The p.H63D allele of the HFE gene protects against low iron stores in Sri Lanka.

In hereditary hemochromatosis, iron overload is associated with homozygosity for the p.C282Y mutation. A second mutation, p.H63D, occurs at significant frequencies in Europe, North Africa, the Middle East and Asia. Early studies in Sri Lanka indicated that the variant had arisen independently, suggesting that it had been the subject of selective pressure. However, its role in iron absorption is unclear. In a survey of 7526 Sri Lankan secondary school students, we determined hemoglobin genotype and measured red cell indices, serum ferritin, transferrin receptor, iron zinc protoporphyrin and hepcidin. These variables were compared according to the presence or absence of the p.H63D variant in a subset of 1313 students for whom DNA samples were available. Students were classified as having low red cell indices if they had an MCV <80 fl and/or MCH <27 pg. Hetero and/or homozygosity for the p.H63D variant was more common in students with normal than low red cell indices (16.4% and 11.9% respectively; p = 0.019). Iron biomarkers and red cell indices were greater in children with the p.H63D variant than in normal and this was statistically significant for MCV (p = 0.046). Our findings suggest that selective pressure by mild iron deficiency contributes to the high frequencies of the p.H63D variant.

Detection of pathogenic Leptospira with rapid extraction followed by recombinase polymerase amplification (RPA) and quantitative polymerase chain reaction (qPCR) assay-A comprehensive study from Sri Lanka.

Leptospirosis is the most widespread zoonosis in the world. The disease is more prevalent in tropical regions where the majority of developing countries are located. Leptospirosis is considered a protean manifestation zoonosis with severity of the disease ranging from a mild febrile illness to a severe and life-threatening illness. Clinical symptoms of leptospirosis overlap with other tropical febrile illnesses. Early, rapid, and definitive diagnosis is important for effective patient management. Since Polymerase Chain Reaction (PCR)-based assays are not readily available in most clinical settings, there is a need for an affordable, simple, and rapid diagnostic test. Quantitative PCR (qPCR) and Recombinase Polymerase Amplification (RPA) were implemented at the Faculty of Medicine, University of Kelaniya, and a prospective study to evaluate RPA for diagnosis of acute phase of leptospirosis was conducted. Results indicate that RPA and qPCR were positive in 81% (98/121) of the total positive and acute clinical samples. Of the 81 positive MAT confirmed patients 60 (74%) and 53 (65%) were positive with qPCR and RPA respectively. Retrospective evaluation revealed a high diagnostic accuracy (sensitivity-70% and specificity-87%) of RPA compared to MAT as the reference gold standard. Results further suggest that there is no significant difference between the two assays, qPCR and RPA-SwiftX (P = 0.40). Laboratory procedures for the extraction and detection by qPCR in the laboratory have been optimized to obtain results within 6 hours. However, the RPA-SwiftX method under field conditions took 35 minutes. The RPA-SwiftX method could replace the qPCR which shows similar sensitivity and specificity. Therefore, RPA established under the current study presents a powerful tool for the early and rapid diagnosis of leptospirosis at point-of-care.

Subclinical mucosal inflammation in diarrhea-predominant irritable bowel syndrome (IBS) in a tropical setting.

