Characterization of antibody-dependent cellular phagocytosis in patients infected with hepatitis C virus with different clinical outcomes.

Early neutralizing antibodies against hepatitis C virus (HCV) and CD8 + T cell effector responses can lead to viral clearance. However, these functions alone are not sufficient to protect patients against HCV infection, thus undefined additional antiviral immune mechanisms are required. In recent years, Fc-receptor-dependent antibody effector functions, particularly, antibody-dependent cellular phagocytosis (ADCP) were shown to offer immune protection against several RNA viruses. However, its development and clinical role in patients with HCV infection remain unknown. In this study, we found that patients with chronic GT1a or GT3a HCV infection had significantly higher concentrations of anti-envelope 2 (E2) antibodies, predominantly IgG1 subclass, than patients that cleared the viruses while the latter had antibodies with higher affinities. 97% of the patients with HCV had measurable ADCP of whom patients with chronic disease showed significantly higher ADCP than those who naturally cleared the virus. Epitope mapping studies showed that patients with antibodies that target antigenic domains on the HCV E2 protein that are known to associate with neutralization function are also strongly associated with ADCP, suggesting antibodies with overlapping/dual functions. Correlation studies showed that ADCP significantly correlated with plasma anti-E2 antibody levels and neutralization function regardless of clinical outcome and genotype of infecting virus, while a significant correlation between ADCP and affinity was only evident in patients that cleared the virus. These results suggest ADCP was mostly driven by antibody titer in patients with chronic disease while maintained in clearers due to the quality (affinity) of their anti-E2 antibodies despite having lower antibody titers.

Longitudinal Characterization of Phagocytic and Neutralization Functions of Anti-Spike Antibodies in Plasma of Patients after Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Phagocytic responses by effector cells to opsonized viruses have been recognized to play a key role in antiviral immunity. Limited data on coronavirus disease 2019 suggest that the role of Ab-dependent and -independent phagocytosis may contribute to the observed immunological and inflammatory responses; however, their development, duration, and role remain to be fully elucidated. In this study of 62 acute and convalescent patients, we found that patients with acute coronavirus disease 2019 can mount a phagocytic response to autologous plasma-opsonized Spike protein-coated microbeads as early as 10 d after symptom onset, while heat inactivation of this plasma caused 77-95% abrogation of the phagocytic response and preblocking of Fc receptors showed variable 18-60% inhibition. In convalescent patients, phagocytic response significantly correlated with anti-Spike IgG titers and older patients, while patients with severe disease had significantly higher phagocytosis and neutralization functions compared with patients with asymptomatic, mild, or moderate disease. A longitudinal subset of the convalescent patients over 12 mo showed an increase in plasma Ab affinity toward Spike Ag and preservation of phagocytic and neutralization functions, despite a decline in the anti-Spike IgG titers by >90%. Our data suggest that early phagocytosis is primarily driven by heat-liable components of the plasma, such as activated complements, while anti-Spike IgG titers account for the majority of observed phagocytosis at convalescence. Longitudinally, a significant increase in the affinity of the anti-Spike Abs was observed that correlated with the maintenance of both the phagocytic and neutralization functions, suggesting an improvement in the quality of the Abs.

Adaptation of Oxford Nanopore technology for hepatitis C whole genome sequencing and identification of within-host viral variants.

