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.

Long-term efficacy and safety of a tetravalent dengue vaccine (TAK-003): 4·5-year results from a phase 3, randomised, double-blind, placebo-controlled trial.

BACKGROUND: About half of the world's population lives in dengue-endemic areas. We aimed to evaluate the long-term efficacy and safety of two doses of the tetravalent dengue vaccine TAK-003 in preventing symptomatic dengue disease of any severity and due to any dengue virus (DENV) serotypes in children and adolescents. METHODS: In this ongoing double-blind, randomised, placebo-controlled trial, we enrolled healthy participants aged 4-16 years at 26 medical and research centres across eight dengue-endemic countries (Brazil, Colombia, Dominican Republic, Nicaragua, Panama, Philippines, Sri Lanka, and Thailand). The main exclusion criteria were febrile illness (body temperature ≥38°C) at the time of randomisation, hypersensitivity or allergy to any of the vaccine components, pregnancy or breastfeeding, serious chronic or progressive disease, impaired or altered immune function, and previous receipt of a dengue vaccine. Participants were randomly assigned 2:1 (stratified by age and region) using an interactive web response system and dynamic block assignment to receive two subcutaneous doses of TAK-003 or placebo 3 months apart. Investigators, participants, and their parents or legal guardians were blinded to group assignments. Active febrile illness surveillance and RT-PCR testing of febrile illness episodes were performed for identification of virologically confirmed dengue. Efficacy outcomes were assessed in the safety analysis set (all randomly assigned participants who received ≥1 dose) and the per protocol set (all participants who had no major protocol violations), and included cumulative vaccine efficacy from first vaccination to approximately 4·5 years after the second vaccination. Serious adverse events were monitored throughout. This study is registered with ClinicalTrials.gov, NCT02747927. FINDINGS: Between Sept 7, 2016, and March 31, 2017, 20 099 participants were randomly assigned (TAK-003, n=13 401; placebo, n=6698). 20 071 participants (10 142 [50·5%] males; 9929 [49·5%] females; safety set) received TAK-003 or placebo, with 18 257 (91·0%) completing approximately 4·5 years of follow-up after the second vaccination (TAK-003, 12 177/13 380; placebo, 6080/6687). Overall, 1007 (placebo: 560; TAK-003: 447) of 27 684 febrile illnesses reported were virologically confirmed dengue, with 188 cases (placebo: 142; TAK-003: 46) requiring hospitalisation. Cumulative vaccine efficacy was 61·2% (95% CI 56·0-65·8) against virologically confirmed dengue and 84·1% (77·8-88·6) against hospitalised virologically confirmed dengue; corresponding efficacies were 53·5% (41·6-62·9) and 79·3% (63·5-88·2) in baseline seronegative participants (safety set). In an exploratory analysis, vaccine efficacy was shown against all four serotypes in baseline seropositive participants. In baseline seronegative participants, vaccine efficacy was shown against DENV-1 and DENV-2 but was not observed against DENV-3 and low incidence precluded evaluation against DENV-4. During part 3 of the trial (approximately 22-57 months after the first vaccination), serious adverse events were reported for 664 (5·0%) of 13 380 TAK-003 recipients and 396 (5·9%) of 6687 placebo recipients; 17 deaths (6 in the placebo group and 11 in the TAK-003 group) were reported, none were considered study-vaccine related. INTERPRETATION: TAK-003 demonstrated long-term efficacy and safety against all four DENV serotypes in previously exposed individuals and against DENV-1 and DENV-2 in dengue-naive individuals. FUNDING: Takeda Vaccines. TRANSLATIONS: For the Portuguese, Spanish translations and plain language summary of the abstract see Supplementary Materials section.

Epidemiological evidence of acute transmission of Zika virus infection in dengue suspected patients in Sri-Lanka.

