Chrysomya chani Kurahashi (Diptera: Calliphoridae), a blow fly species of forensic importance: morphological characters of the third larval instar and a case report from Thailand.

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.

Single nucleotide polymorphisms minisequencing in hypervariable regions for screening of Thais.

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.

Nucleotide sequence analysis of the hypervariable region III of mitochondrial DNA in Thais.

This study analyzed the nucleotide sequences of the hypervariable region III (HVRIII) of mitochondrial DNA in Thai individuals. Buccal swab samples were randomly obtained from 100 healthy, unrelated, adult (18-60 years old), volunteer donors living in Thailand. Eighteen different haplotypes were found, of which 11 haplotypes were unique. The most frequent haplotypes observed were 522D-523D. Nucleotide transition from Thymine (T) to Cytosine (C) at position 489 (43%) was the most frequent substitution. Nucleotide transversions were also observed at position 433 (Adenine (A) to C, 1%) and position 499 (Guanine (G) to C, 1%). Fifty-three samples presented nucleotide insertion and deletion of C and A (CA) at position 514-523. Insertion of 1AC (3%) and 2AC (2%) were observed. Deletion of 1CA (53%) and 2CA (2%) at position 514-523 were revealed. The deletion of T at position 459 was observed. The haplotype diversity, random match probability, and discrimination power were calculated to be 0.7770, 0.2308, and 0.7692, respectively.

Relationship of cell bearing EBER and p24 antigens in biopsy-proven lymphocytic interstitial pneumonia in HIV-1 subtype E infected children.

Lymphocytic interstitial pneumonia (LIP) is an uncommon histopathologic entity characterized by infiltration of the interstitium and alveolar spaces of the lung by lymphocytes and other lymphoid elements. An increased incidence of LIP has been seen in the pediatric population, especially in children with acquired immune deficiency syndrome. Our previous study supports the notion that Langerhans cells (LCs) are reservoirs for Epstein-Barr virus (EBV) in lungs of human immunodeficiency virus (HIV) subtype E-infected pediatric LIP. To further understand the pathogenesis of LIP, we studied the relationship between EBV, the suggested causative agent of LIP and HIV-1 capsid protein p24, which play an important role in the interaction with host proteins during HIV-1 adsorption, membrane fusion, and entry in surgical lung biopsy-proven LIP from 9 vertically HIV subtype E-infected pediatric patients. The dominant microscopic feature of LIP demonstrated widespread widening of alveolar septum by mononuclear inflammatory cell infiltrate, mainly composed of mature lymphocytes and plasma cells surrounding airways and expanding to the lung interstitium. EBV-encoded RNA (EBER) in situ hybridization (ISH) and p24 immunohistochemistry, performed on formalin-fixed, paraffin-embedded tissue from open lung biopsy specimens, revealed positive intranuclear EBER signals and intracytoplasmic immunostains for p24 core protein in all 9 LIP cases. By combining ISH and immunohistochemistry, these results suggest that (i) EBV/p24-carrying cells are likely involved in the development of LIP, either directly or indirectly; (ii) LCs and related dendritic cells are the main reservoir of both EBV and HIV subtype E in pediatric LIP and possibly LCs may play an important role in the recruitment of inflammatory cell infiltrates, especially T cells into these tissues; (iii) coexpression of EBV/p24 in bronchioalveolar epithelium supports the hypothesis that these cells serve as a reactivation source for both viruses to achieve greater quantities in alveolar septum and interstitium around bronchioles. These results indicate a strong association between the presence of HIV core protein p24 and expression of EBV RNA transcripts (EBER). Interactions between LCs and related dendritic cells together with T cells are important for effective HIV and EBV replications. The coexpression of both viruses could be related to the evolution of pediatric LIP in HIV subtype E infection.

Hepatoprotective effects of lychee (Litchi chinensis Sonn.): a combination of antioxidant and anti-apoptotic activities.

AIM OF THE STUDY: Gimjeng and Chakapat lychee (Litchi chinensis Sonn.) were evaluated for hepatoprotective activity on CCl(4)-induced hepatotoxicity in rats. MATERIALS AND METHODS: Fruit pulp extracts of the lychees were examined for vitamin C, phenolic contents, anti-lipid peroxidation activity and hepatoprotective effect. Male Wistar albino rats were intraperitoneally injected (ip) with CCl(4) (2 ml/kg), then were orally administered (po) with silymarin (100mg/kg), and Gimjeng or Chakapat extracts (100 and 500 mg/kg). After ten days, the rats were sacrificed and their livers were examined histopathologically and immunohistochemically. Their serum glutamate-pyruvate transaminase, glutamate-oxalate transaminase, and alkaline phosphatase activities were analyzed. Apoptotic activity of the livers was assessed quantitatively. RESULTS: The Gimjeng and Chakapat extracts showed the contents of vitamin C (1.2±0.6 and 4.3±0.1mg/100g) and phenolics like trans-cinnamic acid and pelargonidin-3-O-glucoside (9.80±0.21 and 19.56±0.4 mg GAE/g extract, respectively), and trolox equivalent antioxidant capacity (TEAC) values (11.64 and 9.09 g/mg trolox), respectively. The Gimjeng as compared to the Chakapat demonstrated a better antioxidant activity as revealed by anti-lipid peroxidation activity with the TEAC values. Administration of CCl(4) in rats elevated the serum GPT, GOT, and ALP level whereas silymarin, Gimjeng and Chakapat extracts prevented these increases significantly. Significant decrease of apoptotic cells together with restoration of morphological changes confirmed the hepatoprotective effect in the CCl(4)-induced rats pretreated with the extracts. CONCLUSION: Antioxidant properties of the Gimjeng and Chakapat lychees as evidenced by the vitamin C and phenolic compounds, anti-lipid peroxidation and anti-apoptosis could explain the hepatoprotective effects in CCl(4)-induced hepatotoxicity.

