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In this study, the nucleotide sequences of the mitochondrial DNA hypervariable region II (HVRII) and inter HVR in the Thai population were investigated. Buccal swab samples were randomly collected from 100 unrelated healthy individuals residing in Thailand. The analysis of HVRII revealed a total of 65 different haplotypes of which 47 were unique and 18 were found in more than one individual. The most frequent haplotype observed was A73G-A249D-A263G-309.1C–315.1C (7%). Nucleotide transition from adenine (A) to guanine (G) was the most frequent substitution. Nucleotide transversions were also observed but with a lower frequency compared to nucleotide transitions. Insertions of C (57%), CC (14%), and CCC (1%) were found at position 309. Twenty-six samples were found to exhibit nucleotide deletion at position 249. The haplotype diversity, random match probability, and discrimination power were calculated to be 0.9859, 0.0240, and 0.976 respectively. In addition, the outside HVR fragment within the control region at positions 16381–72 (inter HVRI/HVRII) and 341–437 (inter HVRII/HVRIII) were sequenced in order to examine the polymorphic sites. High polymorphism was found at positions 16519, 150, 249, and 309. It was concluded that the nucleotide sequence variations in HVRII and inter HVRI/HVRII would be useful for forensic identification.

Blow flies are worldwide the most important insects from a forensic point of view. In Thailand, aside from the two most common species, Chrysomya megacephala (F.) and Chrysomya rufifacies (Macquart), Chrysomya chani Kurahashi was also found to be of forensic importance. We present a case of a human female cadaver in its bloated stage of decomposition, discovered at Pachangnoi Subdistrict, northern Thailand. Entomological sampling during the autopsy displayed an assemblage of numerous dipteran larvae. Macroscopic observations showed the coexistence of third instar larvae of the three blow flies C. megacephala, Chrysomya villeneuvi Patton, an unknown blow fly species and one muscid, Hydrotaea sp. The minimum post-mortem interval was estimated to be six days, based on the developmental rate of C. megacephala. The ID of the unknown larva, which is the focus of this report, was revealed later as C. chani by DNA sequencing, using a 1205 bp of cytochrome c oxidase subunit I (COI). The occurrence of C. chani on a human body revealed the need to analyse and describe the morphology of its immature stage, to enable forensic entomologists to identify this fly species in future cases. The morphological examination of the third instar was performed, revealing peculiar characteristics: protuberant tubercles encircling abdominal segments; 9–11 lobes on the anterior spiracle; six prominent pairs of tubercles along the peripheral rim of the eighth abdominal segment; a heavily sclerotized complete peritreme of the posterior spiracles. A key to differentiate the third instar of blow flies of forensic importance in Thailand is provided.

Mitochondrial DNA (mtDNA) analysis has displayed an important role and been considered as a powerful tool in various fields of forensic science applications. Nowadays, single nucleotide polymorphisms (SNPs) on mtDNA have become additional DNA markers when conventional STR typing practically fails. mtDNA sequencing of polymerase chain reaction (PCR) products from the hypervariable region I (HVRI) and II (HVRII) is the standard method of mtDNA analysis. However, mtDNA sequencing is rather expensive, time consuming and technically complex. This study aims to develop the SNPs minisequencing for screening of Thai populations. For this purpose, sixteen SNPs that possess high discriminating power in hypervariable regions were selected. The DNA samples were obtained from 100 buccal swab samples of Thai healthy individuals. All DNA samples were extracted and were subsequently amplified by single duplex PCR technique. The duplex PCR products were genotyped by SNPs minisequencing. Based on 16 SNPs, a total of 63 haplotypes were observed of which 46 haplotypes were unique. The haplotype diversity, discriminating power and random match probability were calculated to be 0.9830, 0.9732 and 0.0268, respectively. The SNPs at 150, 199, 489, 16129, 16189, 16223, and 16304 were highly polymorphic in the studied population. Our results suggested that the SNPs minisequencing can be an alternative method of SNPs genotyping. This method can be used for an exclusion of a large number of mismatch samples and as a presumptive test prior to do confirmatory mtDNA sequencing.

In Thailand, the predominant HIV subtype is E, rather than subtype B as in North America and Europe. Subtype E has the ability to replicate in vitro in Langerhans cells. We hypothesized that this cell type might constitute a reservoir for the HIV virus in infected lymph nodes. We examined lymph nodes from 25 HIV-1 subtype E-infected patients to determine the immunophenotype of HIV-1-infected cells, their numbers and their distribution. The presence of HIV was detected either by in situ reverse transcriptase-polymerase chain reaction or immunoperoxidase. Cell identity was determined by double labelling using alkaline phosphatase-based immunohistochemistry. The majority of HIV-infected cells in the lymph nodes were Langerhans cells (CD1a+S100+) and Langerhans-related dendritic cells (p55+S100+). These cells were located in the paracortical areas of lymph nodes, with a few cells scattered at the edges of germinal centers, but were absent from germinal centers themselves, in contrast to the reported distribution of subtype B virus. In addition, multinucleated giant cells were significantly more common in HIV-infected nodes (64%) compared to controls (4%) (P=0.00002). In conclusion, Langerhans histiocytes and related cells are reservoirs for HIV subtype E in lymph nodes. Disrupting the pathway of infection of Langerhans cells and related cells may be a viable strategy to interfere with transmission of HIV subtype E.

