Diagnostic challenges and case management of the first imported case of Plasmodium knowlesi in Sri Lanka.

BACKGROUND: Sri Lanka has achieved 'malaria-free' status and is now in the phase of prevention of re-introduction of malaria. Imported malaria remains a challenge to resurgence of the disease. The diagnostic challenges encountered and the rapid response initiated to manage a Plasmodium infection, which was later confirmed as Plasmodium knowlesi, the first reported case from Sri Lanka, is discussed. CASE PRESENTATION: An army officer who returned from Malaysia in October 2016 was found to be positive for Plasmodium both by microscopy and rapid diagnostic test (RDT) by the Anti Malaria Campaign Sri Lanka (AMC) during his third visit to a health care provider. Microscopy findings were suspicious of P. knowlesi infection as the smears showed parasite stages similar to both Plasmodium malariae and Plasmodium falciparum. Nested PCR at AMC confirmed Plasmodium genus, but not the species. In the absence of species confirmation, the patient was treated as a case of P. falciparum. The presence of P. knowlesi was later confirmed by a semi-nested PCR assay performed at the Environmental Health Institute, National Environmental Agency in Singapore. The parasite strain was also characterized by sequencing the circumsporozoite gene. Extensive case investigation including parasitological and entomological surveillance was carried out. CONCLUSIONS: Plasmodium knowlesi should be suspected in patients returning from countries in the South Asian region where the parasite is prevalent and when blood smear results are inconclusive.

Lineage Replacement Associated with Fitness Gain in Mammalian Cells and Aedes aegypti: A Catalyst for Dengue Virus Type 2 Transmission.

Shifting of virus serotypes and clade replacement events are known to drive dengue epidemics. However, only a few studies have attempted to elucidate the virus attributes that contribute to such epidemics. In 2007, Singapore experienced a dengue outbreak affecting more than 8000 individuals. The outbreak ensued with the shuffling of dominant clades (from clade I to clade II) of Dengue virus 2 (DENV-2) cosmopolitan genotype, at a time when the Aedes premise index was significantly low. Therefore, we hypothesized that clade II had higher epidemic potential and fitness than clade I. To test this hypothesis, we tested the replication and apoptotic qualities of clade I and II isolates in mammalian cells and their ability to infect and disseminate in a field strain of Ae. Aegypti. Our findings indicated that clade II replicated more efficiently in mammalian cells than clade I and possessed higher transmission potential in local vectors. This could collectively improve the epidemic potential of clade II, which dominated during the outbreak in 2007. The findings exemplify complex interactions between the emergence, adaptation and transmission potential of DENV, and testify the epidemiological importance of a deeper understanding of virus and vector dynamics in endemic regions.

Wolbachia wAlbB remains stable in Aedes aegypti over 15 years but exhibits genetic background-dependent variation in virus blocking.

The ability of the maternally transmitted endosymbiotic bacterium Wolbachia to induce cytoplasmic incompatibility (CI) and virus blocking makes it a promising weapon for combatting mosquito-borne diseases through either suppression or replacement of wild-type populations. Recent field trials show that both approaches significantly reduce the incidence of dengue fever in humans. However, new questions emerge about how Wolbachia-mosquito associations will co-evolve over time and whether Wolbachia-mediated virus blocking will be affected by the genetic diversity of mosquitoes and arboviruses in the real world. Here, we have compared the Wolbachia density and CI expression of two wAlbB-infected Aedes aegypti lines transinfected 15 years apart. We have also assessed wAlbB-mediated virus blocking against dengue (DENV), Zika (ZIKV), and Chikungunya (CHIKV) viruses and examined whether host genetic backgrounds modulate viral blocking effects by comparing ZIKV infection in mosquitoes with a Mexican genetic background to those with a Singaporean background. Our results show that over 15 years, wAlbB maintained the capacity to form a stable association with Ae. aegypti in terms of both density and CI expression. There were variations in wAlbB-induced virus blocking against CHIKV, DENV, and ZIKV, and higher inhibitory effects on ZIKV in mosquitoes on the Singaporean genetic background than on the Mexican background. These results provide important information concerning the robustness and long-term stability of Wolbachia as a biocontrol agent for arbovirus disease control.

Viral and Antibody Kinetics, and Mosquito Infectivity of an Imported Case of Zika Fever Due to Asian Genotype (American Strain) in Singapore.

We report a case of a Singaporean who acquired Zika virus (ZIKV) during a visit to Cuba. The infection was confirmed using molecular and serological methods. This report highlights potential drawbacks of using IgG serology for diagnosis of flavivirus infections in endemic regions. The low viremia detected during the early phase of this case resulted in low mosquito infectivity rates, suggesting the possibility of ZIKV transmission prior to clinical onset. The report also emphasizes the challenges of public health interventions for Zika fever and the importance of sustaining a low vector population to reduce the risk of arbovirus transmission in vulnerable regions.

Membrane feeding of dengue patient's blood as a substitute for direct skin feeding in studying Aedes-dengue virus interaction.

