Abstract Background Strongyloidiasis a neglected tropical disease is known to cause severe disease among immunosuppressed and has not been studied extensively in Sri Lanka. Parasitological diagnostic approaches based on faecal microscopy and culture often fail to detect low-intensity infections. This study investigates the presence of strongyloidiasis among selected immunocompromised individuals using parasitological, molecular and serological techniques. Methods Adult patients with immunocompromising conditions admitted to three tertiary care hospitals in Sri Lanka were recruited. A faecal sample and 2 ml of venous blood were collected. The faecal samples were subjected to direct faecal smear and cultures (agar plate, charcoal and Harada-Mori) and polymerase chain reaction (PCR) using species specific primers designed for Strongyloides stercoralis. The presence of Strongyloides IgG antibodies was tested in the collected serum samples using DRG Strongyloides IgG enzyme-linked immunosorbent assay (ELISA) kits. The PCR products of the positive samples were sequenced using Sanger sequencing method. Results A total of 260 patients were recruited to this study, out of which 160 provided faecal samples and 122 provided blood samples. Out of the 160 faecal samples, none were positive for strongyloidiasis by direct smear, charcoal and Harada-Mori cultures. Only one sample (0.6%) was positive by agar plate culture. Out of the 123 samples subjected to PCR, 14 (11.4%), including the culture positive patient, were positive for S. stercoralis. Sequencing results of the PCR products indicated 100% similarity to S. stercoralis. Out of the 122 serum samples subjected to ELISA, 20 (16.4%), including the culture positive patient, were positive for Strongyloides IgG antibodies. However, sociodemographic, exposure factors, clinical features were not significantly associated with the presence of strongyloidiasis infection. Conclusions Strongyloidiasis is present among the immunocompromised population in Sri Lanka, even in the absence of a significant relationship with associated factors. It is advisable to screen such patients with highly sensitive tests such as PCR for early diagnosis and treatment. Graphical Abstract

Background Localized cutaneous leishmaniasis (LCL) is a chronic ulcerating disease. A literature review identified inconsistencies in clinical trials. The aims of this study were to reach a consensus on the most important domains to measure when assessing LCL, agree on parameters to measure the domains, and develop a tool representing a Core Outcome Set (COS), for use in clinical assessment of LCL. Methodology & principal findings A literature review was conducted to identify any existing COS for LCL embracing agreed Outcome Domains, i.e. what to measure and any Outcome Measurement Instruments (OMIs). As no COS was available, potential outcome domains for assessment of LCL were identified through an international collaborative approach using e-consultations and virtual discussions with expert stakeholders (n = 20) from geographically different LCL endemic countries. Subsequent judgmental validation process included a face-to-face multidisciplinary stakeholders’ meeting adopting the Nominal Group Technique. A final consensual agreement on outcome domains and items required to measure these domains was established. “Clinical Cure” was defined as the ideal overall "General Concept". The five Core Outcome Domains included Signs capturing clinical morphology, diameter, and induration of an index lesion with the aid of a palpability score, Treatment Efficacy assessing percentage change in size of the lesion and re-epithelialization compared to baseline, Treatment Impact which included an investigator and patient visual analogue score, and Clinical Sequelae rating pigment change, atrophic and hypertrophic/keloid scars. It was agreed that two open-ended questions should be included to capture some aspects of Health-Related Quality of Life as a means of capturing a patient-focused approach. Conclusion LeishCOM_LCL was generated to reflect a COS for LCL. This captured demographic details, agreed outcome domains and measures to assess these domains. Validation of LeishCOM_LCL will be reported in a separate paper. Development of a Patient Reported Outcome Measure will be considered in the future.

Cutaneous leishmaniasis (CL) is caused by Leishmania donovani in Sri Lanka. Pentavalent antimonials (e.g., sodium stibogluconate [SSG]) remain first-line drugs for CL with no new effective treatments emerging. We studied whole blood and lesion transcriptomes from Sri Lankan patients with CL at presentation and during SSG treatment. From lesions but not whole blood, we identified differential expression of immune-related genes, including immune checkpoint molecules, after onset of treatment. Using spatial profiling and RNA-FISH, we confirmed reduced expression of programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1) proteins on treatment in lesions of a second validation cohort and further demonstrated significantly higher expression of these checkpoint molecules on parasite-infected compared with noninfected lesional CD68+ monocytes and macrophages. Crucially, early reduction in PD-L1 but not IDO1 expression was predictive of rate of clinical cure (HR = 4.88) and occurred in parallel with reduction in parasite load. Our data support a model whereby the initial anti-leishmanial activity of antimonial drugs alleviates checkpoint inhibition on T cells, facilitating immune-drug synergism and clinical cure. Our findings demonstrate that PD-L1 expression can be used as a predictor of rapidity of clinical response to SSG treatment in Sri Lanka and support further evaluation of PD-L1 as a host-directed therapeutic in leishmaniasis.