BACKGROUND: Hepatitis C (HCV) and many other RNA viruses exist as rapidly mutating quasi-species populations in a single infected host. High throughput characterization of full genome, within-host variants is still not possible despite advances in next generation sequencing. This limitation constrains viral genomic studies that depend on accurate identification of hemi-genome or whole genome, within-host variants, especially those occurring at low frequencies. With the advent of third generation long read sequencing technologies, including Oxford Nanopore Technology (ONT) and PacBio platforms, this problem is potentially surmountable. ONT is particularly attractive in this regard due to the portable nature of the MinION sequencer, which makes real-time sequencing in remote and resource-limited locations possible. However, this technology (termed here 'nanopore sequencing') has a comparatively high technical error rate. The present study aimed to assess the utility, accuracy and cost-effectiveness of nanopore sequencing for HCV genomes. We also introduce a new bioinformatics tool (Nano-Q) to differentiate within-host variants from nanopore sequencing. RESULTS: The Nanopore platform, when the coverage exceeded 300 reads, generated comparable consensus sequences to Illumina sequencing. Using HCV Envelope plasmids (~ 1800 nt) mixed in known proportions, the capacity of nanopore sequencing to reliably identify variants with an abundance as low as 0.1% was demonstrated, provided the autologous reference sequence was available to identify the matching reads. Successful pooling and nanopore sequencing of 52 samples from patients with HCV infection demonstrated its cost effectiveness (AUD$ 43 per sample with nanopore sequencing versus $100 with paired-end short read technology). The Nano-Q tool successfully separated between-host sequences, including those from the same subtype, by bulk sorting and phylogenetic clustering without an autologous reference sequence (using only a subtype-specific generic reference). The pipeline also identified within-host viral variants and their abundance when the parameters were appropriately adjusted. CONCLUSION: Cost effective HCV whole genome sequencing and within-host variant identification without haplotype reconstruction are potential advantages of nanopore sequencing.

The severity of the pathogen-induced acute sickness response is affected by polymorphisms in genes of the NLRP3 inflammasome pathway.

The acute sickness response (ASR) is a stereotyped set of symptoms including fatigue, pain, and disturbed mood, which are present in most acute infections. The immunological mechanisms of the ASR are conserved, with variations in severity determined partly by the pathogen, but also by polymorphisms in host genes. The ASR was characterised in three different serologically-confirmed acute infections in Caucasians (n = 484) across four symptom domains or endophenotypes (termed 'Fatigue', 'Musculoskeletal pain', 'Mood disturbance', and 'Acute sickness'). Correlations were sought with functional single nucleotide polymorphisms in the NLRP3 inflammasone pathway and severity of the endophenotypes. Individuals with severe Fatigue, Musculoskeletal pain, or Mood endophenotypes were more likely to have prior episodes of significant fatigue (11.4 vs. 3.8%, p = 0.07), pain (14.3 vs. 1.2%, p = 0.001), or Mood disturbance (13 vs 1%, p=0.001), suggesting trait characteristics. The high functioning allele of the rs35829419 SNP in NLRP3 was more common in those with severe Fatigue (OR = 13.3, 95% CI: 1.7-104), particularly in a dominant inheritance pattern (OR = 13.4, 95% CI: 1.8-586.3). In a multivariable analysis assuming dominant inheritance, both rs35829419 and the rs4848306 SNP in Interleukin(IL)-1ß, were independently associated with severe Fatigue (OR = 29.6, 95% CI: 2.6-330.9 and OR = 13, 95% CI: 2.7-61.8, respectively). The severity of fatigue in acute infection is influenced by genetic polymorphisms in NLRP3 and IL-1ß.

Rational use of ultrasonography with triaging of patients to detect dengue plasma leakage in resource limited settings: a prospective cohort study.

OBJECTIVES: To compare the traditional haematocrit-based criteria (>20% rise above baseline) with ultrasonography for diagnosing plasma leakage in dengue fever and to identify clinical indicators for triaging patients in resource-limited settings when the demand for ultrasonography is high. METHODS: The Colombo Dengue Study is a prospective observational cohort study recruiting dengue patients in the first three days of dengue fever, before plasma leakage. Serial haematocrit assessments and ultrasonography were performed in patients recruited from October 2017 to February 2020. Clinical signs/symptoms and laboratory investigation results independently associated with ultrasound detected plasma leakage were identified with a derivation cohort and confirmed in a validation cohort. RESULTS: 129 of 426 patients had ultrasonography-confirmed plasma leakage while 146 had a haematocrit rise >20%. Those positive on ultrasonography were also likely to fulfil the haematocrit-based criteria (OR: 4.42, 95% CI: 2.85-6.86), but the two groups did not overlap fully. In the derivation cohort (n = 317), platelet count <97 000/µl, AST/ALT > 51 IU/l and having abdominal pain in the first three days of fever were independent predictors of ultrasound-detected plasma leakage. In the validation cohort (n = 109), the combination of low platelet count and high aminotransferase level had better predictive capacity in terms of sensitivity and specificity. CONCLUSION: Dengue patients should be monitored with both serial haematocrit and ultrasonography whenever possible and plasma leakage should be diagnosed by either one of these criteria. If accessibility to scans is limited, platelet count, serum transaminase levels and presence of abdominal pain are useful to triage patients.

