Evaluation of a novel rapid one-step immunochromatographic assay for detection of monoclonal Helicobacter pylori antigen in stool samples from children

A new rapid one-step immunochromatographic test using monoclonal antibodies for detection of Helicobacter pylori antigen in stool in children was evaluated on coded stool samples from 159 children (mean age, 9.7 +/- 5.0 years; 118 from Munich, 41 from Vienna): 86 children were H. pylori infected defined by positive culture and/or > or =2 other positive tests ([13C]urea breath test, histology, rapid urease test), and 73 children showed concordant negative results. Seventy-nine patients (12.1 +/- 3.8 years; 42 from Munich; 37 from Vienna) were tested 6 to 8 weeks after anti-Helicobacter pylori therapy with urea breath test and stool test. In Munich, all 160 tests (118 pre- and 42 posttreatment) were independently read by two observers. Equivocal results were excluded for calculation of sensitivity and specificity but were considered as false to assess accuracy. The two observers in Munich agreed in 63 out of 65 positive and 89 out of 95 negative results, while eight times (5.0%) they judged the test as equivocal. Pretreatment and posttreatment results for sensitivity were 88.1% (79.2 to 94.1) and 88.9% (51.8 to 99.7), specificity 88.1% (77.8 to 94.1) and 93.9% (85.2 to 98.3), and accuracy 83.5% and 81.5%, respectively. We conclude that the new monoclonal immunochromatographic quick test shows a good interobserver agreement, but equivocal results occur in 5%. Performance is comparable before and after therapy. The test may become a good alternative in children in settings where a [13C]urea breath test or a reliable enzyme immunoassay stool test are not available.     

Evaluation of immunochromatography and commercial enzyme-linked immunosorbent assay for rapid detection of norovirus antigen in stool samples

The efficiency of immunochromatography and commercial enzyme-linked immunosorbent assay (ELISA) kit (Denka Seiken Co. Ltd., Tokyo, Japan) were evaluated for rapid detection of norovirus (NoV) from stool specimens. A total of 503 stool specimens collected from infants and young children who suffered from acute gastroenteritis were tested for NoV by the NoV-immunochromatography kit, Denka ELISA kit, and by a monoplex RT-PCR method. The NoV-immunochromatography revealed 78.9% sensitivity, 96.4% specificity, and 92.4% efficiency with the monoplex RT-PCR method. The Denka ELISA kit had a sensitivity of 90.4%, specificity of 96.4%, and an efficiency level of 95%. The findings indicate that the newly developed NoV-immunochromatography kit provides the specificity equal to that of the Denka ELISA kit, even through the sensitivity of detection was lower. However, the advantage of the NoV-immunochromatography kit is less time consuming and simpler. The data show that both the Denka ELISA and the NoV-immunochromatography kits may be used as an alternative method for screening of NoV in stool samples.     

LcrV capture enzyme-linked immunosorbent assay for detection of Yersinia pestis from human samples

In the United States, there is currently a major gap in the diagnostic capabilities with regard to plague. To address this, we developed an antigen capture assay using an essential virulence factor secreted by Yersinia spp., LcrV, as the target antigen. We generated anti-LcrV monoclonal antibodies (MAbs) and screened them for the ability to bind bacterially secreted native Yersinia pestis LcrV. Anti-LcrV MAb 19.31 was used as a capture antibody, and biotinylated MAb 40.1 was used for detection. The detection limit of this highly sensitive Yersinia LcrV capture enzyme-linked immunosorbent assay is 0.1 ng/ml. The assay detected LcrV from human sputum and blood samples treated with concentrations as low as 0.5 ng/ml of bacterially secreted native Y. pestis LcrV. This assay could be used as a tool to help confirm the diagnosis of plague in patients presenting with pneumonia.     

Diagnosis of Helicobacter pylori infection in adults and children by using the Malakit Helicobacter pylori, a commercially available enzyme-linked immunosorbent assay.