Leishmania donovani causes both cutaneous and visceral leishmaniasis (CL and VL) in Sri Lanka, where chronic kidney disease (CKD) and kidney transplant recipients’ (KTR) geographical areas overlap. This study aimed to determine the risk of exposure to Leishmania infection among renal patients. This cross-sectional study in a renal unit assessed clinical symptoms and signs of CL and VL in recipients of blood/kidney or immunosuppressives. Sera were tested with Leishmania-specific DAT and rK-39 ELISA. There were 170 participants. A total of 84.1% (n = 143) were males (CKD: 101, KTR; 42, mean age 45) and 27 were females (females: CKD: 23, KTR: 4, mean age 39 years). Recipients of blood transfusion/s within last 2 years: 75.9% (CKD: 115, KTR: 14), on immunosuppressive therapy: 34.1% (CKD: 13, KTR: 45). Two CKD patients repeatedly showed clear positive titres (1: 12,800 and 1: 3200) with Leishmania-DAT and another two (CKD) became marginally positive with rK39-ELISA. Prevalence of anti-Leishmania antibodies: 2.4% (4/170). All four patients were clinically asymptomatic and were recipients of recent blood transfusions. Attributable risk of exposure to Leishmania infection through blood transfusions was 0.032, OR 2.99 (95% CI = 0.16 to 56.45, p = 0.47). Therefore, routine screening of kidney/blood donors and CKD and KTR patients in Sri Lanka may not be necessary.

Cutaneous leishmaniasis (CL) lesions are chronic and result in disfiguring scars. The microbiological aspects of these wounds have not been systematically investigated. We have recently reported that 61.5% of CL wounds in a Sri Lankan cohort harboured bacterial biofilms, mainly composed of bacilli, Enterobacteriaceae, and Pseudomonas, which could delay wound healing. We have additionally reported that biofilms were significantly associated patients over 40 years of age, discharge, pain and/or itching of the wound, and high pus cell counts. Using this as background knowledge and other relevant literature, we highlight the importance of investigating the role of biofilms in CL wound healing, clinical indicators, cost-effective laboratory tests involving less invasive sampling techniques for diagnosing biofilms and potential therapeutic options for biofilm-containing CL wounds, such as adjunctive application of wound debridement and antimicrobial treatment along with anti-parasitic drugs.

The endemic strain of Leishmania donovani in Sri Lanka causes cutaneous leishmaniasis (CL) rather than more common visceral form. We have visualized biofilms and profiled the microbiome of lesions and unaffected skin in thirty-nine CL patients. Twenty-four lesions (61.5%) were biofilm-positive according to fluorescence in situ hybridization. Biopsies of biofilm-positive lesions were dominated by Pseudomonas , class Bacilli and Enterobacteriaceae and distinguished by significantly lower community evenness. Higher relative abundance of a class Bacilli OTU was detected in wound swabs versus contralateral skin. Wound swabs and biopsies had significantly distinct microbiome profiles and lower diversity compared to unaffected skin. Greater abundances of potentially pathogenic organisms were observed in wet ulcers, lesions with high parasite loads and large wounds. In summary, more than half of L. donovani associated CL wounds harboured biofilms and the wounds exhibited a distinct, less diverse, microbiome than unaffected skin.

