overcome these limitations, several commercial enzyme immunoassays (EIAs) have been developed. We report the results of our evaluation of the ANA Microplate EIA (Sanofi Diagnostics Pasteur, Chaska, MN). For the evaluation, 808 serum samples were tested by EIA and IFA; 52 specimens were positive by both assays, 561 were negative by both assays, 91 were positive by EIA only, and 3 were positive by IFA only. Borderline results (not positive or negative) were obtained for 101 specimens, which were excluded when calculating the sensitivity, specificity, and positive and negative predictive values of this assay, which were 94.6%, 86.0%, 36.4%, and 99.5%, respectively. Because of its high negative predictive value, this assay can be used reliably to detect ANA-negative samples; however, the low positive predictive value indicates that EIA-positive specimens should be retested by an IFA to determine the final result.     

Evaluation of the sensitivities of DNA extraction and PCR methods for detection of Giardia duodenalis in stool specimens

Sensitivities of DNA extraction methods and PCR methods for Giardia duodenalis were evaluated. A combination of the most sensitive methods, i.e., FTA filter paper and a PCR protocol using RH11/RH4 and GiarF/GiarR primers, showed no significant differences compared to immunofluorescence assay in terms of their sensitivities and specificities.     

Comparison of microscopy, rapid immunoassay, and molecular techniques for the detection of Giardia lamblia and Cryptosporidium parvum

Giardia lamblia and Cryptosporidium parvum are recognized as the most common protozoan infections in Saudi Arabia. Microscopic examination of stool samples, either direct or concentrated, for the recovery of G. lamblia cysts and trophozoites and C. parvum oocysts is still the most commonly used for the diagnosis of both parasites. We compared the conventional parasitological techniques of iodine-stained wet mount for G. lamblia and Kinyoun's acid-fast for C. parvum against ImmunoCard STAT® Cryptosporidium/Giardia and real-time polymerase chain reaction (PCR) detecting the 18S rRNA gene of G. lamblia and conventional PCR detecting the same gene of C. parvum at a tertiary hospital in Dhahran, Saudi Arabia. Out of 148 stool samples, 19 and 12 true positives were identified for G. lamblia and C. parvum, respectively, using a composite reference standard. In this case, true positives and negatives were considered as those with at least two positive or negative results out of the three tests. Both ImmunoCard STAT! and PCR methods were more sensitive than the microscopic tests of a single stool specimen of 85.7 % (CI?=?62.6–96.2 %) and 85.7 % (CI?=?56.2–97.5 %) for G. lamblia and C. parvum, respectively. However, specificity of microscopic tests was higher than other techniques for both parasites. Although PCR seems to be most sensitive for both G. lamblia and C. parvum, its low specificity may render its superiority over other techniques. When a single stool sample is used for detection of G. lamblia and C. parvum, better results can be obtained when coupled with serological testing. Although PCR is the most sensitive method for the detection of both G. lamblia and C. parvum, its use requires attention in relation to the increased possible false positives.