Malakit Helicobacter pylori (Biolab, Limal, Belgium) is a second-generation enzyme-linked immunosorbent assay (ELISA) for the detection of Helicobacter pylori infection. We evaluated its ability to diagnose H. pylori infection in 489 asymptomatic pregnant women, 427 asymptomatic children, and 95 symptomatic children. 87 asymptomatic adults (17.8%), 31 asymptomatic children (7.3%), and 27 symptomatic children (28.4%) were seropositive. We observed an increase in H. pylori infection with age. 13C-urea breath tests were performed for all seropositive and 100 randomly selected seronegative asymptomatic adults. They were also performed for all seropositive and 65 randomly chosen seronegative asymptomatic children. Breath tests were positive for 86 of 87 (98.9%) seropositive adults, 30 of 31 seropositive children (96.8%), and no seronegative individual. Compared with those of culture, the sensitivity and specificity of the Malakit Helicobacter pylori were both 96%. We conclude that the Malakit Helicobacter pylori is equally suitable for adults and children. Therefore, this ELISA can be proposed as an important alternative to other more time-consuming and/or more expensive diagnostic tests for the detection of H. pylori.     

Detection of clarithromycin-resistant Helicobacter pylori in stool samples

The recognition of the role of Helicobacter pylori in gastric diseases has led to the widespread use of antibiotics in the eradication of this pathogen. The most advocated therapy, triple therapy, often includes clarithromycin. It is well known that clarithromycin resistance is one of the major causes of eradication failure. The development of a rapid noninvasive technique that could easily be performed on fecal samples and that could also provide information about the antibiotic resistance of this microorganism is therefore advisable. Previous findings have demonstrated that clarithromycin resistance is due to a single point mutation in the 23S rRNA. All the mutations described have been associated with specific restriction sites, namely BsaI (A2143G), MboII (A2142C/G), and HhaI (T2717C). On this basis we have developed a new method, a seminested PCR, allowing screening for clarithromycin resistance of H. pylori directly on stool samples. This method furnished a 783-bp fragment of the 23S rRNA, which was subsequently digested by MboII, BsaI, and HhaI, in order to identify single point mutations associated with clarithromycin resistance. Of a total of 283 stool samples examined, 125 were H. pylori positive and two of them were shown to contain clarithromycin-resistant strains due to the presence of a mutation at position 2717, whereas no PCR products contained mutations at position 2142 or 2143. In order to evaluate the reliability of the new system, we compared the results of restriction analysis of the PCR products with the MICs shown by the H. pylori isolates by culturing gastric biopsies from the same patients.     

Indirect immunofluorescence test and enzyme-linked immunosorbent assay for detection of Campylobacter pylori

An indirect immunofluorescence test (IIF) has been developed for detecting Campylobacter pylori in gastroduodenal biopsies. This test was compared with standard methods of C. pylori diagnosis, namely Gram staining and urease test, in a study population of 226 patients; 121 of the biopsy specimens were cultured for C. pylori as well. C. pylori colonization was detected in 154 of 226 patients (68%) by at least one of these methods (IIF, 96%; Gram staining, 78%; urease test, 60%; cultivation, 55%). Serum samples from 191 patients of the study population were screened for circulating antibodies to C. pylori by an indirect enzyme-linked immunosorbent assay with whole, untreated bacteria as antigen. Of these serum specimens, 140 (73%) revealed absorbance readings above the limit of positivity, which was determined as an optical density of greater than 0.35 at 405/620 nm. Of 132 serum specimens, 128 (97%) from patients with C. pylori detected in biopsies, but only 12 (20%) of 59 specimens from those without C. pylori detection showed elevated specific antibody levels. Our data revealed that IIF proved to be the superior rapid, sensitive, and specific diagnostic method. The correlation between microbiological findings and the immune response favors our enzyme-linked immunosorbent assay as an additional tool in C. pylori diagnosis.     