Cutaneous leishmaniasis (CL) is endemic in Sri Lanka. Giemsa-stained slit-skin-smears (SSS-Giemsa) and histology are routinely used in diagnosis with a sensitivity of 40-70%. PCR currently has limited accessibility. Therefore, we assessed the sensitivity and specificity of a previously described fluorescence in situ hybridization assay, on skin smears and biopsy samples to overcome the limitations encountered with routine diagnostic methods.Samples from a total of 123 suspected CL patients were collected and subjected to SSS-Giemsa, fluorescence in situ hybridization (FISH) on slit skin smears (SSS-FISH), formalin-fixed-paraffin-embedded-tissues stained with Hematoxylin & Eosin staining (FFPE-H&E) and FISH on formalin-fixed-paraffin-embedded-tissues (FFPE-FISH). Negative controls of 61 patient samples were collected from a CL non-endemic area and subjected to the same procedures. The gold standard PCR was used as a comparator. For FISH, two previously described cyanine 3 tagged Leihsmania genus-specific probes were used.Compared to PCR, SSS-Giemsa, SSS-FISH, FFPE-H&E, and FFPE-FISH had sensitivities of 76.5%, 79.1%, 50.4% and 80.9%, respectively. Routine diagnostic tests (SSS-Giemsa and FFPE-H&E) had a specificity of 100%. SSS-FISH and FFPE-FISH had specificities of 96.7% and 93.4%, respectively. FFPE-FISH had a statistically significant higher diagnostic performance than FFPE-H&E (p < 0.001). The relative performance of SSS-Giemsa, SSS-FISH and FFPE-FISH was similar (p > 0.05 for all comparisons).We conclude that FFPE-FISH is a more accurate diagnostic tool than FFPE-H&E. SSS-FISH did not have an additional advantage over SSS-Giemsa in diagnosis. However, SSS-FISH could be recommended as a minimally invasive method in studies assessing wound healing where immunological probes are used.

Prevention of re-establishment (POR) refers to the prevention of malaria outbreak/epidemic occurrence or preventing re-establishment of indigenous malaria in a malaria-free country. Understanding the effectiveness of the various strategies used for POR is, therefore, of vital importance to countries certified as “malaria-free” or to the countries to be thus certified in the near future. This review is based on extensive review of literature on both the POR strategies and elimination schemes of countries, (i) that have reached malaria-free status (e.g. Armenia, Mauritius, Sri Lanka), (ii) those that are reaching pre-elimination stage (e.g. South Korea), and (iii) countries at the control phase (e.g. India). History has clearly shown that poorly implemented POR programmes can result in deadly consequences (e.g. Sri Lanka); conversely, there are examples of robust POR programmes that have sustained malaria free status that can serve as examples to countries working toward elimination. Countries awaiting malaria elimination status should pre-plan their POR strategies. Malaria-free countries face the risk of resurgence mostly due to imported malaria cases; thus, a robust passenger screening programme and cross border collaborations are crucial in a POR setting. In addition, sustained vigilance, and continued funding for the national anti-malarial campaign programme and for related research is of vital importance for POR. With distinct intrinsic potential for malaria in each country, tailor-made POR programmes are built through continuous and robust epidemiological and entomological surveillance, particularly in countries such as Sri Lanka with increased receptivity and vulnerability for malaria transmission. In summary, across all five countries under scrutiny, common strengths of the POR programmes are (i) a multipronged approach, (ii) strong passive, active, and activated passive case detection, (iii) Indoor residual spraying (IRS), and (iv) health education/awareness programmes.

Cutaneous leishmaniasis (CL) caused by Leishmania donovani MON-37 is becoming a major public health problem in Sri Lanka, with 100 new cases per month being reported in endemic regions. Diagnosis of CL is challenging for several reasons. Due to relative specificity and rapidity we propose Fluorescence in Situ Hybridization as a diagnostic tool for CL. The objective was to evaluate the potential of Fluorescence in Situ Hybridization as a diagnostic method for Cutaneous leishmaniasis in Sri Lanka. Literature on current laboratory tests used to diagnose Cutaneous leishmaniasis in Sri Lanka and globally was reviewed. Sri Lankan data were reviewed systematically following the PRISMA guidelines. A narrative of the results is presented. There is currently no gold standard diagnostic method for Cutaneous leishmaniasis. Fluorescence in Situ Hybridization has been previously applied to detect dermal pathologies including those involving infectious agents, and its use to detect the Leishmania parasite in human cutaneous lesions reported in small number of studies, generally with limited numbers of subjects. Advantages of FISH has been specificity, cost and ease-of-use compared to the alternatives. Based on the available literature and our current work, FISH has potential for diagnosing CL and should now be evaluated in larger cohorts in endemic regions. FISH for CL diagnosis could find application in countries such as Sri Lanka, where laboratory facilities may be limited in rural areas where the disease burden is highest.

