Epidemiology and risk factors of respiratory syncytial virus associated acute respiratory tract infection in hospitalized children younger than 5 years from Sri Lanka

BackgroundRespiratory syncytial virus (RSV) is the leading cause of acute respiratory tract infections (ARTI) and a major cause of morbidity and mortality in children worldwide.AimThis study aimed to describe the prevalence and seasonal patterns of RSV and to determine the actual and predictive association of RSV-associated ARTI and clinical, socio-demographic, and climatic risk factors in children < 5 years.MethodsNasopharyngeal aspirates were collected from 500 children < 5 years admitted to the Kegalle General Hospital, Sri Lanka between May 2016 to July 2018. RSV and RSV subtypes were detected using immunofluorescence assay and real time RT-PCR, respectively. Descriptive and inferential statistics were done for the data analysis using Chi-square, Fisher’s exact, Kruskal–Wallis test, and multiple binary logistic regression in the statistical package for social sciences (SPSS), version 16.0.ResultsPrevalence of RSV-associated ARTI was 28% in children < 5 years. Both RSV subtypes were detected throughout the study period. RSV-B was the dominant subtype detected with a prevalence of 72.14%. RSV infection in general caused severe respiratory disease leading to hypoxemia. Compared to RSV-B, RSV-A infection had more symptoms leading to hypoxemia. Factors increasing the risk of contracting RSV infection included number of people living (n > 6), having pets at home and inhaling toxic fumes. The inferential analysis predicts RSV infection in children < 5 years with ARTI, with a 75.4% probability with clinical and socio-demographic characteristics like age < 1 year, fever for > 4 days, cough, conjunctivitis, stuffiness, fatigue, six or more people at home, having pets at home and inhaling toxic fumes. Climatic factors like increases in temperature (°C), wind speed (Km/h), wind gust (Km/h), rainfall (mm) and atmospheric pressure (mb) showed a strong correlation with the RSV infection in children

Identifying the research, advocacy, policy and implementation needs for the prevention and management of respiratory syncytial virus lower respiratory tract infection in low- and middle-income countries

Introduction: The high burden of respiratory syncytial virus (RSV) infection in young children disproportionately occurs in low- and middle-income countries (LMICs). The PROUD (Preventing RespiratOry syncytial virUs in unDerdeveloped countries) Taskforce of 24 RSV worldwide experts assessed key needs for RSV prevention in LMICs, including vaccine and newer preventive measures. Methods: A global, survey-based study was undertaken in 2021. An online questionnaire was developed following three meetings of the Taskforce panellists wherein factors related to RSV infection, its prevention and management were identified using iterative questioning. Each factor was scored, by non-panellists interested in RSV, on a scale of zero (very-low-relevance) to 100 (very-high-relevance) within two scenarios: (1) Current and (2) Future expectations for RSV management. Results: Ninety questionnaires were completed: 70 by respondents (71.4% physicians; 27.1% researchers/scientists) from 16 LMICs and 20 from nine high-income (HI) countries (90.0% physicians; 5.0% researchers/scientists), as a reference group. Within LMICs, RSV awareness was perceived to be low, and management was not prioritised. Of the 100 factors scored, those related to improved diagnosis particularly access to affordable point-of-care diagnostics, disease burden data generation, clinical and general education, prompt access to new interventions, and engagement with policymakers/payers were identified of paramount importance. There was a strong need for clinical education and local data generation in the lowest economies, whereas upper-middle income countries were more closely aligned with HI countries in terms of current RSV service provision. Conclusion: Seven key actions for improving RSV prevention and management in LMICs are proposed

Development of antiviral therapies for chronic hepatitis B virus infection.