BACKGROUND AND AIMS: There is evidence for low-grade inflammation in the pathophysiology of post-infectious irritable bowel syndrome (IBS). We assessed the degree of subclinical intestinal mucosal inflammation in diarrhea-predominant IBS (IBS-D) in a tropical setting. MATERIAL AND METHODS: In a prospective study over 1 year, we investigated 49 patients with IBS-D (cases; median age 34 years (range 18-59); M:F 36:13), diagnosed on Rome III criteria. 14 individuals with a family history of colon cancer (median age 46.5 years (range 23-56); M:F 6:8) were selected as controls. Stools of cases and controls were tested for calprotectin. During colonoileoscopy, serial biopsies were obtained. Mucosal mast cells, neutrophils, eosinophils and lymphocytes/plasma cell infiltrate were quantified. Tissue expression of IL-8 and IL-10 was assessed in biopsies by semi-quantitative RT-PCR. RESULTS: A history suggestive of an episode of infectious diarrhea (ID) was present in 16/49 cases and 0/14 controls (p = 0.013). In cases, there were significantly more mucosal mast cells in the ileum and all segments of colon and significantly more eosinophils in the cecum. Tissue expression of IL-8 was significantly higher and IL-10 significantly lower in cases compared with controls (target/standard cDNA ratio, median (range) IL-8: 1.25 (0.75-2) vs. 0.85 (0.63-1.3), p < 0.0001, Mann-Whitney U test; IL-10: 0.33 (0-0.63) vs. 0.55 (0.5-0.7), p < 0.0001). There was a significant inverse correlation between IL-8 and IL-10 expression (Pearson correlation, (-) 0.509; p < 0.01). CONCLUSION: There was evidence of subclinical intestinal mucosal inflammation in patients with IBS-D. The finding of increased eosinophils is novel, and may be of special relevance to IBS-D in the tropics.

Geographic information system (GIS) mapping of lymphatic filariasis endemic areas of Gampaha District, Sri Lanka based on epidemiological and entomological screening.

The objective of this study was to develop a site directed geographic information system (GIS) map of lymphatic filariasis (LF) in Gampaha District, Sri Lanka as a guide for targeted control activities. Epidemiological and entomological screening of LF was carried out in nine pre-identified endemic areas in Gampaha District, using night blood screening and pool-screening PCR-ELISA. In total, 1,073 subjects (286 children, 787 adults) from 9 sites were examined. Positive cases were detected at 2 sites, with prevalence rates of 0.5% (Hekiththa) and 3.4% (Peliyagoda); the prevalence of microfilaria (mf) among adult Culex quinquefasciatus mosquitoes surveyed was 30%. The overall prevalence of mosquitoes with L1-L2 larvae of W. bancrofti ranged from 0% to 8.31% using dissection and point estimates of infection prevalence, and ranged from 0 to 32.4% using PCR-ELISA. The largest number of human cases was found at altitudes of 2.5-3.5 min highly populated areas, where transmission appears to have taken place. Questionnaires indicated that limited community awareness of LF may be a reason for the fairly static infection prevalent among the local population. The GIS mapping of LF cases shows a considerable prevalence of LF and marked variability by geographic site in Gampaha.

Hantavirus Hemorrhagic Fever with Renal Syndrome - Suspected Cases in Sri Lanka: Clinical Picture and Epidemiology from 2013-2021.

Hantavirus hemorrhagic fever with renal syndrome (HFRS) is an emerging zoonotic disease in Europe and Asia, which is clinically indistinguishable from leptospirosis. A total of 1,032 patients with clinical suspicion of HFRS-like illness were included in the analysis from March 2013 to March 2021. Of these, 168 were positive for hantavirus immunoglobulin M (IgM) antibodies. Thirty-one of 35 patients had a 4-fold increase in IgG antibody titer with paired serum, confirming acute hantavirus infections. The detected antibodies showed a diverse pattern, strongly cross-reacting with the Seoul, Hantaan, and Puumala virus antigens. All the IgM-positive patients had no serological evidence of acute dengue or leptospirosis and had classical features of HFRS, including fever, thrombocytopenia, and renal involvement. More than 90% of patients had a history of rodent exposure 2-3 weeks prior to the onset of the fever. The highest number of positive cases was diagnosed in the Western and North Central Provinces of Sri Lanka during the paddy harvesting seasons. A significant number of patients develop severe complications with high mortality rates. Therefore, hantavirus infection should be considered as a differential diagnosis for leptospirosis-like illnesses in Sri Lanka.

A randomised double-blind placebo-controlled clinical trial of oral hydroxyurea for transfusion-dependent ß-thalassaemia.

