Real-time loop-mediated isothermal amplification (real-time LAMP) assay for rapid diagnosis of amoebic liver abscess.

Among the parasitic diseases, amoebic liver abscess (ALA) ranks second to malaria in terms of mortality. Due to the poor sensitivity of conventional diagnostic methods, there is a need for the development of effective and rapid diagnostic methods for ALA. Thus, the purpose of this work was to develop a real-time loop-mediated isothermal amplification (RT-LAMP) assay specific to Entamoeba histolytica. Further, we compared the performance of real-time LAMP with conventional and real-time PCR (RT-PCR) targeting 18S small subunit ribosomal RNA (18S SSU rRNA) gene of E. histolytica in patients with ALA. A total of 126 liver samples were obtained for the study. Of these, 96 aspirated pus samples were obtained from patients suffering from an ALA (serology confirmed, anti-amoebic immunoglobulin IgG positive), 19 aspirated pus samples from patients with pyogenic liver abscess (PLA, 16S RNA gene positive) and 11 autopsy liver tissues. The results showed that the DNA of E. histolytica was detected in 81 samples by conventional PCR, 93 by RT-PCR and 95 by RT-LAMP. The analytical sensitivity of the RT-LAMP assay was much higher than the other two techniques. RT-LAMP assay was able to amplify up to one copy of the targeted gene of E. histolytica while conventional PCR and RT-PCR could amplify up to 103 and 102 copies of the targeted gene of E. histolytica, respectively. In conclusion, RT-LAMP proved to be a sensitive, specific and rapid test which can be utilised as an effective tool for the diagnosis of ALA.

A rapid multiplex loop-mediated isothermal amplification (mLAMP) assay for detection of Entamoeba histolytica and Giardia duodenalis.

We developed a rapid multiplex loop-mediated isothermal amplification (mLAMP) assay for two common intestinal parasites-Entamoeba histolytica and Giardia duodenalis, where early detection may be helpful. The mLAMP assay was optimized for the detection of DNA of E. histolytica (18S rRNA gene) and G. duodenalis (Elongation factor 1 alpha gene) from standard strains by using six specific primers FIP (forward inner primer), BIP (backward inner primer), F3 (forward outer primer), B3 (backward outer primer), loopF (forward loop primer), and loopB (backward loop primer) for each gene target. The amplification time was 16-26 min for E. histolytica and 10-15 min for G. duodenalis, and the parasites could be distinguished based on melting-curve analysis for specific annealing temperatures (Tm) of 84°C-86°C and 88°C-90°C for E. histolytica and G. duodenalis, respectively. The analytical sensitivity was one fg, and no cross-reactivity with other intestinal pathogens was observed. Thus, the mLAMP assay could detect and clearly distinguish E. histolytica and G. duodenalis with a rapid turnaround time and excellent analytical sensitivity and specificity.

Occurrence of Giardia, Cryptosporidium, and Entamoeba in wild rhesus macaques (Macaca mulatta) living in urban and semi-rural North-West India.

Giardia duodenalis, Cryptosporidium spp., and Entamoeba spp. are intestinal protozoa capable of infecting a range of host species, and are important causes of human morbidity and mortality. Understanding their epidemiology is important, both for public health and for the health of the animals they infect. This study investigated the occurrence of these protozoans in rhesus macaques (Macaca mulatta) in India, with the aim of providing preliminary information on the potential for transmission of these pathogens between macaques and humans. Faecal samples (n = 170) were collected from rhesus macaques from four districts of North-West India. Samples were analysed for Giardia/Cryptosporidium using a commercially available direct immunofluorescent antibody test after purification via immunomagnetic separation. Positive samples were characterised by sequencing of PCR products. Occurrence of Entamoeba was investigated first by using a genus-specific PCR, and positive samples further investigated via species-specific PCRs for Entamoeba coli, Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii. Giardia cysts were found in 31% of macaque samples, with all isolates belonging to Assemblage B. Cryptosporidium oocysts were found in 1 sample, however this sample did not result in amplification by PCR. Entamoeba spp. were found in 79% of samples, 49% of which were positive for E. coli. Multiplex PCR for E. histolytica, E. dispar and E. moshkovskii, did not result in amplification in any of the samples. Thus in 51% of the samples positive at the genus specific PCR, the Entamoeba species was not identified. This study provides baseline information on the potential for transmission of these zoonotic parasites at the wildlife-human interface.