A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples

<p>Abstract</p> <p>Background</p> <p><it>E. histolytica</it>, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic <it>E. moshkovskii </it>and <it>E. dispar </it>by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of <it>E. histolytica</it>, <it>E. moshkovskii </it>and <it>E. dispar </it>simultaneously in stool samples.</p> <p>Results</p> <p>The species specific product size for <it>E. histolytica</it>, <it>E. moshkovskii </it>and <it>E. dispar </it>was 439, 553 and 174 bp respectively, which was clearly different for all the three <it>Entamoeba </it>species. The nested multiplex PCR showed a sensitivity of 94% and specificity of 100% for the demonstration of <it>E. histolytica</it>, <it>E. moshkovskii </it>and <it>E. dispar </it>DNA in stool samples. The PCR was positive for <it>E. histolytica</it>, <it>E. moshkovskii </it>and <it>E. dispar </it>in a total of 190 out of 202 stool specimens (94% sensitive) that were positive for <it>E. histolytica/E. dispar</it>/<it>E. moshkovskii </it>by examination of stool by microscopy and/or culture. All the 35 negative control stool samples that were negative for <it>E. histolytica/E. dispar</it>/<it>E. moshkovskii </it>by microscopy and culture were also found negative by the nested multiplex PCR (100% specific). The result from the study shows that only 34.6% of the patient stool samples that were positive for <it>E. histolytica/E. dispar</it>/<it>E. moshkovskii </it>by examination of stool by microscopy and/or culture, were actually positive for pathogenic <it>E. histolytica </it>and the remaining majority of the stool samples were positive for non-pathogenic <it>E. dispar </it>or <it>E. moshkovskii </it>as demonstrated by the use of nested multiplex PCR.</p> <p>Conclusion</p> <p>The present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of <it>E. histolytica</it>, <it>E. dispar </it>and <it>E. moshkovskii </it>DNA in stool specimens. The test is highly specific, sensitive and also rapid, providing the results within 12 hours of receiving stool specimens.</p

Detection of excretory Entamoeba histolytica DNA in the urine, and detection of E. histolytica DNA and lectin antigen in the liver abscess pus for the diagnosis of amoebic liver abscess

<p>Abstract</p> <p>Background</p> <p>Amoebic liver abscess (ALA) and pyogenic liver abscesses (PLA) appear identical by ultrasound and other imaging techniques. Collection of blood or liver abscess pus for diagnosis of liver abscesses is an invasive procedure, and the procedure requires technical expertise and disposable syringes. Collection of urine is a noninvasive procedure. Therefore, there has been much interest shown towards the use of urine as an alternative clinical specimen for the diagnosis of some parasitic infections. Here, we report for the first time the detection of <it>E. histolytica </it>DNA excreted in the urine for diagnosis of the cases of ALA.</p> <p>Results</p> <p><it>E. histolytica </it>DNA was detected in liver abscess pus specimen of 80.4% of ALA patients by a nested multiplex polymerase chain reaction (PCR) targeting 16S-like r RNA gene. The nested PCR detected <it>E. histolytica </it>DNA in all 37 (100%) liver abscess pus specimens collected prior to metronidazole treatment, but were detected in only 53 of 75 (70.6%) pus specimens collected after therapy with metronidazole. Similarly, the PCR detected <it>E. histolytica </it>DNA in 21 of 53 (39.6%) urine specimens of ALA patients. The test detected <it>E. histolytica </it>DNA in only 4 of 23 (17.4%) urine specimens collected prior to metronidazole treatment, but were detected in 17 of 30 (56.7%) urine specimens collected after treatment with metronidazole. The enzyme-linked immunosorbent assay (ELISA) for the detection of lectin <it>E. histolytica </it>antigen in the liver abscess pus showed a sensitivity of 50% and the indirect haemagglutination (IHA) test for detection of amoebic antibodies in the serum showed a sensitivity of 76.8% for the diagnosis of the ALA.</p> <p>Conclusion</p> <p>The present study for the first time shows that the kidney barrier in ALA patients is permeable to <it>E. histolytica </it>DNA molecule resulting in excretion of <it>E. histolytica </it>DNA in urine which can be detected by PCR. The study also shows that the PCR for detection of <it>E. histolytica </it>DNA in urine of patients with ALA can also be used as a prognostic marker to assess the course of the diseases following therapy by metronidazole. The detection of <it>E. histolytica </it>DNA in urine specimen of ALA patients provides a new approach for the diagnosis of ALA.</p