BACKGROUND: Zika Virus (ZIKV) is a re-emerging, arthropod-borne flavivirus transmitted by Aedes mosquitoes (Ae. aegypti and Ae. albopictus). The coexistence of dengue virus (DENV) and ZIKV concurrently has been associated with a wide array of neurological complications, which may influence the clinical outcomes of infections. Sri Lanka witnessed a severe dengue epidemic in 2017, characterized by extraordinary and severe disease manifestations with considerable morbidity. Therefore, this study assessed the potential occurrence of ZIKV infection during DENV outbreak in Sri Lanka from 2017 to 2019, which could bear substantial implications for public health. METHODS: Five hundred ninety-five serum samples were procured from individuals suspected of dengue and admitted to Kandy National Hospital between 2017 and 2018 and the Negombo District General Hospital between 2018 and 2019. These samples underwent quantitative real-time RT-PCR (qRT-PCR) to identify the presence of the ZIKV gene, while enzyme-linked immunosorbent assay was employed to detect ZIKV-specific IgM and IgG antibodies. Focus reduction neutralization tests were subsequently conducted to confirm ZIKV infection. RESULTS: Among the 595 serum samples, 6 (1.0%) tested positive for ZIKV using qRT-PCR. Anti-ZIKV IgM and IgG were identified in 18.0% and 38.6% patients. Sixty-six (11.0%) samples demonstrated the presence of anti-ZIKV IgM and IgG. Within ZIKV IgM-positive samples, 2.2% exhibited neutralizing antibodies against ZIKV. Through the implementation of qRT-PCR, ZIKV IgM detection, and neutralization testing, 2% and 3.7% cases of ZIKV infections were confirmed in the Kandy and Negombo regions, respectively. CONCLUSION: This study is the inaugural endeavor to substantiate the existence of ZIKV infection in Sri Lanka utilizing molecular and serological analysis. The findings of this investigation imply that ZIKV was circulating throughout the 2017-2019 DENV outbreak. These results underscore the necessity for improved preparedness for future outbreaks, fortifying governmental policies on public health, and establishing effective early warning systems regarding the emergence of these viruses.

Effect of the Tetravalent Dengue Vaccine TAK-003 on Sequential Episodes of Symptomatic Dengue.

In the pivotal phase 3 efficacy trial (NCT02747927) of the TAK-003 dengue vaccine, 5 of 13,380 TAK-003 recipients and 13 of 6,687 placebo recipients experienced two episodes of symptomatic dengue between the first dose and the end of the study, ∼57 months later (patients received the second dose 3 months after the first dose). Two of these participants experienced repeat infection with the same serotype (i.e., homotypic reinfection). In comparison with placebo, the relative risk of a subsequent episode of symptomatic dengue was 0.19 (95% CI, 0.07-0.54) in TAK-003 recipients. Based on the small number of subsequent episodes, these data suggest a potential incremental effect of TAK-003 beyond prevention of the first episode of symptomatic dengue after vaccination.

Three-year Efficacy and Safety of Takeda's Dengue Vaccine Candidate (TAK-003).

BACKGROUND: Takeda's live attenuated tetravalent dengue vaccine candidate (TAK-003) is under evaluation in a long-term clinical trial across 8 dengue-endemic countries. Previously, we have reported its efficacy and safety in both seronegative and seropositive participants and that its performance varies by serotype, with some decline in efficacy from first to second year postvaccination. This exploratory analysis provides an update with cumulative and third-year data. METHODS: Healthy 4-16 year olds (n = 20099) were randomized 2:1 to receive TAK-003 or placebo (0, 3 month schedule). The protocol included baseline serostatus testing of all participants and detection of all symptomatic dengue throughout the trial with a serotype specific reverse transcriptase-polymerase chain reaction. RESULTS: Cumulative efficacy after 3 years was 62.0% (95% confidence interval, 56.6-66.7) against virologically confirmed dengue (VCD) and 83.6% (76.8-88.4) against hospitalized VCD. Efficacy was 54.3% (41.9-64.1) against VCD and 77.1% (58.6-87.3) against hospitalized VCD in baseline seronegatives, and 65.0% (58.9-70.1) against VCD and 86.0% (78.4-91.0) against hospitalized VCD in baseline seropositives. Efficacy against VCD during the third year declined to 44.7% (32.5-54.7), whereas efficacy against hospitalized VCD was sustained at 70.8% (49.6-83.0). Rates of serious adverse events were 2.9% in TAK-003 group and 3.5% in placebo group during the ongoing long-term follow-up (ie, second half of the 3 years following vaccination), but none were related. No important safety risks were identified. CONCLUSIONS: TAK-003 was efficacious against symptomatic dengue over 3 years. Efficacy declined over time but remained robust against hospitalized dengue. A booster dose evaluation is planned.

Genetic Variation in the Domain II, 3' Untranslated Region of Human and Mosquito Derived Dengue Virus Strains in Sri Lanka.

Genetic variations in dengue virus (DENV) play a distinct role in epidemic emergence. The DENV 3' UTR has become a recent interest in research. The objective of the study was to examine the genetic variation in the domain II, 3' UTR region of human and mosquito-derived DENV. DENV-infected human sera were orally infected to laboratory reared Aedes aegypti mosquitoes. The domain II, 3' UTR of each human- and mosquito-derived sample was amplified. The nucleotide sequence variation, phylogenetic and secondary structure analysis was carried out incorporating respective regions of so far recorded Sri Lankan and the reference genotype strains of the DENV3 and DENV1 serotypes. The human- and mosquito-derived domain II, 3' UTR were identical in nucleotide sequences within the serotypes isolated, indicating the conserved nature of the region during host switch. The sequence analysis revealed distinct variations in study isolates compared to so far recorded Sri Lankan isolates. However, despite single nucleotide variations, the maintenance of structural integrity was evident in related strains within the serotypes in the secondary structure analysis. The phylogenetic analysis revealed distinct clade segregation of the study sequences from so far reported Sri Lankan isolates and illustrated the phylogenetic relations of the study sequences to the available global isolates of respective serotypes.