Outcomes of stroke patients undergoing thrombolysis in Sri Lanka; an observational prospective study from a low-middle income country.

BACKGROUND: Stroke related deaths are relatively higher in low- and middle-income countries where only a fraction of eligible patients undergo thrombolysis. There is also limited evidence on post-thrombolysis outcomes of patients from Asian countries in these income bands. METHODS: This is a single center prospective observational study of a patient cohort with acute ischaemic stroke, undergoing thrombolysis with alteplase (low and standard dose), over a 24-month period in 2019/2020. Modified Rankin scale (mRS) for dependency at 3 months (primary outcome), duration of hospital stay, incidence of symptomatic intracranial haemorrhages and all-cause mortality at 3 months (secondary outcomes) were recorded. Demographic, clinical and treatment related factors associated with these outcomes were explored. RESULTS: Eighty-nine patients (males - 61, 69%, mean age: 60 years ±12.18) were recruited. Time from symptom onset to reperfusion was 174 min ± 56.50. Fifty-one patients were independent according to mRS, 11 (12.4%) patients died, and 11 (12.5%) developed symptomatic intracranial haemorrhages by 3 months. Functional independence at 3 months was independently associated with National Institutes of Health Stroke Scale (NIHSS) on admission (p < 0.05). Thrombolysis with low dose alteplase did not lead to better or worse outcomes compared to standard dose. CONCLUSIONS: On admission NIHSS is predictive of functional independence at 3 months post-thrombolysis. Low dose alteplase may be as efficacious as standard dose alteplase with associated cost savings, but this needs to be confirmed by a prospective clinical trial for the Sri Lankan population.

Plasma leakage in dengue: a systematic review of prospective observational studies.

Plasma leakage is a precursor to life-threatening complications of dengue, but this group is poorly defined and not often reported in literature. Patients with Dengue haemorrhagic fever (DHF) as defined in the 1997 World Health Organization classification are often reported, and they all have plasma leakage, but some patients with plasma leakage do not meet the definition of DHF. The study aims to estimate the frequency of plasma leakage and DHF (as a surrogate of plasma leakage) in dengue and its variations based on virus serotype, geography, patient gender and pre-existing immunity to dengue. PUBMED, Scopus, EMBASE, CINAHL and Web of Science were searched for prospective observational studies reporting on plasma leakage or DHF. Quality of data was assessed using the NIH quality assessment tool for cohort studies. Forty-three studies that recruited 15,794 confirmed dengue patients were eligible. Cumulative frequency of plasma leakage was 36.8% (15 studies, 1642/4462, 95% CI 35.4-38.2%), but surprisingly the estimated cumulative frequency of DHF was higher (45.7%, 32 studies, 4758/10417, 95% CI 44.7-46.6%), indicating that current medical literature over-reports DHF or under-reports plasma leakage. Therefore, a reliable estimate for the proportion of dengue patients developing plasma leakage cannot be derived from existing medical literature even after applying rigorous inclusion criteria to select homogenous studies. Plasma leakage is an important marker of "at-risk" dengue patients and standardizing its definition, diagnosis and reporting should be a priority in research and global policy.

Attitudes on euthanasia among medical students and doctors in Sri Lanka: a cross sectional study.