Hydroxyurea is an antimetabolite drug that induces fetal haemoglobin in sickle cell disease. However, its clinical usefulness in ß-thalassaemia is unproven. We conducted a randomised, double-blind, placebo-controlled clinical trial to evaluate the efficacy and safety of hydroxyurea in transfusion-dependent ß-thalassaemia. Sixty patients were assigned 1:1 to oral hydroxyurea 10-20 mg/kg/day or placebo for 6 months by stratified block randomisation. Hydroxyurea treatment did not alter the blood transfusion volume overall. However, a significantly higher proportion of patients on hydroxyurea showed increases in fetal haemoglobin percentage (89% vs. 59%; p < 0.05) and reductions in erythropoietic stress as measured by soluble transferrin receptor concentration (79% vs. 40%; p < 0.05). Based on fetal haemoglobin induction (> 1.5%), 44% of patients were identified as hydroxyurea-responders. Hydroxyurea-responders, required significantly lower blood volume (77 ± SD27ml/kg) compared to hydroxyurea-non-responders (108 ± SD24ml/kg; p < 0.01) and placebo-receivers (102 ± 28ml/kg; p < 0.05). Response to hydroxyurea was significantly higher in patients with HbE ß-thalassaemia genotype (50% vs. 0%; p < 0.01) and Xmn1 polymorphism of the γ-globin gene (67% vs. 27%; p < 0.05). We conclude that oral hydroxyurea increased fetal haemoglobin percentage and reduced erythropoietic stress of ineffective erythropoiesis in patients with transfusion-dependent ß-thalassaemia. Hydroxyurea reduced the transfusion burden in approximately 40% of patients. Response to hydroxyurea was higher in patients with HbE ß-thalassaemia genotype and Xmn1 polymorphism of the γ-globin gene.

Efficacy and safety of oral hydroxyurea in transfusion-dependent ß-thalassaemia: a protocol for randomised double-blind controlled clinical trial.

INTRODUCTION: Despite being one of the first diseases to be genetically characterised, ß-thalassaemia remains a disorder without a cure in a majority of patients. Most patients with ß-thalassaemia receive only supportive treatment and therefore have a poor quality of life and shorter life spans. Hydroxyurea, which has shown to induce fetal haemoglobin synthesis in human erythroid cells, is currently recommended for the treatment of sickle cell disease. However, its clinical usefulness in transfusion-dependent ß-thalassaemia is unclear. Here, we present a protocol for a randomised double-blind controlled clinical trial to evaluate the efficacy and safety of oral hydroxyurea in transfusion-dependent ß-thalassaemia. METHODS AND ANALYSIS: This single-centre randomised double-blind placebo-controlled clinical trial is conducted at the Thalassaemia Centre of Colombo North Teaching Hospital, Ragama, Sri Lanka. Adult and adolescent patients with haematologically and genetically confirmed transfusion-dependent ß-thalassaemia are enrolled and randomised into the intervention or control group. The intervention group receives oral hydroxyurea 10-20 mg/kg daily for 6 months, while the control group receives a placebo which is identical in size, shape and colour to hydroxyurea without its active ingredient. Transfused blood volume, pretransfusion haemoglobin level, fetal haemoglobin percentage and adverse effects of treatment are monitored during treatment and 6 months post-treatment. Cessation or reduction of blood transfusions during the treatment period will be the primary outcome measure. The statistical analysis will be based on intention to treat. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the Ethics Committee of Faculty of Medicine, University of Kelaniya (P/116/05/2018) and the trial is approved by the National Medicinal Regulatory Authority of Sri Lanka. Results of the trial will be disseminated in scientific publications in reputed journals. TRIAL REGISTRATION NUMBER: SLCTR/2018/024; Pre-results.

Sickle cell disease in Sri Lanka: clinical and molecular basis and the unanswered questions about disease severity.

