Commentary: Whetting a journalist’s appetite for investigative reporting

Interest in investigative journalism has spiked in Asia and elsewhere, especially in new democracies, and along with it the demand for training in this field. The challenge for trainers in investigative reporting is to help journalists nimbly navigate what is often uncharted territory that demands dogged pursuit and unraveling of the truth. How to do it? This article shares with journalism trainers a few useful tips on getting journalists hooked on muckraking

Rapid Detection and Identification of Human Hookworm Infections through High Resolution Melting (HRM) Analysis

Background: Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world’s poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species. Methods: Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.2460.05uC and 83.0060.04uC for Necator americanus, 79.1260.10uC for Ancylostoma duodenale, 79.4060.10uC for Ancylostoma ceylanicum, 79.6360.05uC for Ancylostoma caninum and 79.7060.14uC for Ancylostoma braziliense. An evaluation of the method’s sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/ml hookworm DNA and amplification was only recorded for hookworm positive samples. Conclusion: The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species

Genetic characterization of the partial mitochondrial cytochrome oxidase c subunit I (cox 1) gene of the zoonotic parasitic nematode, Ancylostoma ceylanicum from humans, dogs and cats

Ancylostoma ceylanicum is the only zoonotic hookworm species that is able to produce patent infections in humans with the majority of cases reported in South East Asia. Over the pastfew years,there have been an increasing number of studies investigating the prevalence of this parasitic zoonosis using molecular diagnostic tools and a single genetic locus as marker for species identification. As there can be limitations in using a single genetic locus for epidemiological studies and genetic discrimination, the complementary use of a more variable locus will provide additional evidence to support the zoonotic exchange of hookworm species between humans and animals. In the present study, the cytochrome c oxidase subunit 1 (cox 1) sequence of A. ceylanicum from positive human and animal fecal samples were determined and compared with published reference sequences. Phylogenetic analysis demonstrated that isolates of A. ceylanicum were divided into two clusters, one consisting 3 human isolates and the other comprising 19 isolates of human and animal origin from different geographical locations within Malaysia. The two groups of A. ceylanicum could be distinguished from one another through five fixed nucleotide differences at locations 891, 966, 1008, 1077 and 1083. The detection of genetically distinct groups and considerable level of genetic variation within the cox 1 sequence of A. ceylanicum might suggest potential haplotypelinked differences in zoonotic, epidemiological and pathobiological characteristics, a hypothesis that still needs further investigation

Molecular detection of human Plasmodium species in Sabah using PlasmoNextm multiplex PCR and hydrolysis probes real-time PCR

BACKGROUND: Malaria is a vector borne-parasitic disease transmitted through the bite of the infective female Anopheles mosquitoes. Five Plasmodium species have been recognized by World Health Organization (WHO) as the causative agents of human malaria. Generally, microscopic examination is the gold standard for routine malaria diagnosis. However, molecular PCR assays in many cases have shown improvement on the sensitivity and specificity over microscopic or other immunochromatographic assays. METHODS: The present study attempts to screen 207 suspected malaria samples from patients seeking treatment in clinics around Sabah state, Malaysia, using two panels of multiplex PCRs, conventional PCR system (PlasmoNex™) and real-time PCR based on hydrolysis probe technology. Discordance results between two PCR assays were further confirmed by sequencing using 18S ssu rRNA species-specific primers. RESULTS: Of the 207 malaria samples, Plasmodium knowlesi (73.4% vs 72.0%) was the most prevalent species based on two PCR assays, followed by Plasmodium falciparum (15.9% vs 17.9%), and Plasmodium vivax (9.7% vs 7.7%), respectively. Neither Plasmodium malariae nor Plasmodium ovale was detected in this study. Nine discrepant species identification based on both the PCR assays were further confirmed through DNA sequencing. Species-specific real-time PCR only accurately diagnosed 198 of 207 (95.7%) malaria samples up to species level in contrast to PlasmoNex™ assay which had 100% sensitivity and specificity based on sequencing results. CONCLUSIONS: Multiplex PCR accelerate the speed in the diagnosis of malaria. The PlasmoNex™ PCR assay seems to be more accurate than real-time PCR in the speciation of all five human malaria parasites. The present study also showed a significant increase of the potential fatal P. knowlesi infection in Sabah state as revealed by molecular PCR assays

