Evaluation of Chikungunya Diagnostic Assays: Differences in Sensitivity of Serology Assays in Two Independent Outbreaks

Chikungunya is a mounting public health concern in many parts of the world. Definitive diagnosis is critical in differentiating the diseases, especially in dengue endemic areas. There are some commercial chikungunya kits and published molecular protocols available, but no comprehensive comparative evaluation of them was performed. Using sera collected in outbreaks caused by two variants of Chikungunya virus (A226 and 226V), we tested 2 commercial IgM tests (CTK lateral flow rapid test and EUROIMMUN IFA) alongside our in-house IgM assays (using both variants of the virus). Sensitivities of 2 published PCR protocols were also evaluated based on RNA standards derived from cell-cultured viruses. The commercial assays had different performances in each outbreak, with CTK's lateral flow test having the best performance in the first outbreak and EUROIMMUN IFA being more sensitive in the second outbreak. Use of the current circulating virus in a test assay improves sensitivity of the MAC-ELISAs. For PCR, a probe-based real time RT-PCR method was found to be 10 times more sensitive than the SYBR Green method. Despite this, the latter protocol is found to be more suitable and cost-effective for our diagnostic laboratory. This evaluation demonstrates the importance of appraisal of commercial kits and published protocols before application of a diagnostic tool in the clinical and operational setting

Singapore’s 5 decades of dengue prevention and control—Implications for global dengue control

This paper summarises the lessons learnt in dengue epidemiology, risk factors, and prevention in Singapore over the last half a century, during which Singapore evolved from a city of 1.9 million people to a highly urban globalised city-state with a population of 5.6 million. Set in a tropical climate, urbanisation among green foliage has created ideal conditions for the proliferation of Aedes aegypti and Aedes albopictus, the mosquito vectors that transmit dengue. A vector control programme, largely for malaria, was initiated as early as 1921, but it was only in 1966 that the Vector Control Unit (VCU) was established to additionally tackle dengue haemorrhagic fever (DHF) that was first documented in the 1960s. Centred on source reduction and public education, and based on research into the bionomics and ecology of the vectors, the programme successfully reduced the Aedes House Index (HI) from 48% in 1966 to <5% in the 1970s. Further enhancement of the programme, including through legislation, suppressed the Aedes HI to around 1% from the 1990s. The current programme is characterised by 4 key features: (i) proactive inter-epidemic surveillance and control that is stepped up during outbreaks; (ii) risk-based prevention and intervention strategies based on advanced data analytics; (iii) coordinated inter-sectoral cooperation between the public, private, and people sectors; and (iv) evidence-based adoption of new tools and strategies. Dengue seroprevalence and force of infection (FOI) among residents have substantially and continuously declined over the 5 decades. This is consistent with the observation that dengue incidence has been delayed to adulthood, with severity highest among the elderly. Paradoxically, the number of reported dengue cases and outbreaks has increased since the 1990s with record-breaking epidemics. We propose that Singapore’s increased vulnerability to outbreaks is due to low levels of immunity in the population, constant introduction of new viral variants, expanding urban centres, and increasing human density. The growing magnitude of reported outbreaks could also be attributed to improved diagnostics and surveillance, which at least partially explains the discord between rising trend in cases and the continuous reduction in dengue seroprevalence. Changing global and local landscapes, including climate change, increasing urbanisation and global physical connectivity are expected to make dengue control even more challenging. The adoption of new vector surveillance and control tools, such as the Gravitrap and Wolbachia technology, is important to impede the growing threat of dengue and other Aedes-borne diseases. Author summary A densely populated, highly urban tropical city-state with long-established populations of Aedes aegypti and Ae. albopictus mosquitoes, plus travel and trade links to all corners of the world, Singapore is ideally suited for dengue transmission. Singapore’s experience and rich surveillance data provide important insights for an increasingly large number of territories at risk of dengue epidemics. Decades of vector control efforts, focused on source reduction and surveillance, have successfully lowered the vector population, with accompanying reductions in the resident population’s seroprevalence to dengue. We propose that Singapore’s vulnerability to outbreaks is due to low levels of immunity in the population, constant introduction of viral variants, expanding urban centres, and increasing human density. The discord between the rising trend in reported cases and falling seroprevalence could be at least partly attributed to improved diagnostics and surveillance. Singapore’s evidence-based vector-control programme, which involves strong partnership between the public and private sectors, will continue to adapt to future challenges in this space

Singapore's 5 decades of dengue prevention and control-implications for global dengue control