BACKGROUND: Euthanasia is a topic of intense ethical debate and it is illegal in most countries at present, including Sri Lanka. The aim of this descriptive cross-sectional study of medical students and practicing doctors was to explore the acceptance of euthanasia and physician assisted suicide (PAS), and factors influencing this opinion. METHODS: A customised online questionnaire which explored opinions on euthanasia was administered to first and final year medical undergraduates in University of Colombo and practicing doctors with more than 5 years of work experience at The National Hospital of Sri Lanka. Attitudes on euthanasia and PAS were also assessed with the attitudes towards euthanasia (ATE) Scale, which is a 10-item questionnaire. RESULTS: A total of 425 individuals responded (males: 178, 42%, age: median - 27 years), which included 143 (33.6%) first-year medical undergraduates, 141 (33.2%) final-year medical undergraduates and 141 (33.2%) practicing doctors. More participants (200, 47.1%) favoured legalizing euthanasia than those directly opposing it (110, 25.9%), but a significant proportion (27%) remained undecided. The mean scores of ATE questionnaire from the whole sample were generally unfavourable towards euthanasia/PAS. Accepting euthanasia as an option for oneself (p = < 0.001) was the strongest predictor of favouring euthanasia/PAS or supporting its legalization. CONCLUSION: In this cross-sectional survey, more respondents supported legalisation of euthanasia in Sri Lanka than those openly opposing it. Yet, a significant minority that responded as "undecided" for legalisation, were more likely to have unfavourable ATE.

Severe Dengue Epidemic, Sri Lanka, 2017.

In 2017, a dengue epidemic of unexpected magnitude occurred in Sri Lanka. A total of 186,101 suspected cases and 440 dengue-related deaths occurred. We conducted a comprehensive analysis of this epidemic by comparing national surveillance data for 2017 with data from the preceding 5 years. In all Sri Lanka districts, dengue incidence in 2017 increased significantly over incidence during the previous 5 years. Older schoolchildren and young adults were more clinically symptomatic than those at extremes of age. Limited virologic surveillance showed the dominant circulating variant was dengue virus type 2 cosmopolitan genotype in the most affected district. One quarter of total annual cases were reported 5 weeks after the southwest monsoon started. Changes in vector abundance were not predictive of the increased incidence. Direct government expenditures on dengue control activities in 2017 were US $12.7 million. The lessons learned from this outbreak are useful for other tropical nations facing increasing dengue incidence.

B cell immunodominance in primary hepatitis C virus infection.

BACKGROUND & AIMS: Neutralising antibodies (NAbs) play a key role in clearance of HCV. NAbs have been isolated and mapped to several domains on the HCV envelope proteins. However, the immunodominance of these epitopes in HCV infection remains unknown, hindering efforts to elicit optimal epitope-specific responses. Furthermore, it remains unclear which epitope-specific responses are associated with broad NAb (bNAb) activity in primary HCV infection. The aim of this study was to define B cell immunodominance in primary HCV, and its implications on neutralisation breadth and clearance. METHODS: Using samples from 168 patients with primary HCV infection, the antibody responses targeted 2 immunodominant domains, termed domains B and C. Genotype 1 and 3 infections were associated with responses targeted towards different bNAb domains. RESULTS: No epitopes were uniquely targeted by clearers compared to those who developed chronic infection. Samples with bNAb activity were enriched for multi-specific responses directed towards the epitopes antigenic region 3, antigenic region 4, and domain D, and did not target non-neutralising domains. CONCLUSIONS: This study outlines for the first time a clear NAb immunodominance profile in primary HCV infection, and indicates that it is influenced by the infecting virus. It also highlights the need for a vaccination strategy to induce multi-specific responses that do not target non-neutralising domains. LAY SUMMARY: Neutralising antibodies will likely form a key component of a protective hepatitis C virus vaccine. In this work we characterise the predominant neutralising and non-neutralising antibody (epitope) targets in acute hepatitis C virus infection. We have defined the natural hierarchy of epitope immunodominance, and demonstrated that viral genotype can impact on this hierarchy. Our findings highlight key epitopes that are associated with broadly neutralising antibodies, and the deleterious impact of mounting a response towards some of these domains on neutralising breadth. These findings should guide future efforts to design immunogens aimed at generating neutralising antibodies with a vaccine candidate.

Tafenoquine for preventing relapse in people with Plasmodium vivax malaria.

