Detection of Giardia lamblia antigens in human fecal specimens by a solid-phase qualitative immunochromatographic assay

The SIMPLE-READ Giardia rapid assay (Medical Chemical Corporation) is a solid-phase qualitative immunochromatographic assay that detects Giardia lamblia in aqueous extracts of human fecal specimens. Testing 106 Giardia-positive and 104 Giardia-negative stool specimens yielded a sensitivity of 97.2% and a specificity of 100% for the SIMPLE-READ Giardia rapid assay.     

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.     

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

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

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.     

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.     

Simultaneous detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR

Entamoeba histolytica, Giardia lamblia, and Cryptosporidium are three of the most important diarrhea-causing parasitic protozoa. For many years, microscopic examination of stool samples has been considered to be the "gold standard" for diagnosis of E. histolytica, G. lamblia, and C. parvum infections. Recently, more specific and sensitive alternative methods (PCR, enzyme-linked immunosorbent assay, and direct fluorescent-antibody assay) have been introduced for all three of these parasitic infections. However, the incorporation in a routine diagnostic laboratory of these parasite-specific methods for diagnosis of each of the respective infections is time-consuming and increases the costs of a stool examination. Therefore, a multiplex real-time PCR assay was developed for the simultaneous detection of E. histolytica, G. lamblia, and C. parvum in stool samples. The multiplex PCR also included an internal control to determine efficiency of the PCR and detect inhibition in the sample. The assay was performed on species-specific DNA controls and a range of well-defined stool samples, and it achieved 100 percent specificity and sensitivity. The use of this assay in a diagnostic laboratory would provide sensitive and specific diagnosis of the main parasitic diarrheal infections and could improve patient management and infection control.     

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 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.     

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.     

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.     

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。     

Evaluation of the immunochromatographic CORIS Giardia-Strip test for rapid diagnosis of Giardia lamblia

The aim of this study was to evaluate the performance of the CORIS Giardia-Strip test (CORIS Bioconcept, Gembloux, Belgium) as a rapid initial method for the routine diagnosis of giardiasis. Compared to a commercial ELISA-coproantigen test (ProSpect Giardia-ELISA-microplate assay; Remel, Lenexa, KS, USA), the commercial strip test had a sensitivity of 58%, a specificity of 99%, a positive predictive value of 93% and a negative predictive value of 93% (n=158). These results are comparable to those obtained using microscopy of direct wet-mounted stool. Since the CORIS Giardia-Strip test is simpler to perform, it can replace direct wet-mounted stool microscopy for the rapid diagnosis of giardiasis; however, its sensitivity is inferior to that of other immunochromatographic antigen detection tests and fresh stool samples are required for its use. Nevertheless, the results suggest that a positive CORIS Giardia-Strip test outcome does not need confirmation, while samples with negative results should be re-examined using another, more sensitive, test.     

Detection and genotyping of Entamoeba histolytica, Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum by oligonucleotide microarray

Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum are the most frequently identified protozoan parasites causing waterborne disease outbreaks. The morbidity and mortality associated with these intestinal parasitic infections warrant the development of rapid and accurate detection and genotyping methods to aid public health efforts aimed at preventing and controlling outbreaks. In this study, we describe the development of an oligonucleotide microarray capable of detecting and discriminating between E. histolytica, Entamoeba dispar, G. lamblia assemblages A and B, and C. parvum types 1 and 2 in a single assay. Unique hybridization patterns for each selected protozoan were generated by amplifying six to eight diagnostic sequences/organism by multiplex PCR; fluorescent labeling of the amplicons via primer extension; and subsequent hybridization to a set of genus-, species-, and subtype-specific covalently immobilized oligonucleotide probes. The profile-based specificity of this methodology not only permitted for the unequivocal identification of the six targeted species and subtypes, but also demonstrated its potential in identifying related species such as Cryptosporidium meleagridis and Cryptosporidium muris. In addition, sensitivity assays demonstrated lower detection limits of five trophozoites of G. lamblia. Taken together, the specificity and sensitivity of the microarray-based approach suggest that this methodology may provide a promising tool to detect and genotype protozoa from clinical and environmental samples.     

Evaluation of seven immunochromatographic assays for the rapid detection of human rotaviruses in fecal specimens

Seven commercially available immunochromatographic assays were tested for the rapid detection of group A rotaviruses in fecal samples compared to a enzyme immunoassay (Argene). Detection of rotaviruses in 80 ELISA positive frozen stool samples showed rates superior to 90% for three reagents (Rota Strip (Cypress Diagnostics), 98.8%; Rotascreen (Microgen), 95.0%; VIKIA Rota/Adeno (bioMérieux), 92.5%); from 82.5% to 88.8% for three others (Diarlex with centrifugation (Orion Diagnostica), 88.8%; Combo Rota/Adeno (All Diag), 87.5%; Rota/Adeno Combi Stick (bmd), 82.5%) and only 70.0% for Diarlex with filtration vial (Orion Diagnostica). The evaluation of the specificity, performed on one hundred fresh rotavirus negative stools, did not show any false positives with any assay. Analysis of the different technical features of these tests showed that they are quick and suitable for a clinical laboratory and do not require expensive equipment.     