Differential detection of , and by nested multiplex PCR on stool samples

<p><b>Copyright information:</b></p><p>Taken from "A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of , and DNA in stool samples"</p><p>http://www.biomedcentral.com/1471-2180/7/47</p><p>BMC Microbiology 2007;7():47-47.</p><p>Published online 24 May 2007</p><p>PMCID:PMC1888694.</p><p></p> The (EM), (EH) and (ED) bands are 553, 439 and 174 bp, respectively. Lane-1, (mono infection); Lane-2, , and (mixed infection); Lane-3, and (mixed infection); Lane-4 and (mixed infection); Lane-5, and (mixed infection); Lane-6 (mono infection), Lane-7 (mono infection); Lane-8, 100 bp DNA ladder (Bangalore genei, Bangalore)

Sensitivity of PCR for detection of minimum number of (EM), (EH) and (ED) cells

<p><b>Copyright information:</b></p><p>Taken from "A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of , and DNA in stool samples"</p><p>http://www.biomedcentral.com/1471-2180/7/47</p><p>BMC Microbiology 2007;7():47-47.</p><p>Published online 24 May 2007</p><p>PMCID:PMC1888694.</p><p></p> 0.05 gm of negative control stool specimen seeded with serially diluted cells corresponding to 10cells (lane 1), 10cells (lane 2), 10cells (lane 3), 10cells (lane 4), 10cells (lane 5), and 10 cells (lane 6) were subjected to DNA extraction followed by PCR amplification. Amplified products were analyzed by agarose gel electrophoresis. The sizes of the amplification products are indicated on the left (in base pairs). Lanes 7, negative control (PCR without DNA

Result of nested multiplex PCR on representative liver abscess pus and urine specimen

<p><b>Copyright information:</b></p><p>Taken from "Detection of excretory DNA in the urine, and detection of DNA and lectin antigen in the liver abscess pus for the diagnosis of amoebic liver abscess"</p><p>http://www.biomedcentral.com/1471-2180/7/41</p><p>BMC Microbiology 2007;7():41-41.</p><p>Published online 18 May 2007</p><p>PMCID:PMC1885440.</p><p></p> The (EH) and Internal amplification control (IAC) bands are 439 and 305 bp respectively. Lane-1 and 4 are positive for DNA in liver abscess pus and urine specimen respectively; Lane-2, 3 and 5 are negative for DNA; Lane-M, 100 bp DNA ladder (Bangalore genei, Bangalore)

Original Article Clinical and serological evaluation of leptospirosis in Puducherry, India

Background: Leptospirosis is a zoonotic disease with a worldwide distribution. There is a paucity of available data about prevalence of this disease in Pondicherry. Our aim was to investigate the seropositivity rate of leptospirosis in suspected cases and also to identify the predominant serogroups present by performing Microscopic Agglutination Test (MAT). The other aim of this study was to compare the results of a commercially available IgM ELISA with that of MAT. Methodology: A total of 110 blood samples from patients suspected of leptospirosis were sent for diagnosis. These samples were subjected to IgM ELISA and the microscopic agglutination test (MAT). MAT was done using a panel of 12 Leptospira serovars. Results: MAT analysis of the 110 samples showed 40 (36%) to be positive. Antibodies were predominantly seen against serogroup Leptospira Icterohemorrhagiae (27%), followed by Pomona (17%), and Pyrogenes (12%). IgM ELISA done on these samples showed a positivity of 37% compared to MAT. Conclusion: This study reveals that the MAT test can be standardized in a diagnostic laboratory and used in conjunction with an IgM ELISA