Evaluation of the bead enzyme-linked immunosorbent assay for detection of cholera toxin directly from stool specimens.

A highly sensitive bead enzyme-linked immunosorbent assay (bead ELISA) for detection of cholera toxin (CT) was evaluated for direct detection of CT from stool specimens of patients with acute secretory diarrhea. Of the 75 stool samples examined, 59 yielded biochemically, and serologically confirmed strains of Vibrio cholerae O1. The bead ELISA was positive for CT in stool supernatants in 50 (84.7%) of the 59 samples from which V. cholerae O1 was isolated. In addition, the bead ELISA was positive for three stool specimens which were negative by culture. The free CT present in 48 of the 50 stool samples positive by culture for V. cholerae O1 and for CT by bead ELISA was completely absorbed by anti-CT immunoglobulin G. All of the 59 strains of V. cholerae O1 biotype eltor isolated in this study produced in vitro CT. The concentration of CT present in the bead ELISA-positive stool samples ranged between 26 pg/ml and greater than 100 ng/ml. This evaluation study demonstrates that the bead ELISA is a sensitive and simple method for direct detection of CT in nonsterile stool samples, and we recommend routine use of this assay for detection of CT in stool samples and culture supernatants in clinical and reference laboratories.     

Serodiagnosis of Helicobacter pylori infection: comparison and correlation between enzyme-linked immunosorbent assay and rapid serological test results.

CLOser is a new, one-step, qualitative anti-Helicobacter pylori immunoglobulin G test having the advantage of convenience and simplicity. We aimed to evaluate its diagnostic accuracy and to compare it with a quantitative enzyme-linked immunosorbent assay (ELISA) (HEL-pTEST II) in a study of 86 adult dyspeptic patients by using the results from histology and urease testing of gastric biopsies as a "gold standard." Forty-six patients were H. pylori positive. The sensitivities, specificities, and positive and negative predictive values were 95.7, 72.5, 80.0, and 93.5%, respectively, for CLOser and 93.5, 92.5, 93.5, and 92.5%, respectively, for HEL-pTEST II. The grade of the colored test bands in CLOser was correlated with antibody titers in HEL-pTEST II (r = 0.71; p < 0.001). The mean antibody titers were 13, 74, 186, and 328 U/ml for the negative, faint, thin, and thick bands, respectively, of CLOser. We concluded that the CLOser rapid serological test yielded sensitivity similar to that of the conventional ELISA. Although CLOser is not suitable for epidemiologic screening for H. pylori infection on account of lower specificity, it is particularly convenient and very easy to perform. Therefore, it may eventually become widely used in the office-based care of patients and lead to more cost-effective patient management decisions.     

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

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

Development of an enzyme-linked immunosorbent assay for the detection of pentachlorophenol residues in water samples

An indirect competitive enzyme-linked immunosorbent assay (ELISA) for pentachlorophenol (PCP) was developed with a polyclonal antiserum produced against a hapten, 4-(2,3,5,6-tetrachloro-4-hydroxyphenoxy)-butyric acid, conjugated to bovine serum albumin (BSA). The ELISA of PCP showed an IC₅₀ of 18 ng/ml and a limit of detection (LOD) of 0.9 ng/ml at the optimal concentration of NaCl (0.2 M) in the assay buffer. This assay was slightly affected by up to 10% (v/v) methanol or acetone. The IC₅₀ and A _max values showed little change in a pH range of 5 and 7. Very low cross reactivities (≤2.5%) were observed for some structurally-related compounds, except for 2,3,4,6-tetrachlorophenol (5.6%) and 2,3,5,6-tetrachlorophenol (9.2%). The average recoveries of PCP from fortified water were in the range of 71–118%. The results by GC and ELISA for the same fortified water samples showed non-significant difference (p>0.05). Finally, this ELISA was applied to monitor PCP in 100 river water samples and no PCP was detected. This ELISA is a good alternative tool for monitoring PCP residues in water samples.     