Acute hepatitis B virus (HBV) infection is self-limiting but leaves a residual infection that can become active in an individual under conditions of immunosuppression. In chronic HBV infection, the virus persistently replicates in hepatocytes and this leads to immune mediated hepatocyte damage. Chronic HBV infection, which occurs worldwide in more than 400 million people, is associated with liver disease, fibrosis, cirrhosis and hepatocellular carcinoma (HCC) (Hepatitis B Fact Sheet 2009). There is a significant need for treatment intervention in chronic HBV infection. Despite the inability to remove the virus in more than 70% of patients, current treatments for chronic HBV infection, which include interferon alpha (IFN-α) and antiviral nucleotide/nucleoside analogues (NAs), aim to reduce levels of viral replication and to prevent or at least delay the progression of disease and the development of cirrhosis and HCC. Current NA therapy involves monotherapy with a conventional NA as a single antiviral agent (Sasadeusz et al. 2007). In the recent past, poor response to monotherapies with NAs and adverse effects to IFN-α have stimulated research into novel therapeutic strategies and enhancing the efficacy of exsisting NA therapy. The duck HBV (DHBV) in its natural host, the Pekin duck (Anas domesticus platyrhynchos), has been used as an animal model to study treatment outcomes and antiviral studies at the pre-clinical level. Much of what is known about viral replication and outcomes of hepadnavirus infection has been discovered using the DHBV model (Schultz et al. 2004; Zoulim et al. 2008) and several immunotherapeutic and antiviral studies have been performed recently in our laboratory (Foster et al. 2003; Miller et al. 2004; Foster et al. 2005; Miller et al. 2006a; Miller et al. 2006b; Miller et al. 2008). The studies described in this Ph.D. thesis focused on the development and testing of novel therapies for chronic HBV infection using the DHBV model. The first approach involved the use of novel amphipathic DNA polymers (APDPs) developed by REPLICor Inc. The second approach tested a combination of NAs developed by Gilead Sciences Pty. Ltd. APDPs developed by REPLICor Inc. have been used as a novel therapeutic approach against human immunodeficiency virus type 1 (HIV-1) and have been shown to inhibit HIV-1-mediated membrane fusion and HIV-1 replication in a size dependent but sequence independent manner (Vaillant et al. 2006). HIV-1 entry is well characterised and involves fusion of the virus to its target cells using a type 1 fusion protein. APDPs are thought to inhibit HIV-1 infection by acting as fusion inhibitors that bind to the V3 loop of the HIV-1 gp41 domain preventing its interaction with the T cell receptor, CD4. Phosphorothioation of oligonucleotides increases their hydrophobicity (amphipathicity) and also makes them more resistant to degradation by nucleases. The amphipathicity of APDPs plays a major role in their antiviral activity. Longer APDPs with lengths of ≥30 bases have a greater amphipathicity and were shown to be more potent in blocking the amphipathic interactions involved in the HIV-1 mediated membrane fusion than shorter APDPs with lengths of <30 bases (Vaillant et al. 2006). This novel antiviral mechanism of action of APDPs with ≥30 bases has future applications for therapy against infection with HIV-1 and other enveloped viruses. In contrast to HIV, the entry mechanisms used by HBV are not well understood although HBV is thought to enter through receptor mediated endocytosis (RME). In RME fusion occurs between the virus and the cell membrane as a late event. We hypothesise that the late fusion of HBV with the cell membrane can be blocked by APDPs that are amphipathic. As a first step in evaluating APDPs as a novel treatment for chronic HBV infection, the APDPs, REP 2006 and REP 2031 and a non-APDP, REP 2086, were tested in primary duck hepatocytes (PDH) for cytotoxicity and antiviral activity. REP 2006, a 40mer PS-ON with a completely degenerate sequence (random ATCG), REP 2031, a 40 mer PS-ON with a poly C sequence, and the non-APDP control, REP 2086, were all found to be noncytotoxic in PDH. Treatment of PDH with REP 2006 inhibited DHBV infection at concentrations as low as 0.01 μM, while REP 2031 had a lower anti-DHBV activity. The antiviral activity of both APDPs, REP 2006 and 2031, was also found to be length and chemistry dependent and sequence independent. Studies were then conducted to test the antiviral efficacy of REP 2006 and REP 2031 in vivo using 14-day-old ducks infected with 5 x 10₈ DHBV DNA genomes. Ducks in 4 Groups were treated with either REP 2006 or REP 2031 or the Bristol-Myers Squibb NA, entecavir (ETV), or normal saline (NS) starting from 1 day prior to DHBV infection for 15 days. REP 2006 showed an excellent anti-DHBV activity but treatment