Cell reservoirs of the Epstein-Barr virus in biopsy-proven lymphocytic interstitial pneumonitis in HIV-1 subtype E infected children: identification by combined in situ hybridization and immunohistochemistry.

Lymphoid interstitial pneumonitis (LIP), a frequent pulmonary complication in human immune deficiency virus (HIV)-infected pediatric patients, is characterized histologically by marked infiltration of lymphoid cells. Several theories have been suggested that LIP may be caused by Epstein-Barr virus (EBV). To identify the reservoir of EBV and pathogenesis of lymphoid infiltrates in HIV subtype E infected pediatric LIP, we examined the distribution and expression of EBV in the inflammatory cell recruitment in surgical lung biopsy-proven LIP from 9 vertically HIV subtype E-infected pediatric patients. The dominant microscopic feature of LIP demonstrated widespread widening of alveolar septum by mononuclear inflammatory cell infiltrate mainly composed of mature lymphocytes and plasma cells surrounding airways and expanding to the lung interstitium. EBV-encoded RNA (EBER) in situ hybridization, performed from paraffin-embedded lung tissues, revealed positive intranuclear signals in all 9 LIP cases. Interestingly, combined immunohistochemical and in situ hybridization analyses in 6 out of 9 LIP cases revealed 30% to 50% of the Langerhans and related dendritic cells were infected with EBV, whereas <30% of the T and B cells were infected with EBV. These results suggested that a chronic antigenic stimulus of EBV played important roles in the pathogenesis of LIP in these patients. This supports the notion that Langerhans cells (LCs) are more readily infected with EBV, indicating that LCs are reservoirs for EBV in lungs of HIV subtype E-infected pediatric LIP. And possibly LCs may play an important role in the recruitment of inflammatory cell infiltrates, especially T cells into these tissues. In addition, HIV may provide a milieu or microenvironment for the evolution of LIP, which represent an immunologic response to EBV infection. Interactions between LCs and related dendritic cells together with T cells are important for effective HIV and EBV replications.

Forensic entomology cases in Thailand: a review of cases from 2000 to 2006.

This paper presents and discusses 30 cases of cadavers that had been transferred for forensic entomology investigations to the Department of Forensic Medicine, Faculty of Medicine, Chiang Mai University, northern Thailand, from 2000 to 2006. Variable death scenes were determined, including forested area and suburban and urban outdoor and indoor environments. The fly specimens found in the corpses obtained were the most commonly of the blow fly of family Calliphoridae, and consisted of Chrysomya megacephala (F.), Chrysomya rufifacies (Macquart) Chrysomya villeneuvi Patton, Chrysomya nigripes Aubertin, Chrysomya bezziana Villeneuve, Chrysomya chani Kurahashi, Lucilia cuprina (Wiedemann), Hemipyrellia ligurriens (Wiedemann), and two unknown species. Flies of the family Muscidae [Hydrotaea spinigera Stein, Synthesiomyia nudiseta (Wulp)], Piophilidae [Piophila casei (L.)], Phoridae [Megaselia scalaris (Loew)], Sarcophagidae [Parasarcophaga ruficornis (F.) and three unknown species], and Stratiomyiidae (Sargus sp.) were also collected from these human remains. Larvae and adults of the beetle, Dermestes maculatus DeGeer (Coleoptera: Dermestidae), were also found in some cases. Chrysomya megacephala and C. rufifacies were the most common species found in the ecologically varied death scene habitats associated with both urban and forested areas, while C. nigripes was commonly discovered in forested places. S. nudiseta was collected only from corpses found in an indoor death scene.

Physical effects of sexually abused children and adolescents at Taksin Hospital.