BACKGROUND: Plasmodium vivax malaria has a persistent liver stage that causes relapse of the disease and continued P vivax transmission. Primaquine (PQ) is used to clear the liver stage of the parasite, but treatment is required for 14 days. Primaquine also causes haemolysis in people with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Tafenoquine (TQ) is a new alternative to PQ with a longer half-life and can be used as a single-dose treatment. OBJECTIVES: To assess the effects of tafenoquine 300 mg (single dose) on preventing P vivax relapse. SEARCH METHODS: We searched the following up to 3 June 2020: the Cochrane Infectious Diseases Group Specialized Register; CENTRAL; MEDLINE; Embase; and three other databases. We also searched the WHO International Clinical Trial Registry Platform and the metaRegister of Controlled Trials for ongoing trials using "tafenoquine" and "malaria" as search terms up to 3 June 2020. SELECTION CRITERIA: Randomized controlled trials (RCTs) that gave TQ to prevent relapse in people with P vivax malaria. We planned to include trials irrespective of whether participants had been screened for G6PD enzyme deficiency. DATA COLLECTION AND ANALYSIS: All review authors independently extracted data and assessed risk of bias. As true relapse and reinfection are difficult to differentiate in people living in endemic areas, studies report "recurrences" of infection as a proxy for relapse. We carried out meta-analysis where appropriate, and gave estimates as risk ratios (RR) with 95% confidence intervals (CI). We assessed the certainty of the evidence using the GRADE approach. MAIN RESULTS: Three individually randomized RCTs met our inclusion criteria, all in endemic areas, and thus reporting recurrence. Trials compared TQ with PQ or placebo, and all participants received chloroquine (CQ) to treat the asexual infection). In all trials, pregnant and G6PD-deficient people were excluded. Tafenoquine 300 mg single dose versus no treatment for relapse prevention Two trials assessed this comparison. TQ 300 mg single dose reduces P vivax recurrences compared to no antihypnozoite treatment during a six-month follow-up, but there is moderate uncertainty around effect size (RR 0.32, 95% CI 0.12 to 0.88; 2 trials, 504 participants; moderate-certainty evidence). In people with normal G6PD status, there is probably little or no difference in any type of adverse events (2 trials, 504 participants; moderate-certainty evidence). However, we are uncertain if TQ causes more serious adverse events (2 trials, 504 participants; very low-certainty evidence). Both RCTs reported a total of 23 serious adverse events in TQ groups (One RCT reported 21 events) and a majority (15 events) were a drop in haemoglobin level by > 3g/dl (or >30% reduction from baseline). Tafenoquine 300 mg single dose versus primaquine 15 mg/day for 14 days for relapse prevention Three trials assessed this comparison. There is probably little or no difference between TQ and PQ in preventing recurrences (proxy measure for relapse) up to six months of follow-up (RR 1.04, 95% CI 0.8 to 1.34; 3 trials, 747 participants; moderate-certainty evidence). In people with normal G6PD status, there is probably little or no difference in any type of adverse events (3 trials, 747 participants; moderate-certainty evidence). We are uncertain if TQ can cause more serious adverse events compared to PQ (3 trials, 747 participants; very low-certainty evidence). Two trials had higher point estimates against TQ while the other showed the reverse. Most commonly reported serious adverse event in TQ group was a decline in haemoglobin level (19 out of 29 events). Some other serious adverse events, though observed in the TQ group, are unlikely to be caused by it (Hepatitis E infection, limb abscess, pneumonia, menorrhagia). AUTHORS' CONCLUSIONS: TQ 300 mg single dose prevents relapses after clinically parasitologically confirmed P vivax malaria compared to no antihypnozoite treatment, and with no difference detected in studies comparing it to PQ to date. However, the inability to differentiate a true relapse from a recurrence in the available studies may affect these estimates. The drug is untested in children and in people with G6PD deficiency. Single-dose treatment is an important practical advantage compared to using PQ for the same purpose without an overall increase in adverse events in non-pregnant, non-G6PD-deficient adults.

Antiretroviral treatment failure and associated factors among HIV patients on first-line antiretroviral treatment in Sekota, northeast Ethiopia.