cause some side effects. In contrast, treatment of ducks with REP 2031 was well tolerated. However, REP 2031 again showed less anti-DHBV activity than REP 2006. We hypothesised that the increased side effects in the REP 2006-treated ducks were due to CpG motifs present in the random ATCG sequence (Krieg 2000; Agrawal and Kandimalla 2001; Shen et al. 2002; Isogawa et al. 2005; Wilson et al. 2006; Plitas et al. 2008; Wang et al. 2008). The lack of side effects with REP 2031 (which has no CpG motifs) was consistent with this hypothesis. The reason for the lower antiviral activity of REP 2031 is unclear. The subsequent testing of REP 2055 (a 40mer PS-ON with a poly AC sequence), which has no CpG motifs and also has an interrupted C nucleotide composition, showed an excellent recovery of antiviral activity without any observable side effects. The antiviral efficacy of REP 2055 was tested in vivo followed by dose optimisation studies using a range of dose regimens (0.5, 2, 3, 5 and 10 mg/kg). REP 2055 demonstrated excellent anti-DHBV activity with a dose as low as 2 mg/kg body weight. The ability of REP 2055 to prevent the rebound of DHBV infection was next tested. Treatment with REP 2055 for 14 days prevented the rebound of DHBV infection after the cessation of treatment. This effect was observed if REP 2055 treatment was initiated one day prior to, or at an early phase (4 days p.i.) of DHBV infection, and continued for 14 days. In these 2 Groups, 4 out of 5 ducks were protected from the rebound of DHBV infection. The therapeutic efficacy and the ability of REP 2055 to prevent the rebound of DHBV infection were then tested. REP 2055 treatment (10 mg/kg) was started at a late stage of DHBV infection when the liver was fully infected and treatment was continued for 28 days. A control Group of DHBV-infected ducks treated with NS was monitored for comparison. Liver enzymes and a complete blood evaluation (CBE) were performed prior to, during, at treatment endpoint and at the end of follow up, 16 weeks after the cessation of treatment. The results showed that 56% of ducks treated with REP 2055 were protected from rebound of DHBV infection and had developed an anti-DHBV surface antibody response, suggesting that they had resolved DHBV their infection. We concluded from this work that the APDP REP 2055 showed excellent anti-DHBV activity and has the ability to prevent the rebound of DHBV infection, making it suitable for further evaluation and possible clinical trials for the treatment of chronic HBV infection in humans. Although the need for combination NA therapy has been suggested by many as a way to combat the development of antiviral resistance, very few studies have investigated the effectiveness of combination therapy using NAs. In a pre-clinical study using HepG2 hepatoma cells, the additive effect of adefovir (AFV) with ETV, emtricitabine (FTC), lamivudine (3TC) and telbivudine (TLB) has been reported (Delaney et al. 2004). AFV with all other NAs in dual combination provided an additive effect. AFV and 3TC combined had a better additive effect than other combinations (Delaney et al. 2004). We hypothesised that the combination of either tenofovir (TFV) or tenofovir disoproxil fumarate (TDF) and FTC is more likely to have a better therapeutic efficacy against chronic DHBV infection than either TDF or FTC alone. As a first step the pharmacokinetics (PK) of TFV and TDF were investigated. This was followed by testing of the antiviral efficacy of TFV and FTC alone and in combination in ducks with persistent DHBV infection. PK studies of TFV and TDF showed that TFV has a half-life of 6 h when administered via the IP route whereas TDF had a half life of 4 h and required twice daily administration. TFV was chosen for the study for practical reasons of once daily administration. Next persistently DHBV infected ducks were treated daily with IP administration of 5 or 25 or 50 mg/kg of TFV or 100 or 200 mg/kg of FTC. The study showed that 5, 25 and 50 mg/kg of TFV suppressed serum levels of DHBV DNA by 3-logs compared to untreated ducks. FTC showed a dose dependent serum DHBV DNA suppression with 1-log reduction for a dose of 100 mg/kg, and a 2-log reduction for a dose of 200 mg/kg. In the next experiment, two different combinations of TFV and FTC were tested. The combination of 5 mg/kg TFV + 200 mg/kg FTC was able to suppress levels of serum DHBV DNA by 5-logs whereas the combination of 5 mg/kg TFV + 100 mg/kg FTC reduced the levels of serum DHBV DNA by 3-logs. In conclusion, a combination of TFV and FTC was superior to either of these drugs alone in suppressing serum DHBV DNA levels in ducks with chronic DHBV infection. Further studies are warranted to test the ability of combinations of TFV and FTC to prevent the rebound of DHBV infection.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 201