OBJECTIVE: To study the incidences and physical effects of sexually abused children and adolescents. MATERIAL AND METHOD: Rape records and records of 250 sexually abused children and adolescents treated at Taksin Hospital between January 1, 1999 and December 31, 2004 from Child-Women Protection Center were studied retrospectively. RESULTS: The incidence of sexual abuse in children and adolescents was 4.74 per 10,000 cases of age-adjusted patients at the OPD and 22.97 at the emergency department. Most of the cases (97.20%) were females. Mean age was 13.74 +/- 4.27 (2-20) years old. Most of the cases were early adolescents (52.40%), late adolescents 32.40%, and children 15.20%. Thirty-nine cases (15.60%) had physical injuries, 36 cases (14.40%) had external genital injuries, 25 cases (10.00%) had gonococcal infections, 15 cases (6.20%) had bleeding in the vaginal canal, and eight cases (3.20%) were pregnant. Two hundred and twenty six cases (90.40%) were reported to the police. There was correlation between age group and hymen tearing (p-value < 0.001), gonococcal infection (p-value < 0.01) and sperm finding (p-value < 0.001). However there was no correlation between age group and gender, physical injury, genital injury, bleeding in the vagina and acid phosphatase finding. In addition, there was correlation between physical injury and bleeding in the vagina (p-value < 0.01) but no correlation between genital injury and hymen findings. CONCLUSION: Sexual abused victims need immediate attention for the traumatic impacts of their physical, psychological, and emotional conditions, as well as on their social impact. Thus, it is imperative that protection be exerted over the treatment to prevent recurrence.

STR loci D10S2325, D16S539 and D19S253: Northern Thai population data.

The STR loci D10S2325, D16S539 and D19S253 were analyzed in 203 unrelated Northern Thai. A power of discrimination of 0.954, 0.923 and 0.921 and a power of exclusion of 0.690, 0.542 and 0.632, respectively were found. The combined power of discrimination and exclusion reached 0.99972 and 0.9478, respectively. No deviation from the Hardy-Weinberg equilibrium was observed for the three loci.

Forensic value of nine STR loci in Northern Thai.

Nine STR loci (D3S1358, D5S818, D7S820, D8S1179, D13S317, TH01, VWA, TPOX, LPL) were studied in a large Northern Thai population sample. All loci meet Hardy-Weinberg expectations. The combined power of discrimination and exclusion is 0.9999999979 and 0.99798 respectively. Mutation rates for STR loci did not exceed 1-4 per 1000 parent/child pairs as derived from disputed paternity cases. Similar mutation rates were also reported from other populations. No mutations were found for D5S818, D7S820, TH01, and TPOX.

Cell reservoirs in lymph nodes infected with HIV-1 subtype E differ from subtype B: identification by combined in situ polymerase chain reaction and immunohistochemistry.

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.

Effectiveness of short-term and long-term zidovudine prophylaxis on detection of HIV-1 subtype E in human placenta and vertical transmission.

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.

Chorioamnionitis is associated with placental transmission of human immunodeficiency virus-1 subtype E in the early gestational period.

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 < 0.01). There were no significant differences between the placentas of the two groups with respect to presence of chorioamnionitis, villitis, villous stromal fibrosis, infarction, abnormal villous maturation, deciduitis or decidual necrosis. HIV-1 subtype E was detected in up to 83% of placentas, either by immunostaining or in situ PCR, in trophoblast, villous stromal cells, Hofbauer cells, decidual and decidual glandular epithelium. Fetal organs were positive for HIV in 30% (6/20) of cases. There was a significant association between transmission of HIV to the fetus and the histologic findings of chorioamnionitis, plasmacellular deciduitis and decidual cell necrosis. This is the first report showing an association of chorioamnionitis with early in utero transmission of HIV-1 subtype E. This may help explain the cases of in utero transmission that persist despite antiretroviral prophylaxis, given that therapy is started in the late gestational period.

STR loci Penta D and Penta E: data from a Northern Thai population sample.

We designed new primers that give shorter amplicons for Penta D and Penta E for easy separation on native polyacrylamide gels. 112 unrelated Northern Thai men were typed. A total of nine and eight-teen alleles was found for Penta D and Penta E, respectively. Both loci showed a high power of discrimination (Penta D: 0.927; Penta E: 0.966; both loci combined: 0.9975) as reported in other populations. Penta D met Hardy-Weinberg expectations, whereas Penta E did not, maybe because of the small sample size in relation to the large number of alleles of this locus.

Identification of DYS385 allele variants by using shorter amplicons and Northern Thai haplotype data.

Primers currently used for amplification of locus DYS385 on the Y chromosome give quite long amplicons that are difficult to resolve on native polyacrylamide gels. We therefore designed new primers that give much shorter products for easy separation. With this system, two allele variants that were characterized by sequencing as 14.1 (GAAA --> GAAAA) and 14.2 (GA insertion), were identified in two unrelated Northern Thai men due to their decreased mobility. Interestingly, initial capillary electrophoresis mistyped the 14.1 variant amplified with the traditional primers as allele 14. Forty different haplotypes were found in 147 Northern Thais with a haplotype diversity of 0.9430. Haplotype 13-18 (frequency 0.136) is the most frequent one: for comparison: haplotype 14-18 (frequency 0.074) was found to be the most common in a population from Bangkok (n = 95) in an earlier study. This striking difference within Thailand's population probably results from genetic differences in the founding populations.