Krishna Khairnar

Multiplex real-time quantitative PCR, microscopy and rapid diagnostic immuno-chromatographic tests for the detection of Plasmodium spp: performance, limit of detection analysis and quality assurance.

BackgroundAccurate laboratory diagnosis of malaria species in returning travelers is paramount in the treatment of this potentially fatal infectious disease.Materials and methodsA total of 466 blood specimens from returning travelers to Africa, Asia, and South/Central America with suspected malaria infection were collected between 2007 and 2009 at the reference public health laboratory. These specimens were assessed by reference microscopy, multipex real-time quantitative polymerase chain reaction (QPCR), and two rapid diagnostic immuno-chromatographic tests (ICT) in a blinded manner. Key clinical laboratory parameters such as limit of detection (LOD) analysis on clinical specimens by parasite stage, inter-reader variability of ICTs, staffing implications, quality assurance and cost analysis were evaluated.ResultsQPCR is the most analytically sensitive method (sensitivity 99.41%), followed by CARESTART (sensitivity 88.24%), and BINAXNOW (sensitivity 86.47%) for the diagnosis of malaria in returning travelers when compared to reference microscopy. However, microscopy was unable to specifically identify Plasmodia spp. in 18 out of 170 positive samples by QPCR. Moreover, the 17 samples that were negative by microscopy and positive by QPCR were also positive by ICTs. Quality assurance was achieved for QPCR by exchanging a blinded proficiency panel with another reference laboratory. The Kappa value of inter-reader variability among three readers for BINAXNOW and CARESTART was calculated to be 0.872 and 0.898 respectively. Serial dilution studies demonstrated that the QPCR cycle threshold correlates linearly with parasitemia (R2 = 0.9746) in a clinically relevant dynamic range and retains a LOD of 11 rDNA copies/μl for P. falciparum, which was several log lower than reference microscopy and ICTs. LOD for QPCR is affected not only by parasitemia but the parasite stage distribution of each clinical specimen. QPCR was approximately 6-fold more costly than reference microscopy.DiscussionThese data suggest that multiplex QPCR although more costly confers a significant diagnostic advantage in terms of LOD compared to reference microscopy and ICTs for all four species. Quality assurance of QPCR is essential to the maintenance of proficiency in the clinical laboratory. ICTs showed good concordance between readers however lacked sensitivity for non-falciparum species due to antigenic differences and low parasitemia.ConclusionMultiplex QPCR but not ICTs is an essential adjunct to microscopy in the reference laboratory detection of malaria species specifically due to the superior LOD. ICTs are better suited to the non-reference laboratory where lower specimen volumes challenge microscopy proficiency in the non-endemic setting.

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

BackgroundE. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar 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 E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples.ResultsThe species specific product size for E. histolytica, E. moshkovskii and E. dispar was 439, 553 and 174 bp respectively, which was clearly different for all the three Entamoeba species. The nested multiplex PCR showed a sensitivity of 94% and specificity of 100% for the demonstration of E. histolytica, E. moshkovskii and E. dispar DNA in stool samples. The PCR was positive for E. histolytica, E. moshkovskii and E. dispar in a total of 190 out of 202 stool specimens (94% sensitive) that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture. All the 35 negative control stool samples that were negative for E. histolytica/E. dispar/E. moshkovskii 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 E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture, were actually positive for pathogenic E. histolytica and the remaining majority of the stool samples were positive for non-pathogenic E. dispar or E. moshkovskii as demonstrated by the use of nested multiplex PCR.ConclusionThe present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii DNA in stool specimens. The test is highly specific, sensitive and also rapid, providing the results within 12 hours of receiving stool specimens.

A Possible Cluster of Sexually Transmitted Entamoeba histolytica: Genetic Analysis of a Highly Virulent Strain