Comparison of a rapid immuno-chromatography assay with a standard ELISA for the detection of IgM and IgG antibodies against dengue viruses

A total of 765 blood samples collected from dengue suspected patients admitted to a Teaching Hospital in Sri Lanka were used to compare a rapid ICT assay with a standard ELISA for the detection of anti-dengue virus (DENV) IgM and IgG. Detection accuracy indices including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), Chi square and Cohen’s kappa values were determined for the ICT assay using the ELISA as a comparator for the detection of anti-DENV IgM and IgG. The rapid ICT assay showed a sensitivity of 64%, specificity of 75%, NPV of 68% and PPV of 72% for anti-DENV IgM detection. However, all the accuracy indices were relatively higher for the anti-DENV IgG detection by the ICT assay than those for anti-DENV IgM detection. Despite the low sensitivity for anti-DENV IgM detection by the ICT assay, considering the limitations in using ELISAs in resource limited regions, rapid ICT assays would be useful for the detection of more recent DENV infections. As many patients present after fever days 5 in the study area, anti-DENV IgM/IgG would be the suitable marker to be detected by rapid ICT assays in such areas

Comparison of a rapid immuno-chromatography assay with a standard ELISA for the detection of IgM and IgG antibodies against dengue viruses

A total of 765 blood samples collected from dengue suspected patients admitted to a Teaching Hospital in Sri Lanka were used to compare a rapid ICT assay with a standard ELISA for the detection of anti-dengue virus (DENV) IgM and IgG. Detection accuracy indices including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), Chi square and Cohen’s kappa values were determined for the ICT assay using the ELISA as a comparator for the detection of anti-DENV IgM and IgG. The rapid ICT assay showed a sensitivity of 64%, specificity of 75%, NPV of 68% and PPV of 72% for anti-DENV IgM detection. However, all the accuracy indices were relatively higher for the anti-DENV IgG detection by the ICT assay than those for anti-DENV IgM detection. Despite the low sensitivity for anti-DENV IgM detection by the ICT assay, considering the limitations in using ELISAs in resource limited regions, rapid ICT assays would be useful for the detection of more recent DENV infections. As many patients present after fever days 5 in the study area, anti-DENV IgM/IgG would be the suitable marker to be detected by rapid ICT assays in such areas

Demographic and clinical characteristics of human bocavirus-1 infection in patients with acute respiratory tract infections during the COVID-19 pandemic in the Central Province of Sri Lanka

Abstract Background Human bocavirus-1 (hBoV-1) was first detected in respiratory specimens in 2005. Due to high co-infection rates and prolonged shedding of the virus, the pathogenic role of hBoV-1 as a primary causative agent of respiratory infections is still under discussion. This study aimed to determine the prevalence of hBoV-1 infection in patients with acute respiratory tract infections (ARTIs) during the COVID-19 pandemic in the Central Province of Sri Lanka. Methods A total of 1021 patients (Age 12 days to ≤ 85 years) with ARTI symptoms including fever, cough, cold, sore throat and shortness of breath within first 7 days of the illness were included. The study was carried out at the National Hospital, Kandy, Sri Lanka from January 2021 to October 2022. Respiratory specimens were tested to detect 23 pathogens including hBoV-1 using a real time PCR. Prevalence of hBoV-1 co-infections with other respiratory pathogens and distribution of hBoV-1 infection among different age groups were determined. Moreover, clinical and demographic characteristics of hBoV-1 mono-infection associated ARTI were compared with that of the hBoV-1 co-infections. Results Respiratory infections were detected in 51.5% (526/1021) of the patients and of these 82.5% were mono- and 17.1% were co-infections. hBoV-1 was detected in 66 patients and this was the most prevalent respiratory virus associated with 40% co-infections. Of the 66 hBoV-1 positive patients, 36 had co-infections and of these 33 had dual and 3 had triple infections. Most of the hBoV-1 co-infections were identified in children aged 2-<5 years. hBoV-1 co-infections were most frequently detected with respiratory syncytial virus (RSV) and Rhino/ Entero viruses (Rh/EnV). No differences were observed in age, gender and clinical presentations in those with hBoV-1 mono- compared to co-infections. Intensive care admissions were less among hBoV-1 mono-infected than hBoV-1 co-infected patients. Conclusion This study shows a prevalence of 12.5% for hBoV-1 infections in patients with ARTI. RSV and Rh/EnV were the most common co-infecting pathogens with hBoV-1. Clinical features of hBoV-1 mono-infections were not different to that of the hBoV-1 co-infections. Interactions between hBoV-1 and other respiratory pathogens need investigation to identify the role of hBoV-1 in clinical severity of co-infections

