W. Abeyewickreme

Re-emergence of Chikungunya virus in South-east Asia: virological evidence from Sri Lanka and Singapore.

Chikungunya fever swept across many South and South-east Asian countries, following extensive outbreaks in the Indian Ocean Islands in 2005. However, molecular epidemiological data to explain the recent spread and evolution of Chikungunya virus (CHIKV) in the Asian region are still limited. This study describes the genetic Characteristics and evolutionary relationships of CHIKV strains that emerged in Sri Lanka and Singapore during 2006-2008. The viruses isolated in Singapore also included those imported from the Maldives (n=1), India (n=2) and Malaysia (n=31). All analysed strains belonged to the East, Central and South African (ECSA) lineage and were evolutionarily more related to Indian than to Indian Ocean Islands strains. Unique genetic characteristics revealed five genetically distinct subpopulations of CHIKV in Sri Lanka and Singapore, which were likely to have emerged through multiple, independent introductions. The evolutionary network based on E1 gene sequences indicated the acquisition of an alanine to valine 226 substitution (E1-A226V) by virus strains of the Indian sublineage as a key evolutionary event that contributed to the transmission and spatial distribution of CHIKV in the region. The E1-A226V substitution was found in 95.7 % (133/139) of analysed isolates in 2008, highlighting the widespread establishment of mutated CHIKV strains in Sri Lanka, Singapore and Malaysia. As the E1-A226V substitution is known to enhance the transmissibility of CHIKV by Aedes albopictus mosquitoes, this observation has important implications for the design of vector control strategies to fight the virus in regions at risk of chikungunya fever.

Single Antigen Detects both Immunoglobulin M (IgM) and IgG Antibodies Elicited by All Four Dengue Virus Serotypes

ABSTRACT The resurgence of dengue (DEN) virus infections in the last few decades coupled with the lack of a preventive vaccine and specific antiviral drugs has jointly contributed to making this a significant global public health problem. Currently, symptomatic supportive treatment and fluid replacement therapy are the only means available to minimize DEN-induced mortality. As the clinical symptoms associated with DEN virus infections are indistinguishable from those of many other viral, bacterial, and parasitic infections, specific diagnostic tests assume critical importance in the unequivocal identification of DEN virus infections. We have designed a novel chimeric antigen based on envelope domain III (EDIII), a critical antigenic region of the major structural protein of DEN viruses. We fused EDIIIs corresponding to each of the four DEN virus serotypes using pentaglycyl linkers, overexpressed the resultant tetravalent chimeric protein in Escherichia coli, and affinity purified it in high yields, obtaining ∼30 mg protein of >95% purity per liter of culture. We show that this tetravalent antigen could specifically recognize anti-DEN virus antibodies of both the immunoglobulin M (IgM) and IgG classes. Using a large panel of IgM antibody capture-enzyme-linked immunosorbent assay- and hemagglutination inhibition-confirmed DEN virus-infected and uninfected patient sera (n = 289), we demonstrate that this tetravalent antigen can function as a diagnostic tool of high sensitivity and specificity.

Revised morphological identification key to the larval anopheline (Diptera: Culicidae) of Sri Lanka

OBJECTIVE To revise morphological identification keys to the anophelines in Sri Lanka. METHOD Samples were collected from selected entomological sites in different districts in the country. Stage III and IV larvae were identified under a light microscope with an objective (×10) using standard larval keys developed for Sri Lankan anophelines. Key larval characters were recorded for each species based on original observations and previous usage in literature. RESULTS This manuscript describes an illustrated key for the identification of 22 of 23 mosquitoes which are currently recognized as local anopheline species in Sri Lanka, as a guide to workers engaged in malaria surveillance and control in the country. CONCLUSIONS Revised morphological keys to the larval of these species may be helpful in easy and accurate identification at the field level.

Species composition and diversity of malaria vector breeding habitats in Trincomalee District of Sri Lanka