BACKGROUND: Antiretroviral treatment has played a pivotal role in the reduction of HIV/AIDS-related morbidity and mortality. However, treatment options can be impaired by the development of antiretroviral treatment failure. Regular monitoring of the Human Immunodeficiency Virus treatment outcome via viral load tests is the key approach. There is a scarcity of information about HIV treatment failure and risk factors in the study area. Therefore, the study was aimed to assess antiretroviral treatment failure and associated factors among patients on first-line antiretroviral treatment at Tefera Hailu Memorial Hospital, Sekota, northeast Ethiopia. METHODS: A hospital-based cross-sectional study was conducted on 295 patients on first-line antiretroviral treatment from Nov. 2018 to Apr. 2019. Socio-demographic and clinical variables were collected using a pretested questionnaire, and blood specimen was collected for PCR viral load and CD4 + cell count estimation. Data were entered into Epi-Info and exported to SPSS for analysis. A binary logistic regression model was used to identify associated factors, and P value < 0.05 was considered as statistically significant. RESULTS: Of the 295 subjects on first-line ART, 49 (16.6%) and 18 (6.1%) experienced virological and immunological failures, respectively. The failure of the former was associated with poor adherence (AOR: 6.367, P < 0.001), CD4 + count < 500 cells/µL (AOR: 4.78, P = 0.031) and shorter (6-24 months) duration on ART (AOR: 0.48, P = 0.048), while poor treatment adherence (AOR: 11.51, P = 0.012) and drug interruption (AOR: 6.374, P = 0.039) were the independent risk factors for latter. Immunological tests to predict virological failures showed as sensitivity, specificity, PPV, and NPV were 20.4%, 96.7%, 55.5%, and 86.0%, respectively. CONCLUSIONS: The rate of ART failure was considerably high. Poor adherence, low CD4 + count, prolonged ART, and drug interruption were found to be the most predictor variables for virological and immunological failures. The discrimination power of the immunological parameter was low in comparison to virological measurements as standard methods. Therefore, the study highlighted the need for more attention and efforts to curb associated factors and maximize virological tests for monitoring treatment failures.

Genomic variability of within-host hepatitis C variants in acute infection.

Interactions between the host immune system and the viral variants determine persistence of hepatitis C virus (HCV) infection after the acute phase of infection. This study describes the genetic variability of within-host HCV viral variants in acute infection and correlates it with host- and virus-related traits and infection outcome. Next generation sequence data (Illumina, MiSeq platform) of viral genomes from 116 incident acute infections (within 180 days of infection) were analysed to determine all the single nucleotide polymorphism (SNP) frequencies above a threshold of 0.1%. The variability of the SNPs for the full open reading frame of the genome as well as for each protein coding region were compared using mean standardized Shannon entropy (SE) values calculated separately for synonymous and nonsynonymous mutations. The envelope glycoproteins regions (E1 and E2) had the highest SE values (indicating greater variability) followed by the NS5B region. Nonsynonymous mutations rather than synonymous mutations were the main contributors to genomic variability in acute infection. The mean difference of Shannon entropy was also compared between subjects after categorizing the samples according to host and virus-related traits. Host IFNL3 allele CC polymorphism at rs12979860 (vs others) and viral genotype 1a (vs 3a) were associated with higher genomic variability across the viral open reading frame. Time since infection, host gender or continent of origin was not associated with the viral genomic variability. Viral genomic variability did not predict spontaneous clearance.

Risk prediction for severe disease and better diagnostic accuracy in early dengue infection; the Colombo dengue study.

BACKGROUND: A major challenge in dengue management in resource limited settings is the confirmation of diagnosis. Clinical features of dengue often overlap with other infections and molecular diagnostic tools are not readily accessible to clinicians at hospitals. In addition, the prediction of plasma leakage in dengue is also difficult. Hematocrit level and ultrasound scans (combined with clinical parameters) are helpful to detect plasma leakage once it has happened, not before. METHODS: Colombo Dengue Study (CDS) is a prospective cohort study of clinically suspected adult dengue patients recruited from the National hospital of Sri Lanka (within the first 3 days of fever) that aimed to a) identify clinical and basic laboratory test parameters to differentiate dengue from non-dengue fever, b) evaluate the comparative efficacy of loop-mediated isothermal amplification (LAMP) for dengue diagnosis (vs. NS1 antigen test and RT-qPCR) and c) identify early associations that are predictive of plasma leakage or severe dengue. The basic laboratory tests considered here included hematological parameters, serum biochemistry and inflammatory markers. RESULTS: Only 70% of clinically suspected patients were confirmed as having dengue by either the NS1 antigen test or RT-qPCR. On a Bayesian latent class model which assumes no "gold standard", LAMP performed equally or better than RT-qPCR and NS1 antigen test respectively. When confirmed dengue patients were compared with others, the earlier group had significantly lower lymphocyte counts and higher aspartate aminotransferase levels (AST) within the first 3 days of fever. Confirmed dengue patients with plasma leakage had a lower mean age and a higher median baseline AST level compared to those without plasma leakage (p < 0.05). CONCLUSION: Clinical suspicion overestimates the true number of dengue patients. RT-LAMP is a potentially useful low-cost diagnostic tool for dengue diagnosis. Confirmed dengue patients had significantly higher AST levels and lower lymphocyte counts in early disease compared to others. In confirmed dengue patients, younger age and a higher AST level in early infection were associated with subsequent plasma leakage.

