Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp. are not only three of the most important and common diarrhea-causing parasitic protozoa, but they often have similar clinical presentations. Microscopic diagnosis of these parasites is neither sensitive nor specific. Recently, more specific and sensitive alternative molecular methods (polymerase chain reaction [PCR] and antigen detection tests) have been introduced for all three of these parasitic infections. The use of these molecular diagnostic tests in routine diagnostic laboratories is still limited. In this study, we developed a multiplex real-time PCR assay for the simultaneous detection of E. histolytica, G. intestinalis, and Cryptosporidium spp. in one reaction using species-specific probes. This assay was evaluated using clinical specimens and was found to be quite sensitive and specific. The reagents used in this multiplex PCR assay can also be used for detection of these parasites individually. The use of this real-time PCR multiplex assay in developing countries at present will have limited scope for routine diagnosis because the cost will be high for a single test, although in the developed world, the test could see immediate application.

BackgroundGeneXpert MTB/RIF (Xpert) and Genotype MTBDRplus (DRplus) are two World Health Organization (WHO) endorsed probe based molecular drug susceptibility testing (DST) methods for rapid diagnosis of drug resistant tuberculosis. Both methods target the same 81 bp Rifampicin Resistance Determining Region (RRDR) of bacterial RNA polymerase β subunit (rpoB) for detection of Rifampicin (RIF) resistance associated mutations using DNA probes. So there is a correspondence of the probes of each other and expected similarity of probe binding.MethodsWe analyzed 92 sputum specimens by Xpert, DRplus and LJ proportion method (LJ-DST). We compared molecular DSTs with gold standard LJ-DST. We wanted to see the agreement level of two molecular methods for detection of RIF resistance associated mutations. The 81bp RRDR region of rpoB gene of discrepant cases between the two molecular methods was sequenced by Sanger sequencing.ResultsThe agreement of Xpert and DRplus with LJ-DST for detection of RIF susceptibility was found to be 93.5% and 92.4%, respectively. We also found 92.4% overall agreement of two molecular methods for the detection of RIF susceptibility. A total of 84 out of 92 samples (91.3%) had agreement on the molecular locus of RRDR mutation by DRplus and Xpert. Sanger sequencing of 81bp RRDR revealed that Xpert probes detected seven of eight discrepant cases correctly and DRplus was erroneous in all the eight cases.ConclusionAlthough the overall concordance with LJ-DST was similar for both Xpert and DRplus assay, Xpert demonstrated more accuracy in the detection of RIF susceptibility for discrepant isolates compared with DRplus. This observation would be helpful for the improvement of probe based detection of drug resistance associated mutations especially rpoB mutation in M. tuberculosis.