Detection of Cryptosporidium oocysts in human fecal specimens by an indirect immunofluorescence assay with monoclonal antibodies.

An indirect fluorescence assay (IFA) with monoclonal antibodies developed for the detection of Cryptosporidium oocysts in fecal smears (Meridian Diagnostics, Inc., Cincinnati, Ohio) was compared with the Zeihl-Neelsen-modified acid-fast stain (MAFS) in 119 human fecal specimens collected between 1984 and 1987. The sensitivity of the IFA was 100%; all 56 specimens positive by MAFS exhibited fluorescence. There were 63 specimens negative for Cryptosporidium sp. by MAFS; of these, 61 were negative by IFA (97% specificity). This discrepancy may reflect an increased sensitivity of the IFA to detect oocysts that were not visualized by MAFS because of faint staining or a paucity of organisms. On average, the IFA required less time than the MAFS (1 versus 5 min, respectively) when only rare or few oocysts were present. Cost comparison of reagents showed the IFA to be three times more expensive. The IFA offers a reasonable alternative to the MAFS because of its high sensitivity and specificity, the simplicity of performing it and interpreting results, and its capability of providing a definitive diagnosis of Cryptosporidium oocysts.     

Identification and determination of the viability of Giardia lamblia cysts and Cryptosporidium parvum and Cryptosporidium hominis oocysts in human fecal and water supply samples by fluorescent in situ hybridization (FISH) and monoclonal antibodies

In the present study, fluorescent in situ hybridization (FISH) and monoclonal antibodies (MAbs) were evaluated for species-specific detection and viability determination of Giardia lamblia, Cryptosporidium parvum, and Cryptosporidium hominis in human fecal and water supply samples. A total of 50 fecal human samples positive for G. lamblia cysts, 38 positive for C. parvum, and 23 positive for C. hominis were studied. Also, 18 water supply samples positive for Giardia spp. and Cryptosporidium spp. by the United States Environmental Protection Agency (USEPA) Method 1623 were studied by FISH and fluorescein isothiocyanate (FITC)-conjugated MAbs. Eighteen percent of the fecal samples parasitologically positive for G. lamblia presented viable and nonviable cysts, and 5% of those positive for Cryptosporidium spp. presented viable and nonviable oocysts. Of the 18 water supply samples analyzed, 6 (33%) presented Giardia spp. viable and nonviable cysts and 2 (11%) presented viable and nonviable Cryptosporidium spp. oocysts. G. lamblia identification was confirmed by polymerase chain reaction (PCR) and sequencing of the β-giardin gene in the fecal and water samples found positive by FISH and FITC-conjugated MAbs. C. parvum and Cryptosporidium muris were identified, by PCR and sequencing of the small subunit of ribosomal RNA gene, in seven and one water samples, respectively. Our results confirm that this technique enables simultaneous visualization, species-specific identification, and viability determination of the organisms present in human fecal and water supply samples.     

Enzyme-linked immunoassay for detection of Cryptosporidium antigens in fecal specimens.

Cryptosporidium sp. is a ubiquitous 4- to 6-micron protozoan parasite infecting the intestinal tract of humans. It causes mild to fulminant diarrhea in patients, especially immunocompromised persons, and it may be hard to detect by microscopic fecal examination. An indirect, double-antibody enzyme-linked immunosorbent assay (ELISA) was developed using specifically produced goat and rabbit antisera to detect Cryptosporidium antigens in human feces. Of 62 frozen stools from patients with cryptosporidiosis, as detected by at least two microscopic diagnostic techniques, 51 were positive by ELISA; all ELISA-negative specimens came from patients with fewer than five oocysts per 0.01 ml of concentrated fecal sample examined after modified acid-fast or fluorescent monoclonal antibody staining. A total of 182 specimens from persons without Cryptosporidium infection were negative by ELISA in 176 instances; 3 ELISA-positive specimens came from patients with cryptosporidiosis diagnosed earlier. The sensitivity of the assay was 82.3%, and specificity was 96.7%. The predictive value of a positive ELISA was 89.5%, and the predictive value of a negative ELISA was 94.2%. The ELISA was not affected by the presence of eight other intestinal parasites but was sometimes affected by repeated freezing and thawing of fecal specimens. All fecal specimens were heated to 100 degrees C for 2 min to reduce proteolytic enzyme activity, although the necessity of this step needs further evaluation. This first-generation ELISA is a simple, rapid, easily standardized test for Cryptosporidium antigens in stool samples which will be useful for diagnosis and for large-scale epidemiologic studies.     

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.     

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.