Cutaneous leishmaniasis (CL) caused by Leishmania donovani MON-37 is becoming a major public health problem in Sri Lanka, with 100 new cases per month being reported in endemic regions. Diagnosis of CL is challenging for several reasons. Due to relative specificity and rapidity we propose Fluorescence in Situ Hybridization as a diagnostic tool for CL. The objective was to evaluate the potential of Fluorescence in Situ Hybridization as a diagnostic method for Cutaneous leishmaniasis in Sri Lanka. Literature on current laboratory tests used to diagnose Cutaneous leishmaniasis in Sri Lanka and globally was reviewed. Sri Lankan data were reviewed systematically following the PRISMA guidelines. A narrative of the results is presented. There is currently no gold standard diagnostic method for Cutaneous leishmaniasis. Fluorescence in Situ Hybridization has been previously applied to detect dermal pathologies including those involving infectious agents, and its use to detect the Leishmania parasite in human cutaneous lesions reported in small number of studies, generally with limited numbers of subjects. Advantages of FISH has been specificity, cost and ease-of-use compared to the alternatives. Based on the available literature and our current work, FISH has potential for diagnosing CL and should now be evaluated in larger cohorts in endemic regions. FISH for CL diagnosis could find application in countries such as Sri Lanka, where laboratory facilities may be limited in rural areas where the disease burden is highest.

Background: The use of lengthy, detailed, and complex informed consent forms (ICFs) is of paramount concern in biomedical research as it may not truly promote the rights and interests of research participants. The extent of information in ICFs has been the subject of debates for decades; however, no clear guidance is given. Thus, the objective of this study was to determine the perspectives of research participants about the type and extent of information they need when they are invited to participate in biomedical research. Methods: This multi-center, cross-sectional, descriptive survey was conducted at 54 study sites in seven Asia-Pacific countries. A modified Likert-scale questionnaire was used to determine the importance of each element in the ICF among research participants of a biomedical study, with an anchored rating scale from 1 (not important) to 5 (very important). Results: Of the 2484 questionnaires distributed, 2113 (85.1%) were returned. The majority of respondents considered most elements required in the ICF to be 'moderately important' to 'very important' for their decision making (mean score, ranging from 3.58 to 4.47). Major foreseeable risk, direct benefit, and common adverse effects of the intervention were considered to be of most concerned elements in the ICF (mean score =4.47, 4. 47, and 4.45, respectively). Conclusions: Research participants would like to be informed of the ICF elements required by ethical guidelines and regulations; however, the importance of each element varied, e.g., risk and benefit associated with research participants were considered to be more important than the general nature or technical details of research. Using a participant-oriented approach by providing more details of the participant-interested elements while avoiding unnecessarily lengthy details of other less important elements would enhance the quality of the ICF.

Background: In special circumstances, establishing public private partnerships for malaria elimination may achieve targets faster than the state sector acting by itself. Following the end of the separatist war in Sri Lanka in 2009, the Anti Malaria Campaign (AMC) of Sri Lanka intensified malaria surveillance jointly with a private sector partner, Tropical and Environmental Diseases and Health Associates Private Limited (TEDHA) with a view to achieving malaria elimination targets by 2014. Methods: This is a case study on how public private partnerships can be effectively utilized to achieve malaria elimination goals. TEDHA established 50 Malaria Diagnostic Laboratories and 17 entomology surveillance sentinel sites in consultation with the AMC in areas difficult to access by government officials (five districts in two provinces affected by war). Results: TEDHA screened 994,448 individuals for malaria, of which 243,867 were screened at mobile malaria clinics as compared to 1,102,054 screened by the AMC. Nine malaria positives were diagnosed by TEDHA, while the AMC diagnosed 103 malaria cases in the same districts in parallel. Over 13,000 entomological activity days were completed. Relevant information was shared with AMC and the data recorded in the health information system. Conclusions: A successful public-private partnership model for malaria elimination was initiated at a time when the health system was in disarray in war ravaged areas of Sri Lanka. This ensured a high annual blood examination rate and screening of vulnerable people in receptive areas. These were important for certification of malaria-free status which Sri Lanka eventually received in 2016.

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.