The noninvasive diagnosis of amebic liver abscess is challenging, as most patients at the time of diagnosis do not have a concurrent intestinal infection with Entamoeba histolytica. Fecal testing for E. histolytica parasite antigen or DNA is negative in most patients. A real-time PCR assay was evaluated for detection of E. histolytica DNA in blood, urine, and saliva samples from amebic liver abscess as well as amebic colitis patients in Bangladesh. A total of 98 amebic liver abscess and 28 amebic colitis patients and 43 control subjects were examined. The real-time PCR assay detected E. histolytica DNA in 49%, 77%, and 69% of blood, urine, and saliva specimens from the amebic liver abscess patients. For amebic colitis the sensitivity of the real-time PCR assay for detection of E. histolytica DNA in blood, urine, and saliva was 36%, 61%, and 64%, respectively. All blood, urine, and saliva samples from control subjects were negative by the real-time PCR assay for E. histolytica DNA. When the real-time PCR assay results of the urine and saliva specimens were taken together (positive either in urine or saliva), the real-time PCR assay was 97% and 89% sensitive for detection of E. histolytica DNA in liver abscess and intestinal infection, respectively. We conclude that the detection of E. histolytica DNA in saliva and urine could be used as a diagnostic tool for amebic liver abscess.Entamoeba histolytica is a protozoan parasite that causes amebic diarrhea, colitis, and amebic liver abscess (ALA), mostly in developing countries (5,7,22,25). Eighty percent of infected individuals remain asymptomatic carriers, while the other 20% develop clinically overt disease (7,9,22,25). About 50 million symptomatic cases of amebiasis occur worldwide each year, resulting in 40,000 to 100,000 deaths annually (25). Mortality from amebiasis is mainly due to extra-amebic colitis, of which ALA is the most common.It is difficult to differentiate ALA from pyogenic liver abscess or other space-occupying lesions of the liver. Imaging techniques such as ultrasound, computed tomography, and magnetic resonance have excellent sensitivities for the detection of liver abscess arising from any cause, but there are no findings specific for ALA (13). Further complicating the diagnosis is the fact that most patients with an ALA do not have coexistent intestinal infection with E. histolytica (11). Therefore, detection of E. histolytica antigen or DNA in stool samples is not very helpful for the diagnosis of ALA (1,6,8,12).The current means for diagnosis of ALA is the detection of antiamebic antibody by serological tests combined with aspiration of the abscess. The presence of serum antibodies against E. histolytica and the absence of bacteria in the abscess fluid are consistent with an ALA. A drawback of serologic tests is that the serum antibody levels in people from areas of endemicity remain positive for years after infection with E. histolytica (3,16,23). Therefore, antiamebic antibodies in the serum may be due to amebiasis in the past, limiting th...

Background: Rifampicin resistance (RR) is a key indicator of multidrug-resistant tuberculosis (MDR-TB) and 95% of the RR is associated with the mutation in the 81-bp rifampicin resistance determining region (RRDR) of the rpoB gene of Mycobacterium tuberculosis complex (MTBC). The Xpert MTB/RIF (Xpert) assay uses five overlapping molecular beacon probes (A-E) complementary to RRDR region that detect MTBC and mutations associated with RR. The objective of the study was to investigate the distribution and frequency of mutations detected by Xpert assay among Beijing and non-Beijing RR-TB isolates. Methods: A total of 205 randomly selected RR-TB specimens detected by Xpert assay were included in this study. A portion of specimens was further subjected to culture, MTBDRplus test and the positive culture isolates were genotyped by spoligotyping. Results: We found that the most frequent mutation occurred at probe E (S531L) binding region in both Beijing and non-Beijing isolates (61.9% and 66.9%, respectively). The Beijing family had higher mutation rates than non-Beijing (19.0% vs 12.4%) at probe B (D516V) while the non-Beijing family had higher mutations at probe D (H526D or H526Y) than the Beijing (13.2% vs 10.7%) family. Mutations at probes Aand C were less common in both Beijing and non-Beijing isolates. There was no significant difference (P=0.36) in the occurrence of mutations at different probes between Beijing and non-Beijing isolates. Conclusions: The study results revealed that the most frequent mutation occurs in the region of probe E and the least common mutations at probe A and C among both Beijing and non-Beijing RR-TB cases. This first insight into the probe mutation variation and frequencies among the RR-TB cases in Bangladesh forms the baseline information for further investigation.

Genotypic methods for drug susceptibility testing of Mycobacterium tuberculosis are desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We developed a microfluidic TaqMan array card (TAC) that utilizes both sequence-specific probes and high-resolution melt analysis (HRM), providing two layers of detection of mutations. Twenty-seven primer pairs and 40 probes were designed to interrogate 3,200 base pairs of critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncA genes. The method was evaluated on 230 clinical M. tuberculosis isolates from around the world, and it yielded 96.1% accuracy (2,431/2,530) in comparison to that of Sanger sequencing and 87% accuracy in comparison to that of the slow culture-based susceptibility testing. This TAC-HRM method integrates assays for 10 genes to yield fast, comprehensive, and accurate drug susceptibility results for the 9 major antibiotics used to treat TB and could be deployed to improve treatment outcomes.