A seven-year retrospective analysis of patch test data in a cohort of patients with contact dermatitis in Sri Lanka.

BACKGROUND: Patch testing with a baseline series is a common tool employed when the sensitizing agent in contact dermatitis is unclear. However, for Asian countries, there are no locally validated baseline series to utilize in screening. METHODS: We completed a retrospective analysis of all patients that had undergone patch testing with the European Baseline series, Shoe Series or Comprehensive International Baseline series, over 7 years from 2012 to 2018 in a tertiary care reference dermatology clinic in Sri Lanka to evaluate the suitability of these investigations to identify causes for contact dermatitis in the local study population. RESULTS: Out of 438 patients tested, 239 (54.8%) reacted to at least one substance in the series. The Shoe Series was significantly more likely to yield a positive result than the European Baseline Series (70.2% vs 46.9%, p < 0.05). The top three sensitizers identified by all series were nickel sulfate (16%, 70/438), p-phenylenediamine (12.3%, 54/438) and 2-mercaptobenzothiazole or mercapto mix (10.5%, 46/438). CONCLUSION: Shoe series has a comparatively high yield in the local population compared to European Baseline series. Since little less than half of the study population did not have any reactivity to any of the allergens tested it is important to develop or modify and validate a locally relevant, more suitable baseline series which is based on the Shoe Series in Sri Lanka. This is further evidence for the continuously changing nature of allergens in the environment and the need to modify existing patch testing standards accordingly.

Carica papaya extract in dengue: a systematic review and meta-analysis.

BACKGROUND: Carica papaya (CP) extract is becoming popular as an unlicensed herbal remedy purported to hasten recovery in dengue infection, mostly based on observations that it may increase platelet counts. This systematic review and meta-analysis aims to critically analyze the evidence from controlled clinical trials on the efficacy and safety of CP extract in the treatment of dengue infection. METHODS: PubMed, LILACS and Google Scholar were searched for randomized or non-randomized trials enrolling patients with suspected or confirmed dengue where CP extract was compared, as a treatment measure, against standard treatment. Recovery of platelet counts as well as other clinical indicators of favourable outcome (duration of hospital stay, prevention of plasma leakage, life threatening complications, and mortality) were assessed. RESULTS: Nine studies (India-6, Pakistan-1, Indonesia-1, Malaysia-1) met the inclusion criteria. Seven studies showed an increase in platelet counts in patients receiving CP extract, while one study showed no significant difference between the two groups, and direct comparison was not possible in the remaining study. Serious adverse events were not reported. CP extract may reduce the duration of hospital stay (mean difference - 1.98 days, 95% confidence interval - 1.83 to - 2.12, 3 studies, 580 participants, low quality evidence), and cause improvement in mean platelet counts between the first and fifth day of treatment (mean difference 35.45, 95% confidence interval 23.74 to 47.15, 3 studies, 129 participants, low quality evidence). No evidence was available regarding other clinical outcomes. CONCLUSIONS: The clinical value of improvement in platelet count or early discharge is unclear in the absence of more robust indicators of favourable clinical outcome. Current evidence is insufficient to comment on the role of CP extract in dengue. There is a need for further well designed clinical trials examining the effect of CP on platelet counts, plasma leakage, other serious manifestations of dengue, and mortality, with clearly defined outcome measures.

Use of a public-private partnership in malaria elimination efforts in Sri Lanka; a case study.

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.

Historical Trends in the Hepatitis C Virus Epidemics in North America and Australia.