Blood group AB is associated with severe forms of dengue virus infection

Several viral and host factors are believed to contribute to the development of severe forms of dengue such as dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) following a dengue virus (DENV) infection. The pathogenesis of DHF/DSS is not fully understood, however, host factors like ABO blood groups have been shown to contribute to the severity of DENV infection. The present study investigated the association of blood groups with severity of DENV infection in the northern region of Sri Lanka. The blood-groups of 405 patients positive for DENV NS1 antigen and anti-DENV IgM/IgG were determined using the standard haemagglutination assay recommended by the national blood bank/s. The occurrence of severe dengue in patients with certain blood groups was significantly different (p < 0.001) to those with other blood groups. Patients with AB blood group had more than 2.5 times higher risk of developing DHF than those with other blood groups. On the other hand, patients with blood group O were significantly under represented for DHF relative to the proportion of this blood group in the general population. Thus dengue patients with blood group O appear to have a low risk of developing DHF than those with other blood groups

Exposure rate of VZV among women attending antenatal care clinic in Sri Lanka - a cross sectional study

Abstract Background Varicella or chickenpox was not a notifiable disease until 2005 in Sri Lanka and only a few studies have been conducted on the epidemiology of VZV infection in the country. The anti­VZV IgG sero-prevalence among antenatal women is extremely limited and thus a selected group of antenatal clinic attendees were chosen to determine the exposure rate to VZV infection. Methods Women attending the antenatal clinic at Teaching Hospital, Peradeniya, Sri Lanka were selected for the study and 3 mL of venous blood was collected from 181 participants and the demographic data was obtained through a pre­tested questionnaire. Sera of the women were then tested for the presence of anti­VZV IgG using ELISA (HUMAN Diagnostics, Germany). Data was analysed using the SPSS statistical software for Windows, Version 12.0. Results Of the 181 antenatal women who took part in the study, 141 were positive for anti­VZV IgG giving a sero-prevalence of 77.9% for the past exposure to VZV. Of the 141 anti­VZV IgG positive women, 43.3% (n = 61) were from urban, 41.8% (n = 59) were from rural and 14.9% (n = 21) were from estate populations (an ethnic population living in small settlements in the tea estates whose ancestors were brought from India during the British colonial period to work in the tea plantations in Sri Lanka). Out of the 88 antenatal women with a positive history for varicella, 85 (96.6%) were positive for anti-VZV IgG. The highest number of anti­VZV IgG positivity was seen in the 31–35 age group, which was 85.0% of the total number of antenatal women included in that category. An increase in the anti­VZV IgG sero-prevalence with increasing age was also noted in the study sample. Conclusion Exposure rate of VZV infection as confirmed by anti­VZV IgG in the present study sample of antenatal women was 77.9%. Age specific, population based future sero-prevalence studies should be conducted in Sri Lanka to understand the anti-VZV IgG status in the country

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

<div><p>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 (<i>p</i><0.0001). No positive correlation was observed between dengue incidence and temperature (<i>p</i> = 0.107) or humidity (<i>p</i> = 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.</p></div

Auto-correlation of series ‘X’ by lag–dengue vs humidity.

<p>Y axis–CCF—Cross correlation function; X axis–Lag time in months.</p