BACKGROUND Transmission of Entamoeba histolytica generally occurs by fecal excretion of cysts followed by oral ingestion of contaminated food or water. However, fecal-oral transmission may occur within households and long-term care institutions, and sexual transmission occurs among men who have sex with men. Epidemiologically linked clusters of E. histolytica infection are rare in industrialized countries. We report such a sexually linked cluster in Canada. METHODS An index case involving a young female with an amebic liver abscess led to an epidemiological investigation of sexual contacts. Anti-amebic serological analysis, stool specimen examinations, and abdominal ultrasounds were done for the contacts. Enzyme-linked immunosorbent assay was done for stool antigen specific to E. histolytica. Genotyping and phylogenetic analysis was performed on 1 stool isolate. RESULTS By tracing sexual contacts related to the index case, we uncovered a cluster of 7 cases of amebiasis (3 with liver abscesses). Oral-anal sex was common in the group; the 5 female individuals were bisexual (4) or homosexual (1). The outbreak strain was genotyped, and cluster analysis indicated that this virulent strain differed substantially from asymptomatic or diarrheal E. histolytica isolates. CONCLUSIONS E. histolytica can be transmitted by heterosexual activity as well as male and female homosexual activity. Patients with amebiasis should be counselled about possible sexual transmission.

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

BackgroundAmoebic 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 E. histolytica DNA excreted in the urine for diagnosis of the cases of ALA.ResultsE. histolytica 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 E. histolytica 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 E. histolytica DNA in 21 of 53 (39.6%) urine specimens of ALA patients. The test detected E. histolytica 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 E. histolytica 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.ConclusionThe present study for the first time shows that the kidney barrier in ALA patients is permeable to E. histolytica DNA molecule resulting in excretion of E. histolytica DNA in urine which can be detected by PCR. The study also shows that the PCR for detection of E. histolytica 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 E. histolytica DNA in urine specimen of ALA patients provides a new approach for the diagnosis of ALA.

Diagnosis of intestinal amoebiasis by using nested polymerase chain reaction-restriction fragment length polymorphism assay.

BackgroundMicroscopy is unreliable to distinguish the pathogenic Entamoeba histolytica from the nonpathogenic Entamoeba dispar or Entamoeba moshkovskii in stool specimens.MethodsNested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was carried out to detect E. histolytica, E. dispar, and E. moshkovskii DNA in stool samples of 202 patients positive for E. histolytica, E. dispar, or E. moshkovskii by microscopy or culture and in 35 controls. The TechLab E. histolytica II enzyme-linked immunosorbent assay (ELISA) was performed to detect Gal/GalNAc lectin in 45 stool samples positive for E. histolytica, E. dispar, or E. moshkovskii by microscopy or culture. Rapid-indirect hemagglutination assay (IHA) was performed to detect serum antiamoebic antibodies in the 85 patients positive for E. histolytica, E. dispar, or E. moshkovskii in their stool specimens and in the 35 controls.ResultsNested PCR-RFLP was positive in 175 of 202 (86.6%) patient stool samples and was negative in all 35 negative control stool samples. ELISA was positive in 29 of 45 (64.4%) patient stool samples. The IHA test was positive in 19 of 85 (22.4%) patient serum samples and in one (2.8%) of the 35 control serum samples. Nested PCR-RFLP detected E. histolytica DNA in stool specimens of 12 (63.2%) of 19 seropositive patients, and in 31 (47%) of 66 seronegative patients. TechLab E. histolytica II ELISA detected E. histolytica antigen in stool specimens of six (54.5%) of 11 seropositive patients, and in 23 (67.6%) of 34 seronegative patients.ConclusionsNested PCR-RFLP was useful for the specific detection of E. histolytica, E. dispar, and E. moshkovskii in stool samples.

Artemether resistance in vitro is linked to mutations in PfATP6 that also interact with mutations in PfMDR1 in travellers returning with Plasmodium falciparum infections

BackgroundMonitoring resistance phenotypes for Plasmodium falciparum, using in vitro growth assays, and relating findings to parasite genotype has proved particularly challenging for the study of resistance to artemisinins.MethodsPlasmodium falciparum isolates cultured from 28 returning travellers diagnosed with malaria were assessed for sensitivity to artemisinin, artemether, dihydroartemisinin and artesunate and findings related to mutations in pfatp6 and pfmdr1.ResultsResistance to artemether in vitro was significantly associated with a pfatp6 haplotype encoding two amino acid substitutions (pfatp6 A623E and S769N; (mean IC50 (95% CI) values of 8.2 (5.7 – 10.7) for A623/S769 versus 623E/769 N 13.5 (9.8 – 17.3) nM with a mean increase of 65%; p = 0.012). Increased copy number of pfmdr1 was not itself associated with increased IC50 values for artemether, but when interactions between the pfatp6 haplotype and increased copy number of pfmdr1 were examined together, a highly significant association was noted with IC50 values for artemether (mean IC50 (95% CI) values of 8.7 (5.9 – 11.6) versus 16.3 (10.7 – 21.8) nM with a mean increase of 87%; p = 0.0068). Previously described SNPs in pfmdr1 are also associated with differences in sensitivity to some artemisinins.ConclusionsThese findings were further explored in molecular modelling experiments that suggest mutations in pfatp6 are unlikely to affect differential binding of artemisinins at their proposed site, whereas there may be differences in such binding associated with mutations in pfmdr1. Implications for a hypothesis that artemisinin resistance may be exacerbated by interactions between PfATP6 and PfMDR1 and for epidemiological studies to monitor emerging resistance are discussed.

