Estimation of post-mortem interval (PMI) is crucial for time of death determination. The advent of DNA-based identification techniques forensic entomology saw the beginning of a proliferation of molecular studies into forensically important Calliphoridae (Diptera). The use of DNA to characterise morphologically indistinguishable immature calliphorids was recognised as a valuable molecular tool with enormous practical utility. The local entomofauna in most cases is important for the examination of entomological evidences. The survey of the local entomofauna has become a fundamental first step in forensic entomological studies, because different geographical distributions, seasonal and environmental factors may influence the decomposition process and the occurrence of different insect species on corpses. In this study, calliphorids were collected from 13 human corpses recovered from indoors, outdoors and aquatic conditions during the post-mortem examination by pathologists from the government hospitals in Malaysia. Only two species, Chrysomya megacephala and Chrysomya rufifacies were recovered from human corpses. DNA sequencing was performed to study the mitochondrial encoded COI gene and to evaluate the suitability of the 1300 base pairs of COI fragments for identification of blow fly species collected from real crime scene. The COI gene from blow fly specimens were sequenced and deposited in GenBank to expand local databases. The sequenced COI gene was useful in identifying calliphorids retrieved from human corpses.
X-chromosome short tandem repeats (X-STRs) are useful for human identification, especially in complex kinship scenarios. Since forensic statistical parameters vary among populations and the X-STRs population data for the diverse population of Peninsular Malaysia's are unavailable, this attempt for Indians (n = 201) appears forensically relevant to support the 12 X-STRs markers' evidential value for human identification in Malaysia. The Qiagen Investigator® Argus X-12 QS kit showed that DXS10135 was the most polymorphic locus with high genetic diversity, polymorphism information richness, heterozygosity, and exclusion power. Based on allele frequencies, the strength of discrimination and mean exclusion chance (MECKrüger, MECKishida, MECDesmarais, and MECDesmaraisDuo) values for the Malaysian Indians were ≥0.999997790686228. As for haplotype frequencies, the overall discrimination power and mean exclusion probability (MECKrüger, MECKishida, MECDesmarais, and MECDesmaraisDuo) were ≥0.9999984801951. The genetic distance, neighbor-joining phylogenetic tree, and principal component analysis also supported the evidential value of the 12 X-STRs markers for forensic practical caseworks in Malaysia.
In this study, PCR-RFLP analysis (PRA) targeting hsp65 and rpoB gene regions was evaluated for the identification of mycobacterial species isolated from Malaysian patients. Overall, the hsp65 PRA identified 92.2 % of 90 isolates compared to 85.6 % by the rpoB PRA. With 47 rapidly growing species, the hsp65 PRA identified fewer (89.4 %) species than the rpoB PRA (95.7 %), but with 23 slow-growing species the reverse was true (91.3 % identification by the hsp65 PRA but only 52.5 % by the rpoB PRA). There were 16 isolates with discordant PRA results, which were resolved by 16S rRNA and hsp65 gene sequence analysis. The findings in this study suggest that the hsp65 PRA is more useful than the rpoB PRA for the identification of Mycobacterium species, particularly with the slow-growing members of the genus. In addition, this study reports 5 and 12 novel restriction patterns for inclusion in the hsp65 and rpoB PRA algorithms, respectively.
Demand for pangolin scales in East Asia has increased dramatically in the past two decades, raising concern to the pangolin survival and bringing them to the brink of local extinction. Enumerating the number of individuals from the seized pangolin scales primarily goes undocumented, mostly due to the unavailability of the appropriate methods. In this study, we developed a Pangolin Indexing System, a multi-locus STR panel of eight dinucleotide microsatellites that showed promising results in individualization and assignment of scales into Chinese and Indian pangolins. The combined power of exclusion was 0.83 and 0.99 for Chinese and Indian pangolin. The select panel of eight polymorphic STRs exhibited the cumulative probability of identity 3.7 × 10-9 for Indian pangolin and 3.6 × 10-7 for Chinese pangolin and identified 51 unique genotypes from the 74 scales selected from the four pangolin seizures. The study demonstrated the first report of cross-species validation of STRs developed from Malayan pangolin to Indian pangolin and showed the potential application of Pangolin Indexing System in screening of large seizures through DNA profiling from the scales of Indian and Chinese pangolin.
The emergence of multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae poses a serious antibiotic management problem as resistance genes are easily transferred from one organism to another. Fifty-one strains of K. pneumoniae isolated from sporadic cases in various hospitals throughout Malaysia were analysed by antimicrobial susceptibility testing, PCR detection of ESBL-encoding genes and DNA fingerprinting. Although 27 of the 51 K. pneumoniae strains were MDR (i.e. resistant to three or more classes of antibiotics), the majority of the strains (98 %) were sensitive to imipenem. PCR detection using ESBL gene-specific primers showed that 46 of the K. pneumoniae strains harboured bla(SHV), 19 harboured bla(CTX-M), 5 harboured bla(OXA-1) and 4 harboured bla(TEM-1). Class 1 integron-encoded intI1 integrase was detected in 21 of the 51 K. pneumoniae strains and amplification of the integron 5'CS region showed the presence of several known antibiotic resistance gene cassettes of various sizes. Results of conjugation and transformation experiments indicated that some of the ESBL-encoding genes (i.e. bla(SHV), bla(CTX-M) and bla(TEM-1)) were transmissible and were likely plasmid-encoded. DNA fingerprinting using PFGE and PCR-based methods indicated that the 51 K. pneumoniae strains were genetically diverse and heterogeneous.
The antibiotic susceptibility profiles and the repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR)-determined genotypes of 109 Acinetobacter strains collected from the University Malaya Medical Center (UMMC), Kuala Lumpur, Malaysia, in 1987 (N=21) and 1996-1998 (N=88) were established. Twelve antibiotic susceptibility profiles of antibiotics used at the UMMC were obtained. In descending order of effectiveness, imipenem, amikacin and ciprofloxacin were the most effective against the Acinetobacter strains. Compared with 1987 isolates, the isolates obtained in 1996-1998 had decreased susceptibility to these antibiotics and were tolerant to the antibiotics up to an MIC90 of > or =256 mg/L. REP-PCR DNA fingerprints of all the isolates revealed the presence of four Acinetobacter spp. lineages; 92% of all the isolates belonged to two dominant lineages (genotypes 1 and 4). Genotype 4 isolates predominant in 1987 showed increased resistance and antibiotic tolerance to imipenem, amikacin and ciprofloxacin compared with the 1996-1998 isolates. In contrast, genotype 1 isolates from 1996-1998 were mainly sensitive to these antibiotics. These findings demonstrate the presence of at least two independent Acinetobacter spp. lineages in the same hospital, and suggest the possibility that genotype 4 Acinetobacter spp. acquired the resistance phenotype in situ, whereas most of the genotype 1 isolates were probably introduced to the hospital in recent years.
We have analysed DNA fingerprinting patterns by pulsed-field gel electrophoresis (PFGE) of 52 unrelated Burkholderia pseudomallei strains isolated from septicemic and localized infections from Malaysian subjects. A total of 38 PFGE types were observed among 36 septicemic and 16 localized strains with no predominant pattern. Type 25 was seen in 2 epidemiologically related strains, suggesting human to human transmission. Twelve PFGE types were shared among 26 strains (21 septicemic and 5 localized) showing close genetic relatedness with coefficient of similarity of 0.81 to 1.0. The other 26 strains (15 septicemic and 11 localized) were unrelated as shown by the similarity coefficient of < 0.8. This study showed that our B. pseudomallei strains in Malaysia were mainly heterogenous with no predominant type both in septicemic or localized strains.