Antiretroviral treatment with zidovudine (ZDV) from the 14th week until the end of pregnancy has markedly reduced the vertical transmission rate of HIV-1 in Europe and North America. A shorter duration of treatment has reduced this rate in Africa and Southeast Asia to a lesser degree. In Southeast Asia, subtype E is the major subtype rather than subtype B as in Western countries. The goals of this study were to determine the optimal duration of ZDV prophylaxis for subtype E and to confirm its effectiveness at the histologic level. Fifty pregnant women seropositive for HIV-1 subtype E were given ZDV prophylaxis consisting of 300 mg administered twice daily, switching to 300 mg administered every 3 hours from the onset of labor until delivery. Twenty-seven received "short-term" ZDV lasting 14 to 35 days before delivery, whereas the other 23 received "long-term" ZDV lasting 62 to 92 days. The effectiveness of ZDV prophylaxis was assessed by detection of HIV-1 in the placenta using in situ polymerase chain reaction (PCR). All babies in this study were tested up to one year of age. Three were not positive until after one month of age, but one was positive as a neonate. Four neonates were positive for HIV-1 as detected by PCR on peripheral blood, including one in the neonatal period. All cases were from the short-term prophylaxis group. Decidual glandular epithelial cells were the only cell type in the placenta that expressed HIV proviral DNA under ZDV prophylaxis. Sixty-seven percent of placentas in the short-term ZDV group showed more than occasional positive cells compared with 22% in the group receiving long-term ZDV prophylaxis (P < 0.02). This is first study to compare the effectiveness of short-term and long-term ZDV prophylaxis with respect to the presence of HIV in the placenta. Our study shows that longer (at least 60 days) prophylaxis is more effective in reducing HIV expression in the placenta and is associated with reduced transmission to neonates.

In Thailand, the predominant HIV subtype is E, rather than Subtype B as in North America and Europe, and the predominant mode of transmission is heterosexual contact. Subtype E has the ability to replicate in vitro in Langerhans cells. We hypothesized that this cell type might constitute a reservoir for the HIV virus in vaginal mucosa of asymptomatic carriers. To examine this hypothesis, we compared vaginal tissue histology in HIV-1-seropositive cases with seronegative cases and determined the immunophenotype of HIV-1-infected cells, their numbers, and their distribution in vaginal mucosa. Vaginal biopsies were performed at four different sites from six asymptomatic HIV-1 Subtype E-infected persons and from six seronegative cases at necropsy and examined histologically. Immunophenotyping was performed using immunoperoxidase for Gag p24 HIV, CD3, CD20, CD68, CD1a, S-100 and p55 antigens and by double labeling, combining immunoperoxidase with alkaline phosphatase using pairs of the above antigens. Twenty of twenty-four vaginal biopsies (83.3%) from HIV-seropositive cases showed definite inflammation compared to five of twenty-four vaginal necropsies (20.8%) from HIV-seronegative cases. One third of HIV-seropositive biopsies (8/24) demonstrated p24-positive cells in the epithelium, whereas three-fourths (18/24) of the biopsies revealed p24-positive cells in the lamina propria. All seropositive patients showed positive cells in at least one biopsy, but not all biopsies contained positive cells. Infected cells were more frequently observed at sites of greater inflammation. The dendritic cell count in HIV-seropositive vaginal epithelium was significantly higher than that observed in the seronegative cases (P =.004). The majority of p24-positive cells in the vaginal epithelium were Langerhans cells (CD1a+/S-100+), whereas in the lamina propria, about half of p24-positive cells were Langerhans-related dendritic cells (p55+ and S-100+) and half were T lymphocytes. In conclusion, the increased propensity for heterosexual transmission of Subtype E may be related to vaginal inflammation, leading to the accumulation of Langerhans cells and related dendritic cells which, once infected with HIV, can act as a reservoir for further virus transmission.

The frequency and the cellular basis for HIV-1 transmission from mother to child in the early gestational period are poorly understood. We compared the placentas of 24 women seropositive for HIV-1 subtype E and who had not received any antiretroviral drugs, to placentas of 25 seronegative women. All placentas were obtained during therapeutic abortion at 6–23 weeks gestation. Placentas and fetal organs were examined by routine light microscopy, immunostaining for p24 capsid protein, and in situ PCR to localize which cells were infected with HIV-1 subtype E. The number of previous abortions was not a factor in placental HIV infection since this number was higher in seronegative women (P

We wish to thank Drs. Kreitchmann Canti Gomes da Silva Barcelos and Fuchs for their letter about our paper and for their interest and comments. We would like to take this opportunity to provide some additional information in response to their questions. The authors asked about whether there was a significant correlation between maternal viral load and HIV expression in placenta. In our series we did not find that there was a significant correlation. Whether this is related to small sample size or differences in subtype E behavior is unclear. We did find examination of the placenta to be informative with respect to HIV expression. We would recommend that this organ be examined in cases of HIV positive mothers for a better understanding of the pathogenesis of HIV transmission to fetuses and infants even if not for any intrinsic prognostic value. (excerpt)