Introduction. Mosquito larval ecology is important in determining larval densities and species assemblage. This in turn influences malaria transmission in an area. Therefore, understanding larval habitat ecology is important in designing malaria control programs. Method. Larval surveys were conducted in 20 localities under five sentinel sites (Padavisiripura, Gomarankadawala, Thoppur, Mollipothana, and Ichchallampaththu) in Trincomalee District, Eastern Province of Sri Lanka, between June 2010 and July 2013. The relationship between seven abiotic variables (temperature, pH, conductivity, Total Dissolved Solid (TDS), turbidity, Dissolved Oxygen (DO), and salinity) was measured. Results. A total of 21,347 anophelines were recorded representing 15 species. Anopheles subpictus 24.72% (5,278/21,347) was the predominant species, followed by 24.67% (5,267/21,347) of An. nigerrimus and 14.56% (3,109/21,347) of An. peditaeniatus. A total of 9,430 breeding habitats under twenty-one categories were identified. An. culcicifacies was noted to be highest from built wells (20.5%) with high salinity (1102.3 ± 81.8 mg/L), followed by waste water collections (20.2%) having low DO levels (2.85 ± 0.03 mg/L) and high TDS (1,654 ± 140 mg/L). Conclusion. This study opens an avenue to explore new breeding habitats of malaria vectors in the country and reemphasizes the requirement of conducting entomological surveillance to detect potential transmission of malaria in Sri Lanka under the current malaria elimination programme.

Entomological investigations on malaria vectors in some war-torn areas in the trincomalee district of sri lanka after settlement of 30-year civil disturbance.

Background. Malaria was an endemic problem in Trincomalee District, Eastern Province of Sri Lanka. Very few recent data concerning Anopheles are available which transmit malaria. Therefore, the aim of this study is to identify various Anopheles species and the dynamics of anophelines including malaria vectors in Trincomalee District for effective vector control under the current malaria elimination program embarked in the country. Method. Entomological surveys were conducted on a monthly basis, using five entomological techniques, namely, indoor hand collection (HC), window trap collection (WTC), cattle-baited net collection (CBNC), and cattle-baited hut collection (CBHC) from June 2010 to June 2012 in 32 study areas under five entomological sentinel sites. Results. Seventeen anopheline species were encountered, of which Anopheles subpictus was the predominant species in all sampling methods. It is noted that A. culicifacies and A. subpictus have adapted to breed in polluted water in urban settings which may cause serious implications on the epidemiology of malaria in the country. Conclusions. It is important to determine the abundance, biology, distribution, and relationship with climatic factors of main and secondary malaria vectors in Sri Lanka in order to initiate evidence based controlling programs under the current malaria elimination program in Sri Lanka.

Determination of the foraging behaviour and blood meal source of malaria vector mosquitoes in Trincomalee District of Sri Lanka using a multiplex real time polymerase chain reaction assay.

BackgroundStudies of host preference patterns in blood-feeding anopheline mosquitoes are crucial to incriminating malaria vectors. However, little information is available on host preferences of Anopheles mosquitoes in Sri Lanka.MethodsAdult Anopheles mosquitoes were collected from five selected sentinel sites in Trincomalee District during June–September 2011. Each blood-fed mosquito was processed on filter papers. DNA was extracted using the dried blood meal protocol of the QIAmp DNA mini kit. A multiplexed, real-time PCR assay targeting eight animals was developed for two panels to identify the host meal of Anopheles. Human blood index (HBI), forage ratio (FR) and host feeding index (HFI) were calculated.ResultsA total of 280 field-caught, freshly engorged female mosquitoes belonging to 12 anopheline species were analysed. The overall HBI and HFI in the present study were low indicating that humans were not the preferred host for the tested anopheline species. Nevertheless, a small proportion engorged Anopheles aconitus, Anopheles culicifacies, Anopheles barbirostris, Anopheles annularis, Anopheles subpictus, Anopheles peditaeniatus, Anopheles pseudojamesi, and Anopheles barbumbrosus contained human blood.ConclusionThe presence of human blood in mosquito species indicates the possibility of them transmitting malaria. Further studies on vector competence are needed to determine the role of each of the above anopheline species as efficient vectors of malaria.

A comparative retrospective study of RT-PCR-based liquid hybridization assay for early, definitive diagnosis of dengue.

Dengue is an important flaviviral infection in tropical and subtropical regions. Early diagnosis of dengue infection helps in monitoring the disease, determining when hospital admission is necessary and reducing case fatalities. The objective of this study was to carry out a retrospective comparison of an RT-PCR-based liquid hybridization (RT-PCR-LH) assay with PCR amplification, virus isolation and serological techniques for laboratory diagnosis of dengue infection. Amplified products of non-structural 3 gene were hybridized with a mixture of four dengue type-specific DNA probes in liquid phase. The assay was validated in a comparative retrospective study using acute serum samples collected from 119 fever patients with or without dengue, confirmed by haemagglutination inhibition (HAI) assay, the gold standard assay for diagnosis of dengue infection. The RT-PCR-LH assay was highly specific for dengue and, as an early laboratory diagnostic method, had 100% sensitivity in detecting dengue patients confirmed by HAI assay. A high analytical sensitivity of two fluorescent focus units of dengue virus/reaction was achieved. This RT-PCR-LH assay using a single serum specimen offers distinct advantages of specificity and sensitivity over other diagnostic techniques for early definitive laboratory diagnosis of dengue infection when serological methods are of little value.