Linking the effects of helminth infection, diet and the gut microbiota with human whole-blood signatures

Helminth infection and dietary intake can affect the intestinal microbiota, as well as the immune system. Here we analyzed the relationship between fecal microbiota and blood profiles of indigenous Malaysians, referred to locally as Orang Asli, in comparison to urban participants from the capital city of Malaysia, Kuala Lumpur. We found that helminth infections had a larger effect on gut microbial composition than did dietary intake or blood profiles. Trichuris trichiura infection intensity also had the strongest association with blood transcriptional profiles. By characterizing paired longitudinal samples collected before and after deworming treatment, we determined that changes in serum zinc and iron levels among the Orang Asli were driven by changes in helminth infection status, independent of dietary metal intake. Serum zinc and iron levels were associated with changes in the abundance of several microbial taxa. Hence, there is considerable interplay between helminths, micronutrients and the microbiota on the regulation of immune responses in humans

Analysis of T cell receptor clonotypes in tumor microenvironment identifies shared cancer-type-specific signatures.

Despite the conventional view that a truly random V(D)J recombination process should generate a highly diverse immune repertoire, emerging reports suggest that there is a certain bias toward the generation of shared/public immune receptor chains. These studies were performed in viral diseases where public T cell receptors (TCR) appear to confer better protective responses. Selective pressures generating common TCR clonotypes are currently not well understood, but it is believed that they confer a growth advantage. As very little is known about public TCR clonotypes in cancer, here we set out to determine the extent of shared TCR clonotypes in the intra-tumor microenvironments of virus- and non-virus-driven head and neck cancers using TCR sequencing. We report that tumor-infiltrating T cell clonotypes were indeed shared across individuals with the same cancer type, where the majority of shared sequences were specific to the cancer type (i.e., viral versus non-viral). These shared clonotypes were not particularly enriched in EBV-associated nasopharynx cancer but, in both cancers, exhibited distinct characteristics, namely shorter CDR3 lengths, restricted V- and J-gene usages, and also demonstrated convergent V(D)J recombination. Many of these shared TCRs were expressed in patients with a shared HLA background. Pattern recognition of CDR3 amino acid sequences revealed strong convergence to specific pattern motifs, and these motifs were uniquely found to each cancer type. This suggests that they may be enriched for specificity to common antigens found in the tumor microenvironment of different cancers. The identification of shared TCRs in infiltrating tumor T cells not only adds to our understanding of the tumor-adaptive immune recognition but could also serve as disease-specific biomarkers and guide the development of future immunotherapies

Genotypic and phenotypic characterization of Escherichia coli isolated from indigenous individuals in Malaysia

Objective(s): The occurrence of asymptomatic verocytotoxin (VT)-producing Escherichia coli (VTEC) infections among humans in recent years is posing a high risk to public health. Thus, the role of asymptomatic human carriers as a source of dissemination should not be underestimated. This study aimed to elucidate the phenotypic and genotypic characteristics of E. coli in the stool samples collected from indigenous individuals in Malaysia. Materials and Methods: E. coli strains (n=108) were isolated from stool samples obtained from 41 indigenous individuals. All strains were subjected to Repetitive Extragenic Palindromic-Polymerase Chain Reaction (REP-PCR) typing and confirmation of VTEC variants. Non-duplicate strains were selected based on REP-PCR profiles and further subjected to antimicrobial susceptibility test (AST). The genotypic and phenotypic characteristics of the strains were then correlated with the demographic data of the subjects. Results: A total of 66 REP-PCR profiles grouped in 53 clusters (F=85%) were obtained. Four genetically distinct strains were confirmed as VTEC (eaeA-positive). The predominant resistance was against ampicillin (34.2%), followed by trimethoprim-sulfamethoxazole (32.9%), ampicillin-sulbactam (5.5%), and ciprofloxacin (1.4%). All isolates were sensitive to amoxicillin-clavulanate, cefuroxime, ceftriaxone, imipenem, and meropenem. Conclusion: Genetically diverse E. coli and VTEC strains were found to colonize the intestines of the indigenous populations. This study is important for the prospective surveillance of E. coli among the indigenous individuals in Malaysia, especially in asymptomatic VTEC infection and antimicrobial resistance phenomenon

