From 2005 to 2009, the Institute for Medical Research (IMR), Kuala Lumpur, Malaysia received 488 serum and blood samples from hospitalized patients on the East Coast of Peninsular Malaysia, suspected of having dengue infection. In this study we determined the prevailing dengue serotypes using a real time polymerase chain reaction assay (RT-PCR). All 4 dengue virus serotypes were found circulating during the study period; however the predominant serotype varied. In 2005 and 2006, the predominant serotypes circulating were DENV-1 and DENV-3, in 2007, DENV-1 and DENV-2 were predominant, and in 2008 and 2009, DENV-3 was the predominant serotype.
Restriction enzymes SpeI and XbaI were used in a pulsed-field gel electrophoresis (PFGE) study for molecular characterization of 146 clinical Burkholderia pseudomallei isolates. The PFGE parameters were optimized to enable comparable, reproducible, and robust results. The optimized parameters for both SpeI and XbaI restriction enzymes used in this study were 200 V and a pulse time of 5 to 65 s for a 28-h runtime. Using SpeI, 9 different clusters were identified, whereas 6 clusters were identified by XbaI digestion, which exhibited 85% similarity to SpeI. SpeI (discrimination index [D]=0.854) showed higher discriminatory power than XbaI did (D=0.464).
Determination of Streptococcus pneumoniae serotypes is essential for epidemiological surveillance. Therefore accurate, reliable and cost effective serotyping method is crucial. In this study, we determined the serotypes of 41 pneumococcal isolates recovered from human anterior nares by multiplex Polymerase Chain Reaction (PCR) utilizing published primers. The data was then compared with conventional serology using latex agglutination (LA) and the Quellung reaction. Based on the PCR-approach, 8 different serogroups/serotypes were detected with one isolate classified as non-typeable (cpsA-negative). In reference to the serology-based data, the results were in agreement except for one isolate. For the latter isolate, the LA and Quellung tests failed to show a reaction but the PCR-approach and sequencing identified the isolate as serogroup 15B/C. Based on this experimental setting, we found that the PCR-approach for pneumococcal serotypes determination is reliable to serve as the alternative for determining the pneumococcal serotyping.
A variable-number tandem-repeat (VNTR) typing assay for the differentiation of Mycobacterium abscessus strains was developed. This assay showed complete reproducibility, locus stability, and a discriminatory power (Hunter-Gaston discriminatory index [HGDI] of 0.9563) that is superior to that of multilocus sequencing. It is a promising tool for the investigation of Mycobacterium abscessus epidemiology and nosocomial outbreaks.
We barcoded 25 in vitro isolates (representing 92 samples) of Giardia duodenalis from humans and other animals, which have been assembled by the Upcroft team at the Queensland Institute of Medical Research over a period of almost three decades. We used mutation scanning-coupled sequencing of loci in the triosephosphate isomerase, glutamate dehydrogenase and β-giardin genes, combined with phylogenetic analysis, to genetically characterise them. Specifically, the isolates (n514) of G. duodenalis from humans from Australia (AD113; BRIS/83/HEPU/106; BRIS/87/HEPU/713; BRIS/89/HEPU/1003; BRIS/92/HEPU/1541; BRIS/92/HEPU/1590; BRIS/92/HEPU/2443; BRIS/93/HEPU/1706), Malaysia (KL/92/IMR/1106) and Afghanistan (WB), a cat from Australia (BAC2), a sheep from Canada (OAS1) and a sulphur-crested cockatoo from Australia (BRIS/95/HEPU/2041) represented assemblage A (sub-assemblage AI-1, AI-2 or AII-2); isolates (n510) from humans from Australia (BRIS/91/HEPU/1279; BRIS/92/HEPU/2342; BRIS/92/HEPU/2348; BRIS/93/HEPU/1638; BRIS/93/HEPU/1653; BRIS/93/HEPU/1705; BRIS/93/HEPU/1718; BRIS/93/HEPU/1727), Papua New Guinea (BRIS/92/HEPU/1487) and Canada (H7) represented assemblage B (sub-assemblage BIV) and an isolate from cattle from Australia (BRIS/92/HEPU/1709) had a match to assemblage E. Isolate BRIS/90/HEPU/1229 from a human from Australia was shown to represent a mixed population of assemblages A and B. These barcoded isolates (including stocks and derived lines) now allow direct comparisons of experimental data among laboratories and represent a massive resource for transcriptomic, proteomic, metabolic and functional genomic studies using advanced molecular technologies.
In the last decades, leptospirosis had gained public health concern due to morbidity and mortality rates caused by pathogenic Leptospira. The need for rapid and robust molecular typing methods to differentiate this zoonotic pathogen is of utmost importance. Various studies had been conducted to determine the genetic relatedness of Leptospira isolates using molecular typing methods. In this study, 29 pathogenic Leptospira isolates from rat, soil, and water samples in Sarawak, Malaysia, were characterized using BOX-PCR and ERIC-PCR. The effectiveness of these two methods with regard to the ease of interpretation, reproducibility, typeability, and discriminatory power was also being evaluated. Using BOX-PCR, six clusters and 3 single isolates were defined at a genetic distance percentage of 11.2%. ERIC-PCR clustered the isolates into 6 clusters and 2 single isolates at a genetic distance percentage of 6.8%. Both BOX-PCR and ERIC-PCR produced comparable results though the discriminatory index for ERIC-PCR (0.826) was higher than that for BOX-PCR (0.809). From the constructed dendrogram, it could be summarized that the isolates in this study were highly heterogeneous and genetically diverse. The findings from this study indicated that there is no genetic relatedness among the pathogenic Leptospira isolates in relation to the locality, source, and identity, with some exceptions. Out of the 29 pathogenic Leptospira isolates studied, BOX-PCR and ERIC-PCR successfully discriminated 4 isolates (2 isolates each) into the same cluster in relation to sample sources, as well as 2 isolates into the same cluster in association with the sample locality. Future studies shall incorporate the use of other molecular typing methods to make a more thorough comparison on the genetic relatedness of pathogenic Leptospira.