BACKGROUND: Though case reports and limited case series of Sickle cell disease in Sri Lanka have been reported previously, no attempt has been made hitherto to undertake a comprehensive genotypic-phenotypic analysis of this "rare" group of patients. RESULTS: All accessible Sickle cell disease patients, totaling 60, including, 51 Sickle ß-thalassaemia and 9 homozygous sickle patients were enrolled from seven thalassaemia treatment centres between December 2016-March 2019. The majority of patients were of Sinhalese ethnicity (n = 52, 86.67%). Geographically, two prominent clusters were identified and the distribution of Sickle haemoglobin in the island contrasted markedly with the other haemoglobinopathies. 3/ 9 homozygous sickle patients and 3/ 51 Sickle ß-thalassaemia patients were receiving regular transfusion. Joint pain was the commonest clinical symptom among all sickle cell disease patients (n = 39, 65.0%). Dactylitis was significantly more common in homozygous sickle patients compared with the Sickle ß-thalassaemia groups (p 0.027). Two genetic backgrounds sickle mutation were identified namely, Arab Indian and Benin. Among the regulators of Foetal hemoglobin in Sickle patients of the present study rs1427407 G > T seemed to be the most prominent modifier, with a significant association with Foetal haemoglobin levels (p 0.04). CONCLUSIONS: Overall, the clinical course of the Asian version of Sickle cell disease in Sri Lanka appears to be milder than that described in India.

STR polymorphisms in Sri Lanka: evaluation of forensic utility in identification of individuals and parentage testing.

OBJECTIVES: This preliminary study was carried out to determine the allele frequencies and forensic efficiency parameters for the short tandem repeat loci CSF1PO, TPOX, THO1, D16S539, D7S820, D13S317, vWA, FESFPS and F13B in a test sample population of Sri Lankans. DESIGN: Test samples were obtained from 305 non-related individuals originating from all 9 provinces of Sri Lanka. DNA was extracted from whole blood using chelex-100 and amplified by PCR using the GenePrint STR kit and silver stained. Final DNA profiles were analysed for forensic efficiency parameters and paternity indices using PowerStats version 12. Possible divergence from Hardy-Weinberg Equilibrium was tested using the chi-square test and exact test. RESULTS: All common alleles in the allelic ladders were found in the test sample studied. PIC values >0.5 for all 9 STR loci indicate this STR system to be informative and useful for identification purposes. The D13S317, vWA and D7S820 loci were found to be the most polymorphic markers of the system studied. CONCLUSIONS: No deviations from Hardy-Weinberg Equilibrium were found for any of the loci examined. The results indicate that the 9 STR loci system described here is suitable for estimating DNA profile frequencies in human identification and forensic and parentage testing for legal purposes among Sri Lankans.

Evaluation of rapid extraction and isothermal amplification techniques for the detection of Leishmania donovani DNA from skin lesions of suspected cases at the point of need in Sri Lanka.

BACKGROUND: Leishmaniasis is a disease caused by vector-borne protozoans. In Sri Lanka, the cutaneous form of the disease is predominant, which is usually diagnosed using Giemsa-stained slit skin smear examination and by histology. However, the sensitivity of slit skin smears and histology are reportedly low. Moreover, facilities for the highly sensitive polymerase chain reaction (PCR) are available only in a few highly-equipped parasitology laboratories. Therefore, there is a need for low cost, sensitive and specific screening tests for diagnosis of leishmaniasis at the point of need. RESULTS: In this study, a mobile suitcase laboratory applying novel extraction (SpeedXtract) and isothermal amplification and detection (recombinase polymerase amplification assay, RPA) methods were evaluated for the diagnosis of cutaneous leishmaniasis in Sri Lanka. First, the developed assay was applied to three different sample types (punch biopsy, slit skin smears and fine needle aspirates) at a local hospital. The results showed that the 2 mm punch biopsy sample produced the best exponential amplification curve and early fluorescence signal in the RPA assay. Secondly, punch biopsies were collected from 150 suspected cutaneous leishmaniasis cases and screened with SpeedXtract/RPA, RNAlater/PCR and ATL buffer/PCR, in addition to Giemsa-stained slit skin smears. Fifty-seven samples were negative in all detection methods. In total 93 samples were positive with assay sensitivities of 65.5% (SpeedXtract/RPA), 63.4% (RNAlater/PCR) and 92.4% (ATL buffer/PCR). The Giemsa-stained slit skin smear delivered the worst clinical sensitivity (32.2%). CONCLUSIONS: The SpeedXtract/RPA method under field conditions took 35 min, while almost 8 h were needed to finalize the extraction and detection by PCR in the laboratory. The SpeedXtract/RPA method produced similar sensitivity to samples preserved in RNAlater and subjected to PCR amplification, but both were less sensitive than ATL-preserved samples subjected to PCR amplification. There is a need for a standardization of sample collection and nucleic acid extraction methods.