This paper summarises the lessons learnt in dengue epidemiology, risk factors, and prevention in Singapore over the last half a century, during which Singapore evolved from a city of 1.9 million people to a highly urban globalised city-state with a population of 5.6 million. Set in a tropical climate, urbanisation among green foliage has created ideal conditions for the proliferation of Aedes aegypti and Aedes albopictus, the mosquito vectors that transmit dengue. A vector control programme, largely for malaria, was initiated as early as 1921, but it was only in 1966 that the Vector Control Unit (VCU) was established to additionally tackle dengue haemorrhagic fever (DHF) that was first documented in the 1960s. Centred on source reduction and public education, and based on research into the bionomics and ecology of the vectors, the programme successfully reduced the Aedes House Index (HI) from 48% in 1966 to <5% in the 1970s. Further enhancement of the programme, including through legislation, suppressed the Aedes HI to around 1% from the 1990s. The current programme is characterised by 4 key features: (i) proactive inter-epidemic surveillance and control that is stepped up during outbreaks; (ii) risk-based prevention and intervention strategies based on advanced data analytics; (iii) coordinated inter-sectoral cooperation between the public, private, and people sectors; and (iv) evidence-based adoption of new tools and strategies. Dengue seroprevalence and force of infection (FOI) among residents have substantially and continuously declined over the 5 decades. This is consistent with the observation that dengue incidence has been delayed to adulthood, with severity highest among the elderly. Paradoxically, the number of reported dengue cases and outbreaks has increased since the 1990s with record-breaking epidemics. We propose that Singapore's increased vulnerability to outbreaks is due to low levels of immunity in the population, constant introduction of new viral variants, expanding urban centres, and increasing human density. The growing magnitude of reported outbreaks could also be attributed to improved diagnostics and surveillance, which at least partially explains the discord between rising trend in cases and the continuous reduction in dengue seroprevalence. Changing global and local landscapes, including climate change, increasing urbanisation and global physical connectivity are expected to make dengue control even more challenging. The adoption of new vector surveillance and control tools, such as the Gravitrap and Wolbachia technology, is important to impede the growing threat of dengue and other Aedes-borne diseases.Published versio

Construction sites as an important driver of dengue transmission: implications for disease control

Abstract Background In 2013 and 2014, Singapore experienced its worst dengue outbreak known-to-date. Mosquito breeding in construction sites stood out as a probable risk factor due to its association with major dengue clusters in both years. We, therefore, investigated the contribution of construction sites to dengue transmission in Singapore, highlighting three case studies of large construction site-associated dengue clusters recorded during 2013–16. Methods The study included two components; a statistical analysis of cluster records from 2013 to 2016, and case studies of three biggest construction site-associated clusters. We explored the odds of construction site-associated clusters growing into major clusters and determined whether clusters seeded in construction sites demonstrated a higher tendency to expand into major clusters. DENV strains obtained from dengue patients residing in three major clusters were genotyped to determine whether the same strains expanded into the surroundings of construction sites. Results Despite less than 5% of total recorded clusters being construction site-associated, the odds of such clusters expanding into major clusters were 17.4 (2013), 9.2 (2014), 3.3 (2015) and 4.3 (2016) times higher than non-construction site clusters. Aedes premise index and average larvae count per habitat were also higher in construction sites than residential premises during the study period. The majority of cases in clusters associated with construction sites were residents living in the surroundings. Virus genotype data from three case study sites revealed a transmission link between the construction sites and the surrounding residential areas. Conclusions Significantly high case burden and the probability of cluster expansion due to virus spill-over into surrounding areas suggested that construction sites play an important role as a driver of sustained dengue transmission. Our results emphasise that the management of construction-site associated dengue clusters should not be limited to the implicated construction sites, but be extended to the surrounding premises to prevent further transmission

SYBR green-based one step quantitative real-time polymerase chain reaction assay for the detection of Zika virus in field-caught mosquitoes