Novel bacteriophage therapy for controlling metallo-beta-lactamase producing Pseudomonas aeruginosa infection in Catfish

BackgroundThe bacteriophage therapy is an effective antimicrobial approach with potentially important applications in medicine and biotechnology which can be seen as an additional string in the bow. Emerging drug resistant bacteria in aquaculture industry due to unrestricted use of antibiotics warrants more sustainable and environmental friendly strategies for controlling fish infections.The isolated bacteria from fish lesions was characterised based on isolation on selective and differential medium like Pseudomonas agar, gram staining, biochemical tests and 16SrRNA sequencing. The metallo-beta-lactamase (MBL) producing bacterial isolate was evaluated using Imipenem - Ethylenediaminetetraacetic acid (EDTA) disk method. The specific bacteriophage was isolated and concentrated using coal bed developed in our lab at CSIR-NEERI. The isolated and enriched bacteriophage was characterised by nucleotide sequencing and electron microscopy. The phage therapy was applied for treating ulcerative lesion in fish.ResultsThe pathogenic bacterium responsible for causing ulcerative lesions in catfish species (Clarias gariepinus) was identified as Pseudomonas aeruginosa. One out of twenty P. aeruginosa isolate showing multi drug resistance (MDR) was incidentally found to be MBL producing as determined by Imipenem-EDTA disk method. The phage therapy effectively cured the ulcerative lesions of the infected fish in 8–10 days of treatment, with a sevenfold reduction of the lesion with untreated infection control.ConclusionBacteriophage therapy can have potential applications soon as an alternative or as a complement to antibiotic treatment in the aquaculture. We present bacteriophage therapy as a treatment method for controlling MDR P. aeruginosa infection in C. gariepinus. To the best of our knowledge this is a first report of application of phage therapy against MBL producing P. aeruginosa isolated from aquatic ecosystem.

Comparison of molecular diagnostic methods for the detection of Acanthamoeba spp. from clinical specimens submitted for keratitis

Acanthamoeba spp. are responsible for a significant annual number of keratitis (AK) cases leading to vision-threatening disease worldwide. Current methods rely on direct examination of specimens by microscopy and/or culture. The former lacks sensitivity and the latter suffers from a poor turnaround time. We undertook a comparison of all published molecular methods, evaluating performance characteristics such as analytical sensitivity, specificity, limit of detection (LOD), reproducibility, accuracy, and cost of test. The study population comprised 128 patients. Eligible specimens were tested prospectively between April 2007 and May 2010 by microscopy and/or culture. Eleven different specimen types were used including corneal scrapings (51.5%), corneal swab (17.9%), and contact lens material (10.9%). Results of 2 published gel-based polymerase chain reaction (PCR) and 2 published real-time quantitative (Q) PCR methods were compared in a blinded manner to direct microscopic examination and/or culture for the detection of Acanthamoeba in clinical specimens. QPCR (Riviere method) had the highest sensitivity at 89.3%, excellent accuracy using ROC analysis (AUC ∼0.90), lowest LOD down to 0.1 organism per microliter, and superior linear correlation with parasite density (R(2) = 0.9965) when compared with microscopy, culture, and other molecular methods. Phylogenetic analysis using a sequence-based typing method revealed that clinical isolates in this population with AK were genetically distinct from granulomatous amebic encephalitis or environmental isolates. The QPCR method was more expensive (

Chloroquine-resistant malaria in travelers returning from Haiti after 2010 earthquake.

14.80) than traditional methods such as culture (

Artesunate misuse and Plasmodium falciparum malaria in traveler returning from Africa.

2.50) or microscopy (