Reliability of Kirby-Bauer disk diffusion method for detecting meropenem resistance among non-fermenting gram-negative bacilli

Background: Meropenem is empirically used as a last resort for the treatment of infections by non-fermenting gram-negative bacilli (NFGNB). Minimum inhibitory concentration (MIC) determined using agar or broth dilution methods is widely used for testing meropenem resistance. However, it is not possible in resource-poor settings. Aim: A prospective study was performed to evaluate the reliability of Kirby-Bauer disk diffusion (KBDD) method for detecting meropenem resistance among NFGNB. Materials and Methods: A total of 146 NFGNB consisting of 56 Acinetobacter baumannii, 24 Acinetobacter lwoffii, 48 Pseudomonas aeruginosa and 18 Pseudomonas spp. were included in the study. All the isolates were tested simultaneously by both KBDD method and agar dilution method. Results: Very major errors were not observed with A. baumannii, A. lwoffii and P. aeruginosa, while other Pseudomonas spp. showed a very major error rate of about 5.6%. The major error rates observed with A. baumannii, A. lwoffii, P. aeruginosa and Pseudomonas spp. were 1.8%, 0%, 2.1% and 28.6%, respectively. All the isolates showed a good correlation between zone diameters (KBDD method) and MICs (agar dilution method). The sensitivity and specificity of KBDD method for detecting meropenem resistance was above 90% for all the NFGNB except Pseudomonas spp. Conclusions: The KBDD method can be reliably used for routine testing of meropenem resistance in A. baumannii, A. lwoffii and P. aeruginosa. However, further studies are needed before employing this technique for detecting meropenem resistance in Pseudomonas spp

Detection of Cysticercus antigens and antibodies in cerbrospinal fluid of patients with chronic meningitis Detecção de antígenos e anticorpos de Cysticercus em fluido cerebrospinal de pacientes com meningite crônica

Chronic meningitism is a less frequent manifestation of neurocysticercosis caused by Taenia solium cysticerci. In the present study we used Co-agglutination (Co-A), a simple and rapid slide agglutination test to detect specific Cysticercus antigen in the 67 cerebrospinal fluid (CSF) samples from patients with chronic meningitis of unknown etiology. The results were compared with that of ELISA for detection of antibodies. Among these samples four (5.97%) were positive for Cysticercus antigen by Co-A test and six (8.95%) were positive for antibodies by ELISA. Two samples were positive by both Co-A and ELISA, two were positive only by Co-A and four were positive only by ELISA. In the present study, although Cysticercus antigen and antibodies were present in CSF samples from eight (11.94%) patients, we cannot affirm that all the cases of chronic meningitis are due to cysticercosis, but for any case of chronic meningitis of unknown origin, it would be useful to consider the possibility of cysticercal meningitis.<br>Meningite crônica é manifestação pouco freqüente de neurocisticercose causada por cisticerco de Taenia solium. No presente estudo utilizamos co-aglutinação (Co-A) um teste simples e rápido de aglutinação para detectar antígeno específico de Cysticercus nas 67 amostras de fluido cerebrospinal (CSF) de pacientes com meningite crônica de etiologia desconhecida. Os resultados foram comparados com os de ELISA para detecção de anticorpos. Dentre estas amostras quatro (5,97%) foram positivas para antígenos de Cysticercus pelo teste Co-A e seis (8,95%) foram positivas para anticorpos por ELISA. Duas amostras foram positivas por ambos Co-A e ELISA, duas foram positivas somente por Co-A e quatro foram positivas somente por ELISA. No presente estudo embora antígenos e anticorpos de Cysticercus estivessem presentes nas amostras de CSF de oito pacientes (11,94%), não podemos afirmar que todos os casos de meningite crônica sejam devidos à cisticercose, mas para qualquer caso de meningite crônica de origem desconhecida seria útil considerar a possibilidade de meningite por cisticerco