BACKGROUND: Bayesian evolutionary analysis (coalescent analysis) based on genetic sequences has been used to describe the origins and spread of rapidly mutating RNA viruses, such as influenza, Ebola, human immunodeficiency virus (HIV), and hepatitis C virus (HCV). METHODS: Full-length subtype 1a and 3a sequences from early HCV infections from the International Collaborative of Incident HIV and Hepatitis C in Injecting Cohorts (InC3), as well as from public databases from a time window of 1977-2012, were used in a coalescent analysis with BEAST software to estimate the origin and progression of the HCV epidemics in Australia and North America. Convergent temporal trends were sought via independent epidemiological modeling. RESULTS: The epidemic of subtype 3a had more recent origins (around 1950) than subtype 1a (around 1920) in both continents. In both modeling approaches and in both continents, the epidemics underwent exponential growth between 1955 and 1975, which then stabilized in the late 20th century. CONCLUSIONS: Historical events that fuelled the emergence and spread of injecting drug use, such as the advent of intravenous medical therapies and devices, and growth in the heroin trade, as well as population mixing during armed conflicts, were likely drivers for the cross-continental spread of the HCV epidemics.

A method for near full-length amplification and sequencing for six hepatitis C virus genotypes.

BACKGROUND: Hepatitis C virus (HCV) is a rapidly evolving RNA virus that has been classified into seven genotypes. All HCV genotypes cause chronic hepatitis, which ultimately leads to liver diseases such as cirrhosis. The genotypes are unevenly distributed across the globe, with genotypes 1 and 3 being the most prevalent. Until recently, molecular epidemiological studies of HCV evolution within the host and at the population level have been limited to the analyses of partial viral genome segments, as it has been technically challenging to amplify and sequence the full-length of the 9.6 kb HCV genome. Although recent improvements have been made in full genome sequencing methodologies, these protocols are still either limited to a specific genotype or cost-inefficient. RESULTS: In this study we describe a genotype-specific protocol for the amplification and sequencing of the near-full length genome of all six major HCV genotypes. We applied this protocol to 122 HCV positive clinical samples, and had a successful genome amplification rate of 90%, when the viral load was greater than 15,000 IU/ml. The assay was shown to have a detection limit of 1-3 cDNA copies per reaction. The method was tested with both Illumina and PacBio single molecule, real-time (SMRT) sequencing technologies. Illumina sequencing resulted in deep coverage and allowed detection of rare variants as well as HCV co-infection with multiple genotypes. The application of the method with PacBio RS resulted in sequence reads greater than 9 kb that covered the near full-length HCV amplicon in a single read and enabled analysis of the near full-length quasispecies. CONCLUSIONS: The protocol described herein can be utilised for rapid amplification and sequencing of the near-full length HCV genome in a cost efficient manner suitable for a wide range of applications.

Contribution of the private sector healthcare service providers to malaria diagnosis in a prevention of re-introduction setting.

BACKGROUND: Sri Lanka is currently in the prevention of re-introduction phase of malaria. The engagement of the private sector health care institutions in malaria surveillance is important. The purpose of the study was to determine the number of diagnostic tests carried out, the number of positive cases identified and the referral system for diagnosis in the private sector and to estimate the costs involved. METHODS: This prospective study of private sector laboratories within the Colombo District of Sri Lanka was carried out over a 6-month period in 2015. The management of registered private sector laboratories was contacted individually and the purpose of the study was explained. A reporting format was developed and introduced for monthly reporting. RESULTS: Forty-one laboratories were eligible to be included in the study and 28 participated by reporting data on a monthly basis. Excluding blood bank samples and routine testing for foreign employment, malaria diagnostic tests were carried out on 973 individuals during the 6-month period and nine malaria cases were identified. In 2015, a total of 36 malaria cases were reported from Sri Lanka. Of these, 24 (67 %) were diagnosed in the Colombo District and 50 % of them were diagnosed in private hospitals. CONCLUSIONS: An equal number of cases were diagnosed from the private sector and government sector in the Colombo District in 2015. The private sector being a major contributor in the detection of imported malaria cases in the country should be actively engaged in the national malaria surveillance system.