Reliable interpretation and long-term stability using SYBRTM safe fluorescent assay for loop-mediated isothermal amplification (LAMP) detection of Leishmania spp

@#Leishmaniasis, a vector-borne disease caused by Leishmania, is the second leading mortality after malaria. Continuously increasing cases of cutaneous and visceral leishmaniasis (CL/VL) have been documented in Thailand. Recently, loop-mediated isothermal amplification (LAMP) based on malachite green (MG) colorimetric assay that detects Leishmania DNA was developed to facilitate epidemiological studies of leishmaniasis in affected areas. However, ambiguous reading interpretation sometimes occurred using the MG-LAMP assay. In this study, the efficiency and effectiveness of the SYBRTM Safe fluorescent assay for LAMP detection of Leishmania siamensis (MON-324) and Leishmania martiniquensis (MON-229) were compared under two different light sources, i.e., blue light and ultraviolet light transilluminators. Regarding the SYBRTM-LAMP assay, the detection limit of DNA of both L. siamensis and L. martiniquensis was 103 parasites/mL. The assay exhibited consistency and reproducibility without requiring any post-reaction preparations. The dye is generally available, affordable and safe while reliable interpretation can be easily visualized under both blue light and ultraviolet light transilluminators. Using buffy coat of VL patients, the SYBRTM-LAMP offers an alternative method for screening samples with high sensitivity and specificity. This cost effective SYBRTM Safe fluorescent assay is simple to use without ambiguous evaluation which could provide another suitable choice of a standard LAMP assay in molecular laboratories as well as further development in field studies.

Autochthonous visceral leishmaniasis: a report of a second case in Thailand.

Visceral leishmaniasis (VL) is a rare disease in Thailand. Only one previous case has been reported in which transmission was likely autochthonous. We conducted an investigation of a case of VL, which included serological and symptom surveys of people who lived near the case in Nan Province and Bangkok, serological surveys of domestic animals in his home village and sand fly surveys in his home village and in Bangkok. No humans interviewed met our case definition for possible VL. One hundred thirty-one villagers were seronegative for Leishmania antibodies. We found three cows and one cat that had positive direct agglutination tests for Leishmania spp, but we were unable to confirm current infection by PCR. Sand fly surveys showed that most of the flies were of the Sergentomyia genus, which has not previously been reported as a competent vector in Thailand. Nonetheless, we conclude, based on the patient's lack of travel outside Thailand and the presence of seropositive domestic animals in his home village, that he was most likely infected by the bite of a sand fly in Thailand. We believe this is the second case of autochthonously transmitted VL in Thailand.

Intestinal microsporidiosis in HIV-infected children with acute and chronic diarrhea.

A prospective study of intestinal microsporidiosis in HIV-positive children was conducted at the Queen Sirikit National Institute of Child Health and Phramongkutklao Hospital, Bangkok, Thailand. Hospitalized HIV-positive children with and without diarrhea were enrolled in this study. Microsporidial spores identified by calcofluor fluorescent and gram-chromotrope stain were confirmed by electron microscopy. As well as Cryptosporidium parvum, Microsporidia was the most common protozoa found in the present study, each was 7.1%. Microsporidia was significantly more common in those who had diarrhea. Intestinal microsporidiosis was found in HIV-positive children with both acute and chronic diarrhea. This study emphasizes the importance of Microsporidia in HIV-infected children. Early detection of microsporidia could be of benefit for the patients, since the infection is treatable.

Transmission of intestinal blastocystosis related to the quality of drinking water.

A cross-sectional study was performed to evaluate the risk factors of Blastocystis hominis infection in the Thai army population of the 11th Infantry Division, Chachoengsao Province, Thailand. 201 army personnel and their family members were enrolled in this study. Intestinal parasitic infections in this population were assessed by stool examination using simple smear, formalin/ether technique and Kato-thick smear. Approximately one third of the specimens were positive for one or more intestinal parasites. With the prevalence of 21.9%, B. hominis was the most common intestinal parasite found in this population. Our data indicated that blastocystosis in this army population was significantly linked to the quality of drinking water. After being adjusted for potential confounders, consuming neither filtered nor boiled water was independently associated with blastocystosis.