Evaluation of PCR-ELISA as a tool for monitoring transmission of Wuchereria bancrofti in District of Gampaha, Sri Lanka.

OBJECTIVE: To compare Wuchereria bancrofti (W. bancrofti) infection rates of Culex quinquefasciatus, using dissection and PCR-ELISA in two consecutive time periods (from 2007 to 2008 and from 2008 to 2009). METHODS: Mosquitoes were collected in 30 sentinel and 15 non-sentinel sites in 15 Medical Officer of Health areas of Gampaha District known for the presence of W. bancrofti transmission in two consecutive time period of 2007 to 2008 and 2008 to 2009. Captured mosquitoes were dissected to determine the W. bancrofti larvae (L1, L2, L3). PCR was carried out using DNA extracted from mosquito pools (15 body parts/pool) utilizing the primers specific for Wb-SspI repeat. PCR products were analyzed by hybridization ELISA using fluorescein-labeled wild type specific probes. The prevalence of infected/infective mosquitoes in PCR pools (3 pools/site) was estimated using the PoolScreen™ algorithm and a novel probability-based method. RESULTS: Of 45 batches of mosquitoes dissected, W. bancrofti infected mosquitoes were found in 19 and 13 batches, with an infection rate of 13.29% and 3.10% with mean larval density of 8.7 and 1.0 larvae per mosquito for two study periods in the Gampaha District. Total of 405 pools of head, thorax and abdomen were processed by PCR-ELISA for each year. Of these, 51 and 31 pools were positive for W. bancrofti in the two study periods respectively. The association of dissection based prevalence rates with PCR based rates as determined by the Pearson correlation coefficient were 0.176 and 0.890 respectively for the two periods. CONCLUSIONS: Data indicate that PCR-ELISA is more sensitive than the traditional dissection techniques for monitoring transmission intensity.

Evaluation of the genetic diversity of domain II of Plasmodium vivax Apical Membrane Antigen 1 (PvAMA-1) and the ensuing strain-specific immune responses in patients from Sri Lanka.