Detection of cytomegalovirus in urine samples by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytomegalovirus (CMV) in urine using monoclonal antibodies directed against CMV as a capture for viral antigen. The assay was capable of detecting virus at 10^2.3TCID₅₀/ml as determined by titration of stock virus, strain Ad169. The assay was found to have a sensitivity of 65% and a specificity of 100% when 73 coded stored urine specimens were examined. Assuming that the poor sensitivity was due to loss of antigen following storage, we proceeded to analyse fresh urine specimens. Surprisingly, the assay gave negative results with 46 fresh urines known to contain CMV; however, following storage at +4°C for two weeks, 35 (76%) of these samples gave ELISA results in the positive range. This detection of CMV, after storage at +4°C, could be due to degradation of virus particles leading to release of soluble glycoproteins into the medium or to the presence of an inhibitory substance in fresh urine that is destroyed during storage.     

Evaluation of the novel Helicobacter pylori ClariRes real-time PCR assay for detection and clarithromycin susceptibility testing of H. pylori in stool specimens from symptomatic children

The aim of the present study was to evaluate the Helicobacter pylori ClariRes assay (Ingenetix, Vienna, Austria) for the detection of H. pylori infection and the simultaneous clarithromycin susceptibility testing of the H. pylori isolates in stool samples from 100 symptomatic children. The results obtained by this novel biprobe real-time PCR method were directly compared with the results obtained from histological examination of gastric biopsy specimens, culturing, the [13C]urea breath test, and a monoclonal antibody-based stool antigen enzyme immunoassay (EIA). Fecal specimens from all 54 children who were shown to be noninfected by "gold standard" tests gave true-negative PCR results (specificity, 100%). Of the remaining 46 individuals with a positive H. pylori status, 29 were found to be positive by real-time PCR (sensitivity, 63%). For these 29 cases, the H. pylori ClariRes assay confirmed all results from phenotypic clarithromycin susceptibility testing by Etest. In summary, this investigation demonstrates that detection of Helicobacter DNA in stool samples by real-time PCR is a difficult task and that this method cannot replace the stool antigen EIA (sensitivity, 95.7%) for the accurate diagnosis of H. pylori infection in children.     

Serodiagnosis of Helicobacter pylori: comparison of enzyme-linked immunosorbent assays.

Enzyme-linked immunosorbent assays (ELISAs) have been developed to diagnose Helicobacter pylori infection. However, the methods are not standardized. We therefore prospectively evaluated the sensitivities and specificities of ELISAs developed in the United States and the United Kingdom in a study population comprising 41 consecutive symptomatic outpatients and 35 volunteers. At endoscopy, multiple biopsies were obtained for histology and culture and stained sections were graded for chronic gastritis, active chronic gastritis, and density of H. pylori. Serum samples were analyzed for H. pylori by ELISA. The first set of assays for immunoglobulin G (IgG) and IgA used a pool of sonicated isolates of H. pylori from five patients in the United States (antigen A). The second set of assays, developed in the United Kingdom, used three different antigens: antigen 1, an acid-extractable surface antigen; antigen 2, an acid-extractable antigen from an aflagellate variant; and antigen 3, a urease-containing fraction. Cutoff scores for positive results were determined a priori on the basis of previous serological studies. There was close agreement between histology and culture. In the study population, 36% of the individuals were H. pylori positive. The diagnostic value of the different ELISAs were highly comparable, and the crude antigens performed as well as the more purified antigens. The antigen A IgG had a sensitivity and specificity of 96 and 94%, respectively; the values for antigen 1 were 93 and 96%, respectively. The antigen A IgA and antigen 3 assays were the least sensitive tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of enzyme-linked immunosorbent assay for detection of Mycoplasma hominis antibodies in infertile women serum samples