Cutaneous leishmaniasis (CL) in Sri Lanka is caused by Leishmania donovani. This study assessed the diagnostic value of a new rapid diagnostic immunochromatographic strip (CL-Detect™ IC-RDT), that captures the peroxidoxin antigen of Leishmania amastigotes.We sampled 74 clinically suspected CL lesions, of which 59 (79.7%) were positive by PCR, 43 (58.1%) by Giemsa stained slit skin smear (SSS) and 21 (28.4%) by the new IC-RDT. All samples which were positive either by SSS or IC-RDT or both were positive by PCR. The sensitivities of the IC-RDT and SSS compared to PCR were 36% and 73%, respectively. Fifteen patients from this endemic region were negative by all three tests. Twenty two clinically non-CL skin lesions from a CL non-endemic region were also negative by all three methods. Specificity and PPV of both IC-RDT and SSS compared to PCR were 100%; the NPVs of IC-RDT and SSS were 37% and 58%, respectively. The median parasite grading of the 59 PCR positive samples was 2+ (1-10 parasites/100 HPFs) and IC-RDT positive lesions was 3+ (1-10 parasites /10HPFs). The duration of the lesion was not associated with IC-RDT positivity.The median parasite grade of Sri Lankan CL lesions is low. The low sensitivities of SSS and CL Detect™ IC-RDT may be due to low parasite counts or low expression of peroxidoxin antigen in amastigotes of the Sri Lankan L. donovani strain. Our results indicate that negative SSS has to be combined with PCR for confirmation of CL in Sri Lanka. The current commercially available IC-RDT is not suitable to diagnose CL in Sri Lanka; an IC-RDT with improved sensitivity to detect L. donovani would be a valuable addition in the diagnostic tool kit for Sri Lanka.

Imported malaria cases continue to be reported in Sri Lanka, which was declared ‘malaria-free’ by the World Health Organization in September 2016. Chemoprophylaxis, a recommended strategy for malaria prevention for visitors travelling to malaria-endemic countries from Sri Lanka is available free of charge. The strategy of providing chemoprophylaxis to visitors to a neighbouring malaria-endemic country within the perspective of a country that has successfully eliminated malaria but is highly receptive was assessed, taking Sri Lanka as a case in point.

Background: Trichomoniasis is a sexually transmitted parasitic infection. The World Health Organization (WHO) advocated flow charts for curable sexually transmitted infections (STIs) to improve the care. In this study, an attempt was made to evaluate the validity and reliability of WHO syndromic algorithm for vaginal discharge against trichomonas immunochromatographic test (ICT). Trichomonas ICT is a test with high validity, reliability, and feasibility. Objectives: The objective was to evaluate the validity and reliability of "WHO syndromic algorithm for vaginal discharge" against "trichomonas ICT" as a screening tool for trichomonas infection among women of reproductive age in the Western Province, Sri Lanka. Materials and Methods: This cross-sectional study was conducted in sexually transmitted disease clinics, well woman clinics, gynecology clinics, and institutional health clinics in the Western Province, Sri Lanka. We enrolled 100 women in the age group of 15-45 years using the stratified random sampling method. They were interviewed and examined and the specimens were collected to identify trichomoniasis by culture and ICT. Two-stage analyses were done to evaluate the performance of the WHO algorithm against Trichomonas ICT. Results: In a two-stage analysis, the specificity of syndromic algorithm improved from 80.9% to 94.4% while false positive rate reduced from 19.1% to 5.6%. The net effect of specificity was 98.7% while the false positive rate was 1.3%. Conclusion: The validity and reliability of WHO syndromic algorithm as a diagnostic tool for trichomoniasis can be improved by adding trichomonas ICT.

Objectives: This study was initiated to determine the sensitivity and specificity of a commercially available rapid diagnostic test (RDT) to detect leishmania antigen in cutaneous leishmaniasis(CL) skin lesions among Sri Lankan patients compared to PCR and Slit skin smear(SSS). Methods: Patients clinically suggestive of CL lesions were subjected to parasitological investigations. The clinical history was collected by the researcher. Samples were collected by a qualified trained medical officer from the suspected CL lesions at the dermatology clinic in the Hambantota hospital and from the patients coming to the Department of Parasitology, Faculty of Medical Sciences, University of Sri Jayewardenepura. RDT was done at the Hambantota hospital and samples were brought to the Department of Parasitology, to perform SSS, PCR and cultures. Results: Fifty samples have been collected thus far and PCR was performed only in 48 samples. Out of the 50 samples only 9 were positive by RDT and 32 by SSS and PCR. The 9 samples that were positive for RDT were positive by SSS and 7 positive by PCR. Of the 41 samples that were negative by RDT, 16 were negative by PCR as well as SSS. Conclusions: From the data collected it can concluded that RDT is not the best method to diagnose CL skin lesions in patients in Sri Lanka. Also it confirms that the best method to diagnose leishmaniasis is PCR.