MALARIA DIAGNOSIS BY LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) IN THAILAND.

The loop-mediated isothermal amplification method (LAMP) is a recently developed molecular technique that amplifies nucleic acid under isothermal conditions. For malaria diagnosis, 150 blood samples from consecutive febrile malaria patients, and healthy subjects were screened in Thailand. Each sample was diagnosed by LAMP, microscopy and nested polymerase chain reaction (nPCR), using nPCR as the gold standard. Malaria LAMP was performed using Plasmodiumgenus and Plasmodium falciparum specific assays in parallel. For the genus Plasmodium, microscopy showed a sensitivity and specificity of 100%, while LAMP presented 99% of sensitivity and 93% of specificity. For P. falciparum, microscopy had a sensitivity of 95%, and LAMP of 90%, regarding the specificity; and microscopy presented 93% and LAMP 97% of specificity. The results of the genus-specific LAMP technique were highly consistent with those of nPCR and the sensitivity of P. falciparum detection was only marginally lower.

Comparative diagnosis of malaria infections by microscopy, nested PCR, and LAMP in northern Thailand.

Three methods, microscopy, nested polymerase chain reaction (nPCR), and loop-mediated isothermal amplification (LAMP) have been applied for malaria diagnosis in 105 human blood samples collected in Northern Thailand. Only Plasmodium falciparum and Plasmodium vivax infections were detected. A total number of 57 positives (54%) could be detected for P. falciparum and 25 (24%) for P. vivax when all samples that were positive in any of the three methods are counted together. The nPCR was used as a reference standard for comparison with the other methods, microscopy and LAMP. The sensitivity of LAMP for P. falciparum was 100%. All nPCR-negative samples for P. falciparum were also negative by both microscopy and LAMP (specificity, 100%). For diagnosis of P. vivax, microscopy detected 15 of 23 nPCR-positive samples (sensitivity, 65%). LAMP detected 22 of 23 nPCR-positives (sensitivity, 96%). Among the 82 nPCR-negative samples microscopy detected two samples (specificity, 98%). All 82 nPCR-negative were also negative by the LAMP method (specificity, 100%). Both Plasmodium genus- and species-specific LAMP primer sets yielded the same results in all samples. There were no significant differences in the prevalence detected by each method. We assume that LAMP was as reliable as nPCR and more reliable than microscopy in the detection of Plasmodium DNA tested in the examined Thai field blood samples. This study further validates LAMP as an alternative molecular diagnostic tool, which can be used in the diagnosis of early infections of malaria cases and together with nPCR can also be used as supplementary methods for clinical and epidemiological use.

Circulation of dengue serotypes in five provinces of northern Thailand during 2002-2006.

disease prevalent in all provinces of Thailand. This study was to determine the circulating dengue serotypes by reverse transcription polymerase chain reaction (RT-PCR). A total of 1116 seropositive acute samples were analysed from DF/DHF patients in five provinces of northern Thailand (Chiangmai, Lampang, Lamphun, Mae Hong Son and Phrae) during the period January 2002 to December 2006. Five hundred and fifty-nine samples were found positive, of which 47.2%, 30.6%, 18.4% and 3.8% were affected with DENV-2, DENV-1, DENV-4 and DENV-3 respectively. From 2002 to 2005, the predominant dengue serotype was DENV-2, whereas DENV-1 was predominant in 2006. There was an apparent increase in the percentage of DENV-4 from 2005 to 2006. Our results indicated that all four dengue serotypes were circulating in this region and the annual change of predominant serotypes was the cause of the severity of the disease.

Determination and geographic distribution of Orientia tsutsugamushi serotypes in Thailand by nested polymerase chain reaction.

Clotted blood samples of 240 scrub typhus patients were collected from 8 Regional Medical Sciences Centers in Thailand during 1999 to 2002. The serotypes of Orientia tsutsugamushi and their geographic distribution were determined. A nested polymerase chain reaction (PCR) was used to identify the serotypes of O. tsutsugamushi. The number of patients with positive results for O. tsutsugamushi was 25.0%. Two serotypes, Karp and Kato, were detected in these samples. No Gilliam serotype was detected from any of the study locations. The PCR products were sequenced using an automated DNA sequencer. The nucleotide sequence of gene encoding 56-kDa protein from these samples showed a high sequence homology with the reference sequence of O. tsutsugamushi Karp and Kato serotypes. O. tsutsugamushi Karp serotype was predominant throughout Thailand with the percentage of 96.8% of the total serotype-positive patients, whereas 3.2% for Kato serotype was observed only in the south. The highest number among the region of Karp serotype-positive patients of 31.6% was found in the northeast.

Early diagnosis of scrub typhus in Thailand from clinical specimens by nested polymerase chain reaction.

The early detection of scrub typhus in Thailand by nested polymerase chain reaction (PCR) is presented. The diagnosis of scrub typhus, from clinical samples obtained from hospitals in the northern part of Thailand, by nested PCR was compared to immunofluorescence (IF) and Weil-Felix (WF) tests. The primer pairs used for the nested PCR were designed on the basis of the nucleotide sequence of the gene that encodes the 56-kDa antigen, and RFLP analysis was used for identification. Clotted blood from 80 patients suspected of scrub typhus infection were tested. With the IF test, antibodies for Orientia tsutsugamushi were observed in 38 patients checking IgM and IgG titers. Only 21 patients showed positive seroconversion while 17 patients were negative. For the WF test, only 13 patients gave a positive seroconversion. In the early stage of infection, 19, 13 and 3 patients were detected with a sensitivity of 90.47% (19/21), 61.90% (13/21) and 14.28% (3/21) by the nested PCR, IF and WF test respectively. Two patients who were negative for seroconvesion by IF and WF were positive by nested PCR. Therefore, this suggests that nested PCR is applicable for specific rapid diagnosis at an early stage of scrub typhus in endemic regions.