BACKGROUND: Besides Chlamydiae trachomatis and Mycoplasma genitalium, Mycoplasma hominis may also cause infertility due to damage of the Fallopian tubes. Therefore serum samples from infertile women were analyzed for antibodies to M. hominis. METHODS: Sera from 304 infertile women were investigated for seropositivity to M. hominis by immunoblotting and a developed ELISA. Women were classified into groups based on the type of infertility: infertile due to lack of passage in Fallopian tubes (TFI, tubal factor infertility), an infertile male partner (MFI, male factor infertility) and unexplained infertility (UFI, unexplained factor infertility). Three M. hominis isolates were used in the immunoblotting analysis and clear differences in patient immunoprofiles were observed between two isolates. For the ELISA we used a mixture of Triton X-114 extracted membrane proteins from those two M. hominis isolates as antigen. RESULTS: Ninety-seven sera (32%) were seropositive to M. hominis when tested by the ELISA. There was a significant correlation between TFI and seropositivity to M. hominis (P = 0.0015, OR = 2.21, CI = 1.35-3.61). We compared the seropositivity of 304 patients to M. hominis with the presence of antibodies against two other bacteria Chlamydiae trachomatis and Mycoplasma genitalium and there was no statistical correlation between those bacteria and M. hominis. CONCLUSION: Our results indicate that M. hominis may be an independent predictor of TFI.     

Comparison of three stool antigen tests for Helicobacter pylori detection

BACKGROUND: Active Helicobacter pylori infection can be diagnosed by invasive (biopsy based) or non-invasive methods, such as stool antigen testing. AIMS: To compare three stool antigen enzyme immunoassay kits--Premier Platinum Hp SA, FemtoLab Cnx, and Hp Ag--with biopsy based methods for the detection of H pylori in previously undiagnosed patients. METHODS: One hundred and eleven adults with dyspepsia referred for endoscopy provided a stool sample for testing and had biopsies taken. Patients were considered H pylori positive if two out of three invasive tests were positive or if culture alone was positive. RESULTS: The sensitivities and specificities of the Premier Platinum Hp SA, FemtoLab Cnx, and Hp Ag stool antigen kits when compared with biopsy based diagnosis were, 63.6%, 88.0%, and 56.0% and 92.6%, 97.6%, and 97.6%, respectively. CONCLUSIONS: FemtoLab Cnx may be considered as an alternative to urea breath testing in the initial diagnosis of patients with dyspepsia who do not require immediate endoscopy. Stool testing has the potential advantages of being simple to perform, relatively cheap, and samples can be submitted directly from primary care.     

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.     

Diagnosis of visceral leishmaniasis by enzyme-linked immunosorbent assay using urine samples

A diagnostic method has been developed to detect anti-Leishmania donovani immunoglobulin G (IgG) in urine by enzyme-linked immunosorbent assay (ELISA). In measuring anti-L. donovani IgG, IgA, and IgM in urine, the method performed best in the detection of IgG. The sensitivity and specificity of the assay were determined with panels of urine samples from 62 visceral leishmaniasis (VL) patients, 59 healthy controls from areas of endemicity, 53 healthy controls from areas of nonendemicity, 59 malaria patients, 13 tuberculosis patients, 23 cutaneous leishmaniasis patients, and 7 patients with other diseases. Using L. donovani promastigote crude antigen, the test had 93.5% sensitivity (58 positives of 62 VL patient samples) and 89.3% specificity (191 negatives of 214 non-VL patient samples). The ELISA with acetone-treated L. donovani promastigote antigen raised the sensitivity and specificity to 95.0 and 95.3%, respectively. Western blot analysis revealed that most of the samples that cross-reacted with crude antigen in ELISA did not recognize any antigenic component of L. donovani crude antigen. We also checked 40 serum samples from the same group of VL patients for anti-L. donovani IgG and got 90.0% sensitivity with both crude and acetone-treated antigens. As collection of urine is much easier than collection of serum, the detection of anti-L. donovani IgG in urine with acetone-treated antigen will be useful in epidemiological studies. It could be an adjunct of laboratory diagnosis.