Modeling the relative role of human mobility, land-use and climate factors on dengue outbreak emergence in Sri Lanka.

BACKGROUND: More than 80,000 dengue cases including 215 deaths were reported nationally in less than 7 months between 2016 and 2017, a fourfold increase in the number of reported cases compared to the average number over 2010-2016. The region of Negombo, located in the Western province, experienced the greatest number of dengue cases in the country and is the focus area of our study, where we aim to capture the spatial-temporal dynamics of dengue transmission. METHODS: We present a statistical modeling framework to evaluate the spatial-temporal dynamics of the 2016-2017 dengue outbreak in the Negombo region of Sri Lanka as a function of human mobility, land-use, and climate patterns. The analysis was conducted at a 1 km × 1 km spatial resolution and a weekly temporal resolution. RESULTS: Our results indicate human mobility to be a stronger indicator for local outbreak clusters than land-use or climate variables. The minimum daily temperature was identified as the most influential climate variable on dengue cases in the region; while among the set of land-use patterns considered, urban areas were found to be most prone to dengue outbreak, followed by areas with stagnant water and then coastal areas. The results are shown to be robust across spatial resolutions. CONCLUSIONS: Our study highlights the potential value of using travel data to target vector control within a region. In addition to illustrating the relative relationship between various potential risk factors for dengue outbreaks, the results of our study can be used to inform where and when new cases of dengue are likely to occur within a region, and thus help more effectively and innovatively, plan for disease surveillance and vector control.

Efficacy of a Tetravalent Dengue Vaccine in Healthy Children and Adolescents.

BACKGROUND: Dengue, a mosquito-borne viral disease, was designated a World Health Organization top 10 threat to global health in 2019. METHODS: We present primary efficacy data from part 1 of an ongoing phase 3 randomized trial of a tetravalent dengue vaccine candidate (TAK-003) in regions of Asia and Latin America in which the disease is endemic. Healthy children and adolescents 4 to 16 years of age were randomly assigned in a 2:1 ratio (stratified according to age category and region) to receive two doses of vaccine or placebo 3 months apart. Participants presenting with febrile illness were tested for virologically confirmed dengue by serotype-specific reverse-transcriptase polymerase chain reaction. The primary end point was overall vaccine efficacy in preventing virologically confirmed dengue caused by any dengue virus serotype. RESULTS: Of the 20,071 participants who were given at least one dose of vaccine or placebo (safety population), 19,021 (94.8%) received both injections and were included in the per-protocol analysis. The overall vaccine efficacy in the safety population was 80.9% (95% confidence interval [CI], 75.2 to 85.3; 78 cases per 13,380 [0.5 per 100 person-years] in the vaccine group vs. 199 cases per 6687 [2.5 per 100 person-years] in the placebo group). In the per-protocol analyses, vaccine efficacy was 80.2% (95% CI, 73.3 to 85.3; 61 cases of virologically confirmed dengue in the vaccine group vs. 149 cases in the placebo group), with 95.4% efficacy against dengue leading to hospitalization (95% CI, 88.4 to 98.2; 5 hospitalizations in the vaccine group vs. 53 hospitalizations in the placebo group). Planned exploratory analyses involving the 27.7% of the per-protocol population that was seronegative at baseline showed vaccine efficacy of 74.9% (95% CI, 57.0 to 85.4; 20 cases of virologically confirmed dengue in the vaccine group vs. 39 cases in the placebo group). Efficacy trends varied according to serotype. The incidence of serious adverse events was similar in the vaccine group and placebo group (3.1% and 3.8%, respectively). CONCLUSIONS: TAK-003 was efficacious against symptomatic dengue in countries in which the disease is endemic. (Funded by Takeda Vaccines; TIDES ClinicalTrials.gov number, NCT02747927.).

Predictive value of persistent NS1 antigen positivity beyond 3rd day for dengue haemorrhagic fever in Sri Lankan children.