Four repetitive element sequence-based polymerase chain reaction (rep-PCR) methods, namely repetitive extragenic palindromic PCR (REP-PCR), enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), polytrinucleotide (GTG)₅ -PCR and BOX-PCR, were evaluated for the molecular differentiation of 12 probiotic Lactobacillus strains previously isolated from the gastrointestinal tract of chickens and used as a multistrain probiotic. This study represents the first analysis of the comparative efficacy of these four rep-PCR methods and their combination (composite rep-PCR) in the molecular typing of Lactobacillus strains based on a discriminatory index (D).
Vibrio cholerae, the causative agent of cholera, is endemic in many parts of the world, especially in countries poor in resources. Molecular subtyping of V. cholerae is useful to trace the regional spread of a clone or multidrug-resistant strains during outbreaks of cholera. Current available PCR-based fingerprinting methods such as Random Amplified Polymorphic DNA (RAPD)-PCR, Enterobacterial Repetitive Intergenic Consensus Sequence (ERIC)-PCR, and Repetitive Extragenic Palindromic (REP)-PCR were used to subtype V. cholerae. However, there are problems for inter-laboratory comparison as these PCR methods have their own limitations especially when different PCR methods have been used for molecular typing. In this study, a Vibrio cholerae Repeats-PCR (VCR-PCR) approach which targets the genetic polymorphism of the integron island of Vibrios was used and compared with other PCR-based fingerprinting methods in subtyping. Forty-three V. cholerae of different serogroups from various sources were tested. The PCR-fingerprinting approaches were evaluated on typeability, reproducibility, stability and discriminatory power. Overall, Malaysian non-O1/non-O139 V. cholerae were more diverse than O1 strains. Four non-O1/non-O139 strains were closely related with O1 strains. The O139 strain in this study shared similarity with strains of both O1 and non-O1/non-O139 serogroups. ERIC-PCR was the most discriminative approach (D value = 0.996). VCR-PCR was useful in discriminating non-O1/non-O139 strains. RAPD-PCR and REP-PCR were less suitable for efficient subtyping purposes as they were not reproducible and lacked stability. The combination of the ERIC-PCR and VCR-PCR may overcome the inadequacy of any one approach and hence provide more informative data.
INTRODUCTION: Uropathogenic virulence factors have been identified by comparing the prevalence of these among urinary tract isolates and environmental strains. The uropathogenic-specific protein (USP) gene is present on the pathogenicity island (PAI) of uropathogenic Escherichia coli (UPEC) and, depending on its two diverse gene types and the sequential patterns of three open reading frame units (orfUs) following it, there is a method to characterize UPEC epidemiologically called PAIusp subtyping.
METHODOLOGY: A total of 162 UPEC isolates from Sabah, Malaysia, were tested for the presence of the usp gene and the sequential patterns of three orfUs following it using polymerase chain reaction (PCR). In addition, by means of triplex PCR, the prevalence of the usp gene was compared with other two VFs of UPEC, namely alpha hemolysin (α-hly) and cytotoxic necrotizing factor (cnf-1) genes encoding two toxins.
RESULTS: The results showed that the usp gene was found in 78.40% of UPEC isolates, indicating that its prevalence was comparable to that found in a previous study in Japan. The two or three orfUs were also associated with the usp gene in this study. All the PAIusp subtypes observed in Japan were present in this study, while subtype IIa was the most common in both studies. The usp gene was observed in a higher percentage of isolates when compared with α-hly and cnf-1 genes.
CONCLUSIONS: The findings in Japan and Sabah, East Malaysia, were similar, indicating that PAIusp subtyping is applicable to the characterization of UPEC strains epidemiologically elsewhere in the world.
Hepatitis B virus infection is a serious health problem worldwide, and more than 350 million people are chronic carriers, constituting a major global threat. Southeast Asia and the Western Pacific have the highest levels of endemicity in the world, with an estimated seroprevalence ranging between 2% and 31%. Mutations in the hepatitis B surface antigen (HBsAg) have been reported in many parts of the world but are most common in Asian infants; such mutants have several clinical effects, such as the development of hepatocellular carcinoma. Diagnostic failures by commercial assays have reduced the diagnostic effectiveness of HBsAg detection. For example the substitution of an amino acid in the major hydrophilic region of the S gene reduces the binding of hepatitis B surface antibodies leading to immune escape. The safety of blood transfusion may be compromised by current screening tests due to escape from being neutralised by antibodies induced by HBsAg mutants, and undetectable levels of viral surface protein. Data on the epidemiology of HBsAg mutation in Asia Pacific are scant; however, this manuscript has reviewed the available information on the epidemiology of HBsAg mutation in Asia Pacific.