RNA positivity rates among anti-HCV reactive blood donors in Sri Lanka: A preliminary study

Since details of sexual practices was not sought in this study, any association with MSM practices could not be ascertained. However, sexual abuse of children is common in Pakistan. According to a report from the Coalition on Child Rights (1998), Pakistan has the dubious distinction of having a large number of boys and young males who are victims of commercial sexual exploitation.[6] Whether child sex abuse is an important factor in the emergence of syphilis in young males in Pakistan is a question that needs to be answered.

Molecular markers of chloroquine resistance in Plasmodium falciparum in Sri Lanka:frequency before revision of the antimalarial drug policy

Drug resistance is now a major obstacle in the control of human malaria, with reports of widespread clinical resistance against all the commonly used antimalarial drugs except the artemisinin derivatives (WHO, 2008). In Sri Lanka, although resistance of Plasmodium falciparum to chloroquine (CQ) was first documented in 1984 and 50% of P. falciparum infections in certain endemic areas were recently found to show such resistance (Handunnetti et al., 1996; Hapuarachchi et al., 2004), CQ remained the first-line therapy for all uncomplicated malarial infections until 2008. The detection of antimalarial resistance is of vital importance for containment of its emergence and spread. The detection of molecular markers has become a popular tool for the recording of such resistance since, compared with conventional in-vivo and in-vitro assays, it is often easier to perform at community level, and less laborious. Evaluation of the prevalence of the molecular markers associated with resistance can be used to identify communities that are most in need of in-vivo assessments (Plowe, 2003). The most commonly used markers for CQ resistance in P. falciparum are variants of the parasite’s CQ resistance transporter (pfcrt) and multiple drug resistance 1 (pfmdr1) genes. The results of several in-vitro and in-vivo studies of P. falciparum have confirmed the association between mutations in pfcrt and CQ resistance (Fidock et al., 2000; Labbé et al., 2001; Maguire et al., 2001). Of the reported mutations, the lycine-tothreonine substitution at codon 76 (K76T) is currently considered the most appropriate molecular marker for predicting CQ resistance, given its high frequency in CQresistant parasites and strong association with CQ resistance (Djimdé et al., 2001). Although the exact role of pfmdr1 in CQ resistance is still controversial, it has been suggested that mutations N86Y, Y184F, S1034C, D1042N and D1246Y may each play a potential role in modulating the level of CQ resistance (Reed et al., 2000).

Empirical optimization of risk thresholds for dengue: an approach towards entomological management of Aedes mosquitoes based on larval indices in the Kandy District of Sri Lanka

BackgroundLarval indices such as Premise Index (PI), Breteau Index (BI) and Container Index (CI) are widely used to interpret the density of dengue vectors in surveillance programmes. These indices may be useful for forecasting disease outbreaks in an area. However, use of the values of these indices as alarm signals is rarely considered in control programmes. Therefore, the current study aims to propose threshold values for vector indices based on an empirical modeling approach for the Kandy District of Sri Lanka.MethodsMonthly vector indices, viz PI, BI and CI, for Aedes aegypti and Aedes albopictus, of four selected dengue high risk Medical Officer of Health (MOH) areas in the Kandy District from January 2010 to August 2017, were used in the study. Gumbel frequency analysis was used to calculate the exceedance probability of quantitative values for each individual larval index within the relevant MOH area, individually and to set up the threshold values for the entomological management of dengue vectors.ResultsAmong the study MOH areas, Akurana indicated a relatively high density of both Ae. aegypti and Ae. albopictus, while Gangawata Korale MOH area had the lowest. Based on Ae. aegypti, threshold values were defined for Kandy as low risk (BIagp > 1.77), risk (BIagp > 3.23), moderate risk (BIagp > 4.47) and high risk (BIagp > 6.23). In addition, PI > 6.75 was defined as low risk, while PI > 9.43 and PI>12.82 were defined as moderate and high risk, respectively as an average.ConclusionsThreshold values recommended for Ae. aegypti (primary vector for dengue) along with cut-off values for PI (for Ae. aegypti and Ae. albopictus), could be suggested as indicators for decision making in vector control efforts. This may also facilitate the rational use of financial allocations, technical and human resources for vector control approaches in Sri Lanka in a fruitful manner.