Development of high resolution melting analysis for the diagnosis of human malaria

Molecular detection has overcome limitations of microscopic examination by providing greater sensitivity and specificity in Plasmodium species detection. The objective of the present study was to develop a quantitative real-time polymerase chain reaction coupled with high-resolution melting (qRT-PCR-HRM) assay for rapid, accurate and simultaneous detection of all five human Plasmodium spp. A pair of primers targeted the 18S SSU rRNA gene of the Plasmodium spp. was designed for qRT-PCR-HRM assay development. Analytical sensitivity and specificity of the assay were evaluated. Samples collected from 229 malaria suspected patients recruited from Sabah, Malaysia were screened using the assay and results were compared with data obtained using PlasmoNexTM, a hexaplex PCR system. The qRT-PCR-HRM assay was able to detect and discriminate the five Plasmodium spp. with lowest detection limits of 1–100 copy numbers without nonspecific amplifications. The detection of Plasmodium spp. in clinical samples using this assay also achieved 100% concordance with that obtained using PlasmoNexTM. This indicated that the diagnostic sensitivity and specificity of this assay in Plasmodium spp. detection is comparable with those of PlasmoNexTM. The qRT-PCR-HRM assay is simple, produces results in two hours and enables high-throughput screening. Thus, it is an alternative method for rapid and accurate malaria diagnosis

A new paradigm for Aedes spp. surveillance using gravid ovipositing sticky trap and NS1 antigen test kit

BACKGROUND:Dengue remains a serious public health problem in Southeast Asia and has increased 37-fold in Malaysia compared to decades ago. New strategies are urgently needed for early detection and control of dengue epidemics. METHODS:We conducted a two year study in a high human density dengue-endemic urban area in Selangor, where Gravid Ovipositing Sticky (GOS) traps were set up to capture adult Aedes spp. mosquitoes. All Aedes mosquitoes were tested using the NS1 dengue antigen test kit. All dengue cases from the study site notified to the State Health Department were recorded. Weekly microclimatic temperature, relative humidity (RH) and rainfall were monitored. RESULTS:Aedes aegypti was the predominant mosquito (95.6%) caught in GOS traps and 23% (43/187 pools of 5 mosquitoes each) were found to be positive for dengue using the NS1 antigen kit. Confirmed cases of dengue were observed with a lag of one week after positive Ae. aegypti were detected. Aedes aegypti density as analysed by distributed lag non-linear models, will increase lag of 2-3 weeks for temperature increase from 28 to 30 °C; and lag of three weeks for increased rainfall. CONCLUSION:Proactive strategy is needed for dengue vector surveillance programme. One method would be to use the GOS trap which is simple to setup, cost effective (below USD 1 per trap) and environmental friendly (i.e. use recyclable plastic materials) to capture Ae. aegypti followed by a rapid method of detecting of dengue virus using the NS1 dengue antigen kit. Control measures should be initiated when positive mosquitoes are detected

Mapping and modelling the geographical distribution of soil-transmitted helminthiases in Peninsular Malaysia : implications for control approaches

Soil-transmitted helminth (STH) infections in Malaysia are still highly prevalent, especially in rural and remote communities. Complete estimations of the total disease burden in the country has not been performed, since available data are not easily accessible in the public domain. The current study utilised geographical information system (GIS) to collate and map the distribution of STH infections from available empirical survey data in Peninsular Malaysia, highlighting areas where information is lacking. The assembled database, comprising surveys conducted between 1970 and 2012 in 99 different locations, represents one of the most comprehensive compilations of STH infections in the country. It was found that the geographical distribution of STH varies considerably with no clear pattern across the surveyed locations. Our attempt to generate predictive risk maps of STH infections on the basis of ecological limits such as climate and other environmental factors shows that the prevalence of Ascaris lumbricoides is low along the western coast and the southern part of the country, whilst the prevalence is high in the central plains and in the North. In the present study, we demonstrate that GIS can play an important role in providing data for the implementation of sustainable and effective STH control programmes to policy-makers and authorities in charge