Antigenic polymorphism displayed by malaria parasites is a skewed schema to escape the host immune system. The prevailing genetic diversity at domain II of the Plasmodium vivax Apical Membrane Antigen-1 (Pvama-1DII) was characterized in 64 single clone P. vivax isolates from Sri Lanka, where unstable malaria prevails with low intensity. In Sri Lanka, the Pvama-1DII gene showed meager meiotic recombination with the enclosure of single nucleotide polymorphisms (SNPs). Eleven amino acid (a.a.) variant positions defined 21 a.a. haplotypes with 9 unique to the island, where the predominant haplotype, H1, was identical to the reference Salvador I strain. A further 376 globally dispersed isolates defined 38 a.a. haplotypes (H22-H59), with 4 and 26 haplotypes exclusive to India and Thailand, respectively. The phylogenetic tree revealed no clustering, where most isolates had a very recent common origin. The polymorphism detected in PvAMA-1DII B and T cell epitopes evidenced an immune evasion mechanism exploited by the parasite. Majority of Sri Lankan patients developed antibody responses to both conformational and linear B cell epitopes. The ensuing strain-specific immunity due to extensive antigenic polymorphism was evaluated by aligning a.a. sequences of PvAMA-1DII with the homologous total (IgM+IgG) antibody responses assayed by in-house established indirect ELISAs against 7 PvAMA-1DII overlapping synthetic peptides, P01-P07. While the antibody responses to P01-P03, P06, P07 harbouring P. vivax clinical isolates with polymorphic a.a. haplotype to Sal I was clearly strain-transcending (cross-reactive), individuals with isolates identical to the Sal I strain observed varying antibody prevalence against the seven PvAMA-1DII Sal-I synthetic peptides, with the highest prevalence detected against P04. Synthetic peptide P04, spanning a.a. positions 302-324 of the PvAMA-1DII of the Sal I strain that included the epitope recognized by the invasion inhibitory 4G2 monoclonal antibody of PfAMA-1, was highly conserved in all 440 local and global P. vivax isolates examined. A functional role for this region is reinforced by the highly immunogenic nature of P04, and could point towards a presumably "protective" anti-P04 antibody response that elicited an isotype switch from IgM to IgG, with increasing exposure to malaria exclusively in endemic residents. Thus the conserved and seemingly "protective" nature of the domain II loop of PvAMA-1 makes it a putative contender to be included in a cocktail vaccine against P. vivax asexual erythrocytic stages in Sri Lanka.

Current developments in genomics and personalized health care: impact on public health.

The knowledge gained from the characterization of genomes, especially the human genome, holds considerable potential for the development of new health care innovations for prevention, diagnosis, and management of many diseases in the coming decade. However, owing to the presence of highly complex scientific, economic, social, and ethical issues associated with this field, societies will need to be better prepared for the era of postgenomics and its consequences. It is important to ensure that the benefits of genomics are distributed fairly among all the countries of the world and that the well-tried and more conventional approaches to medical research and practice are not neglected while the medical potential of genomics is being explored. In this report, the author focuses mainly on human genomics, its applications, development of related technologies and issues related to the dissemination of knowledge derived from genome information, and finally, their impact on global health care.

Evaluation of PCR-ELISA as a tool for monitoring transmission of Wuchereria bancrofti in District of Gampaha, Sri Lanka

Objective: To compare Wuchereria bancrofti (W. bancrofti) infection rates of Culexquinquefasciatus, using dissection and PCR-ELISA in two consecutive time periods (from 2007 to 2008 and from 2008 to 2009). Methods: Mosquitoes were collected in 30 sentinel and 15 non-sentinel sites in 15 Medical Officer of Health areas of Gampaha District known for the presence ofW. bancrofti transmission in two consecutive time period of 2007 to 2008 and 2008 to 2009. Captured mosquitoes were dissected to determine the W. bancrofti larvae (L1, L2, L3). PCR was carried out using DNA extracted from mosquito pools (15 body parts/pool) utilizing the primers specific for Wb-SspI repeat. PCR products were analyzed by hybridization ELISA using fluorescein-labeled wild type specific probes. The prevalence of infected/infective mosquitoes in PCR pools (3 pools/site) was estimated using the PoolScreenTM algorithm and a novel probability-based method.Results:Of 45 batches of mosquitoes dissected, W. bancrofti infected mosquitoes were found in 19 and 13 batches, with an infection rate of 13.29% and 3.10% with mean larval density of 8.7 and 1.0 larvae per mosquito for two study periods in the Gampaha District. Total of 405 pools of head, thorax and abdomen were processed by PCR-ELISA for each year. Of these, 51 and 31 pools were positive for W. bancrofti in the two study periods respectively. The association of dissection based prevalence rates with PCR based rates as determined by the Pearson correlation coefficient were 0.176 and 0.890 respectively for the two periods. Conclusions: Data indicate that PCR-ELISA is more sensitive than the traditional dissection techniques for monitoring transmission intensity.