Indirect immunoperoxidase test for the diagnosis of leptospirosis.

Leptospirosis is a widespread zoonotic disease that affects all mammals, including humans, in different parts of the world. Clinical recognition of leptospirosis is challenging, and the definitive serologic diagnosis assay, the microscopic agglutination test (MAT), is time-consuming and difficult to conduct. In this study, an indirect immunoperoxidase (IIP) test to detect Leptospira-specific antibodies in human serum samples was developed. The efficacy of the IIP was compared with the indirect immunofluorescent assay (IFA) and MAT. A total of 368 human serum samples were analyzed by MAT, IFA, and IIP. Using a MAT titer of > or = 1:100 as the gold standard, the sensitivities for the detection of Leptospiral antibodies at a titer of 1:200 were 94.7% by IFA and 93.6% by IIP; specificities were 95.3% by IFA and 94.9% by IIP; and accuracies were 95.1% by IFA and 94.6% by IIP. With a titer of 1:400, the sensitivity, specificity, and accuracy were 86.2%, 98.9%, and 95.7% by IFA, respectively; whereas, for the IIP, the sensitivity was 85.1%, specificity 98.5%, and accuracy 95.1%. A further evaluation of this test with 80 unknown-febrile-disease sera was also included. We found that the sensitivity and specificity of this test were 100% and 76.8%, respectively. Therefore, the IIP test is a potentially valuable tool for the diagnosis of leptospirosis.

Development of multiplex PCR for the detection of total coliform bacteria for Escherichia coli and Clostridium perfringens in drinking water.

Multiplex PCR amplification of lacZ, uidA and plc genes was developed for the simultaneous detection of total coliform bacteria for Escherichia coli and Clostridium perfringens, in drinking water. Detection by agarose gel electrophoresis yielded a band of 876 bp for the lacZ gene of all coliform bacteria; a band of 147 bp for the uidA gene and a band of 876 bp for the lacZ gene of all strains of E. coli; a band of 280 bp for the p/c gene for all strains of C. perfringens; and a negative result for all three genes when tested with other bacteria. The detection limit was 100 pg for E. coli and C. perfringens, and 1 ng for coliform bacteria when measured with purified DNA. This assay was applied to the detection of these bacteria in spiked water samples. Spiked water samples with 0-1,000 CFU/ml of coliform bacteria and/or E. coli and/or C. perfringens were detected by this multiplex PCR after a pre-enrichment step to increase the sensitivity and to ensure that the detection was based on the presence of cultivable bacteria. The result of bacterial detection from the multiplex PCR was comparable with that of a standard plate count on selective medium (p=0.62). When using standard plate counts as a gold standard, the sensitivity for this test was 99.1% (95% CI 95.33, 99.98) and the specificity was 90.9 % (95% CI 75.67, 98.08). Multiplex PCR amplification with a pre-enrichment step was shown to be an effective, sensitive and rapid method for the simultaneous detection of these three microbiological parameters in drinking water.

Development and evaluation of a latex agglutination test for the rapid diagnosis of scrub typhus.

The purpose of this research was to develop a simple and rapid diagnostic test for scrub typhus using a latex agglutination test (LAT) to detect antibodies against Orientia tsutsugamushi. Five strains of O. tsutsugamushi were propagated in L929 cells. The rickettsiae were purified and concentrated with percoll density gradient centrifugation. A suitable concentration of O. tsutsugamushi soluble antigen was used to sensitize latex to prepare the latex antigen. The specificity, sensitivity, and accuracy of the latex antigen were assessed. The LAT, indirect immunofluorescent antibody test (IFA), and Weil-Felix agglutination test (WF) were compared by testing 109 acute febrile illness cases and 100 confirmed non-scrub typhus cases (50 other febrile disease cases and 50 healthy controls). By using the IFA as the standard reference method, the overall sensitivity, specificity, and accuracy of the LAT were 89.1, 98.2, and 93.6%, respectively. By contrast, the sensitivity of the WF, compared with the IFA, was only 47.3%, while the specificity and accuracy were 92.6 and 69.7%, respectively. Thus, the LAT described here is another important alternative test for the diagnosis of scrub typhus.

The use of flow cytometry as a diagnostic test for malaria parasites.

A total of 453 clinical blood samples were determined for malaria parasites by flow cytometric assay (FCM) and reagents from Sysmex Corporation, Japan. In this study, the FCM greatly simplified and accelerated parasite detection, with sensitivity of 91.26%, specificity 86.28% and accuracy 87.42%. Overall, the parasite counts by flow cytometric measurement correlated well with the parasitemia measured by microscopic assay (regression coefficient = 0.9409). The detection limit was 0.05-0.1% parasitemia. No evidence of malaria parasites in either blood donor volunteers or other disease patients groups was determined by FCM. However, 48 samples who had been treated with antimalarial drugs and whose parasite microscopic counts were negative, showed false-positive results. When the data of these 48 samples were analyzed, they were found to have high levels of reticulocytes, ranging from 2.0-18.9%. This finding suggested that a high reticulocyte concentration in the blood may interfere with the performance of the FCM. Further improvement, by eliminating this interference, will make the FCM one of the most promising tests for malaria diagnosis.

Prevalence of lower genital tract infection among women attending maternal and child health and family planning clinics in Hanoi, Vietnam.

To determine the prevalence of lower genital tract infection (LGTI) with Candida spp, Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and bacterial vaginosis among symptomatic and asymptomatic women attending maternal and child health and family planning (MCH/FP) clinics in Hanoi, Vietnam. A multi-centered, cross-sectional descriptive study stratified by reported symptoms of vaginal discharge was carried out in three MCH/FP clinics among 1,000 women aged 18-44 years in 1998. Of these, 89.1% lived in Hanoi, 97.6% were currently married, and 99.2% had only one sexual partner in the past 12 months. Regarding their contraceptive use, 28.2% did not use any contraception, 25.6% used an intrauterine device (IUD), 22.8% used condoms, and 23.4% used other methods. The overall prevalence of Candida spp was 11.1% (95% CI = 9.1-13.1%); T. vaginalis, 1.3% (95% CI = 0.6-2.0%); no gonococcal infection was found; the prevalence of C. trachomatis was 4.4% (95% CI = 3.1-5.7%); and of bacterial vaginosis, 3.5% (95% CI = 2.4-4.6%). The presence of LGTI was not associated with reported symptom of vaginal discharge. LGTI was common among married and monogamous women attending MCH/FP clinics in Hanoi, of whom many used IUDs and may have an increased risk of complications in the presence of LGTI. The lack of association between symptoms and laboratory-confirmed infection underscores the challenge of diagnosing LGTI when laboratory testing is not available.