Abstract Background The monitoring of vectors is one of the key surveillance measures to assess the risk of arbovirus transmission and the success of control strategies in endemic regions. The recent re-emergence of Zika virus (ZIKV) in the tropics, including Singapore, emphasizes the need to develop cost-effective, rapid and accurate assays to monitor the virus spread by mosquitoes. As ZIKV infections largely remain asymptomatic, early detection of ZIKV in the field-caught mosquitoes enables timely implementation of appropriate mosquito control measures. Results We developed a rapid, sensitive and specific real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for the detection of ZIKV in field-caught mosquitoes. The primers and PCR cycling conditions were optimized to minimize non-specific amplification due to cross-reactivity with the genomic material of Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex tritaeniorhynchus, Culex sitiens and Anopheles sinensis, as well as accompanying microbiota. The performance of the assay was further evaluated with a panel of flaviviruses and alphaviruses as well as in field-caught Ae. aegypti mosquitoes confirmed to be positive for ZIKV. As compared to a probe-based assay, the newly developed assay demonstrated 100% specificity and comparable detection sensitivity for ZIKV in mosquitoes. Conclusions Being a SYBR Green-based method, the newly-developed assay is cost-effective and easy to adapt, thus is applicable to large-scale vector surveillance activities in endemic countries, including those with limited resources and expertise. The amplicon size (119 bp) also allows sequencing to confirm the virus type. The primers flank relatively conserved regions of ZIKV genome, so that, the assay is able to detect genetically diverse ZIKV strains. Our findings, therefore, testify the potential use of the newly-developed assay in vector surveillance programmes for ZIKV in endemic regions

Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in retail food in Singapore

Abstract We characterised 227 Staphylococcus aureus isolates from retail food and food handlers’ gloves samples obtained through food surveillance and risk assessment studies between 2011 and 2014. Of 227 isolates, five (2.2%) were methicillin-resistant and belonged to sequence types ST80 (n = 3) and ST6 (n = 2). All five isolates belonged to SCCmec type IV, were Panton-Valentine leukocidin (pvl)-negative and staphylococcal enterotoxin genes-positive. Resistance to azithromycin was found in ST80 isolates, in addition to resistance to beta-lactams. Our finding of two clinically relevant methicillin-resistant S. aureus (MRSA) strains (ST80 and ST6) in ready-to-eat food and food contact surfaces at retail in Singapore suggests food and food contact surfaces as potential environmental sources of MRSA in the community

Evolution, heterogeneity and global dispersal of cosmopolitan genotype of Dengue virus type 2

10.1038/s41598-021-92783-yScientific Reports1111349

Highly Selective Transmission Success of Dengue Virus Type 1 Lineages in a Dynamic Virus Population: An Evolutionary and Fitness Perspective

Summary: Arbovirus transmission is modulated by host, vector, virus, and environmental factors. Even though viral fitness plays a salient role in host and vector adaptation, the transmission success of individual strains in a heterogeneous population may be stochastic. Our large-scale molecular epidemiological analyses of a dengue virus type 1 population revealed that only a subset of strains (16.7%; n = 6) were able to sustain transmission, despite the population being widely dispersed, dynamic, and heterogeneous. The overall dominance was variable even among the “established” lineages, albeit sharing comparable evolutionary characteristics and replication profiles. These findings indicated that virological parameters alone were unlikely to have a profound effect on the survival of viral lineages, suggesting an important role for non-viral factors in the transmission success of lineages. Our observations, therefore, emphasize the strategic importance of a holistic understanding of vector, human host, and viral factors in the control of vector-borne diseases. : Disease; Virology; Evolutionary Biology; Phylogenetics Subject Areas: Disease, Virology, Evolutionary Biology, Phylogenetic

Flavivirus Cross-Reactivity to Dengue Nonstructural Protein 1 Antigen Detection Assays

Dengue virus (DENV) and Zika virus (ZIKV) are flaviviruses of public health relevance. Both viruses circulate in the same endemic settings and acute infections generally manifest similar symptoms. This highlights the importance of accurate diagnosis for clinical management and outbreak control. One of the commonly used acute diagnostic markers for flaviviruses is nonstructural protein 1 (NS1). However, false positives due to antigenic cross-reactivity have been reported between DENV and ZIKV infections when using DENV NS1 antigen (NS1 Ag) detection assays in acute cases. Therefore, we investigated the lowest detectable virus titres and cross-reactivity of three commercial dengue NS1 Ag rapid assays and two ELISAs for different flaviviruses. Our results showed that substantially high viral titres of ZIKV, Kunjin virus (KUNV) and yellow fever virus (YFV) are required to give false-positive results when using DENV NS1 rapid detection assays. Commercial DENV NS1 ELISAs did not react with ZIKV and YFV. In comparison, tested assays detected DENV at a significantly low virus titre. Given the relatively low viral loads reported in clinical samples, our findings suggest that commercially available dengue NS1 Ag detection assays are less likely to generate false-positive results among clinical samples in areas where multiple flaviviruses cocirculate