OBJECTIVE: Dengue haemorrhagic fever (DHF) is a major public health concern responsible for significant morbidity in both adult and paediatric populations in Sri Lanka. This study examined if persistent non structural protein 1 (NS1) antigen positivity beyond day 3 was predictive of the occurrence of dengue haemorrhagic fever. The patients were followed up during their in-hospital stay and the severity of the illness was classified according to the WHO classification. The NS1 antigen test was repeated after day 3 of the onset of illness, at least 2 days after the initial test. RESULTS: One hundred and fifty-seven patients were enrolled. Persistent NS1 antigen test positivity after day 3 of the illness was not predictive of subsequent development of DHF. Out of multiple other demographic and illness related factors assessed, only having a secondary dengue infection was associated with a high risk of DHF (relative risk = 3.077, 95% CI 1.361, 6.954). Persistent NS1 positivity on day 3 may not be indicative of disease severity. However results need to be confirmed by a larger study with quantitative NS1 testing.

Chiari malformation type 1 presenting as unilateral progressive foot drop: a case report and review of literature.

BACKGROUND: Foot drop is a disabling clinical condition with multiplicity of causes, which requires detailed evaluation to identify the exact aetiology. Here, we report an extremely rare cause of foot drop in a child, which if not recognized early, could lead to multiple complications. CASE PRESENTATION: A 6-year-old girl presented with difficulty in walking and left sided foot droop for1-month duration. On examination she had reduced muscle power in dorsiflexors and plantar flexors and diminished knee and absent ankle jerk in the left side. Sensory loss was noted in L4 and L5 dermatomes on the left side. Superficial abdominal reflex was absent on the left side while preserved in the right. Nerve conduction and electromyography revealed nerve root or spinal cord cause for the foot drop. These results prompted ordering MRI spine and brain which revealed Chiari malformation type-1 with holocord syrinx extending from the cervicomedullary junction to conus medullaris. CONCLUSIONS: This case highlights the importance of considering broad differential diagnosis for foot drop and value of the complete neurological examination including superficial reflexes in arriving at a diagnosis. Prompt diagnosis helped to early neurosurgical referral and intervention which is an important prognostic factor.

Effect of Climatic Factors and Population Density on the Distribution of Dengue in Sri Lanka: A GIS Based Evaluation for Prediction of Outbreaks.

Dengue is one of the major hurdles to the public health in Sri Lanka, causing high morbidity and mortality. The present study focuses on the use of geographical information systems (GIS) to map and evaluate the spatial and temporal distribution of dengue in Sri Lanka from 2009 to 2014 and to elucidate the association of climatic factors with dengue incidence. Epidemiological, population and meteorological data were collected from the Epidemiology Unit, Department of Census and Statistics and the Department of Meteorology of Sri Lanka. Data were analyzed using SPSS (Version 20, 2011) and R studio (2012) and the maps were generated using Arc GIS 10.2. The dengue incidence showed a significant positive correlation with rainfall (p<0.0001). No positive correlation was observed between dengue incidence and temperature (p = 0.107) or humidity (p = 0.084). Rainfall prior to 2 and 5 months and a rise in the temperature prior to 9 months positively correlated with dengue incidence as based on the auto-correlation values. A rise in humidity prior to 1 month had a mild positive correlation with dengue incidence. However, a rise in humidity prior to 9 months had a significant negative correlation with dengue incidence based on the auto-correlation values. Remote sensing and GIS technologies give near real time utility of climatic data together with the past dengue incidence for the prediction of dengue outbreaks. In that regard, GIS will be applicable in outbreak predictions including prompt identification of locations with dengue incidence and forecasting future risks and thus direct control measures to minimize major outbreaks.

NS 1 lasts longer than the dengue virus nucleic acid in the clinically suspected patients with dengue fever and dengue haemorrhagic fever.

The objectives of the study were to compare the DENV NS1 and nucleic acid positivity in dengue fever (DF) and dengue haemorrhagic fever (DHF) patients and to evaluate the usefulness of these parameters in severe dengue. Blood samples were collected from 91 patients with DF or DHF (fever days 3-9) from Gampaha and Negombo General Hospitals, Sri Lanka and tested for DENV NS1 antigen and nucleic acid. On fever day 3, 67% of the DF and 33% of the DHF patients had DENV nucleic acid whereas 50% of the DF and 67% of the DHF patients had NS1. On fever day 4, 67% of the DF and 100% of the DHF patients had DENV nucleic acid whereas 83% of the DF and DHF patients had NS1. From fever day 5 in the DHF group, DENV nucleic acid declined but NS1 remained detectable even on day 9 (p < 0.0001). NS1 positivity did not correlate with the decrease in the WBC count in DF and DHF patients (p > 0.05) whereas NS1 positivity significantly correlated with the decrease in the platelet count in DF and DHF patients (p < 0.0001). NS1 is a useful marker to detect DENV infection even in the latter stages of DHF.