BACKGROUND: Understanding the interaction between Aedes vectors and dengue viruses (DENV) has significant implications in determining the transmission dynamics of dengue. The absence of an animal model and ethical concerns regarding direct feeding of mosquitoes on patients has resulted in most infection studies using blood meals spiked with laboratory-cultured DENV. Data obtained from such studies may not reflect the natural human-mosquito transmission scenario. This study explored the potential of using membrane feeding of dengue patient's blood as a substitute for direct skin feeding. METHODS: Four to six-day old female Ae. aegypti were provided the opportunity to feed via direct exposure to a patient's forearm for 15 min or via exposure to EDTA-treated blood from the same patient through an artificial membrane for 30 min. Mosquitoes from both feeding methods were incubated inside environmental chambers. Mosquitoes were sampled at day 13 post-feeding. Midgut and salivary glands of each mosquito were dissected to determine DENV infection by RT-qPCR and viral titration, respectively. RESULTS: Feeding rates: Direct skin feeding assay (DSFA) consistently showed higher mosquito feeding rates (93.3-100%) when compared with the membrane feeding assay (MFA) (48-98.2%). Midgut infection: Pair-wise comparison between methods showed no significant difference in midgut infection rates between mosquitoes exposed via each method and a strong correlation was observed in midgut infection rates for both feeding methods (r = 0.89, P < 0.0001). Overall midgut viral titers (n = 20) obtained by both methods were comparable (P ≥ 0.06). Salivary gland infection: Pair-wise comparison between both methods revealed no significant difference in salivary gland infection rate. Strong correlation in salivary gland infection was observed between DSFA and MFA (r = 0.81, P < 0.0001). In general, mosquitoes fed directly on dengue patients and those on patients' blood (n = 11) had comparable virus titer (P ≥ 0.09). CONCLUSION: DENV midgut and salivary gland infection rates showed good concordance between DSFA and MFA blood meal exposure methods. Freshly-obtained venous blood in EDTA from dengue patients for MFA can be used as a substitute to DSFA, especially in circumstances where bioethics approval or patient recruitment is difficult to obtain for vector competence studies. Nevertheless, mosquito numbers will need to be increased to compensate for lower feeding rate in MFA.

Aedes (Stegomyia) albopictus (Skuse): a potential vector of Zika virus in Singapore.

BACKGROUND: Zika virus (ZIKV) is a little known arbovirus until it caused a major outbreak in the Pacific Island of Yap in 2007. Although the virus has a wide geographic distribution, most of the known vectors are sylvatic Aedes mosquitoes from Africa where the virus was first isolated. Presently, Ae. aegypti is the only known vector to transmit the virus outside the African continent, though Ae. albopictus has long been a suspected vector. Currently, Ae. albopictus has been shown capable of transmitting more than 20 arboviruses and its notoriety as an important vector came to light during the recent chikungunya pandemic. The vulnerability of Singapore to emerging infectious arboviruses has stimulated our interest to determine the competence of local Ae. albopictus to transmit ZIKV. METHODOLOGY/PRINCIPAL FINDINGS: To determine the competence of Ae. albopictus to ZIKV, we orally infected local mosquito strains to a Ugandan strain virus. Fully engorged mosquitoes were maintained in an environmental chamber set at 29°C and 80-85%RH. Twelve mosquitoes were then sampled daily from day one to seven and on day 10 and 14 post infection (pi). Zika virus titre in the midgut and salivary glands of each mosquito were determined using tissue culture infectious dose50 assay, while transmissibility of the virus was determined by detecting viral antigen in the mosquito saliva by qRT-PCR. High dissemination and transmission rate of ZIKV were observed. By day 7-pi, all mosquitoes have disseminated infection and 73% of these mosquitoes have ZIKV in their saliva. By day 10-pi, all mosquitoes were potentially infectious. CONCLUSIONS/SIGNIFICANCE: The study highlighted the potential of Ae. albopictus to transmit ZIKV and the possibility that the virus could be established locally. Nonetheless, the threat of ZIKV can be mitigated by existing dengue and chikungunya control program being implemented in Singapore.

Oral susceptibility of Singapore Aedes (Stegomyia) aegypti (Linnaeus) to Zika virus.

BACKGROUND: Zika virus (ZIKV) is a little known flavivirus that caused a major outbreak in 2007, in the South-western Pacific Island of Yap. It causes dengue-like syndromes but with milder symptoms. In Africa, where it was first isolated, ZIKV is mainly transmitted by sylvatic Aedes mosquitoes. The virus has also been isolated from Ae. aegypti and it is considered to be the vector involved in the urban transmission of the virus. Transmission of the virus by an African strain of Ae. aegypti has also been demonstrated under laboratory conditions. The aim of the present study is to describe the oral susceptibility of a Singapore strain of Ae. aegypti to ZIKV, under conditions that simulate local climate. METHODOLOGY/PRINCIPAL FINDINGS: To assess the receptivity of Singapore's Ae. aegypti to the virus, we orally exposed a local mosquito strain to a Ugandan strain of ZIKV. Upon exposure, fully engorged mosquitoes were maintained in an environmental chamber set at 29 °C and 70-75% RH. Eight mosquitoes were then sampled daily from day 1 to day 7, and subsequently on days 10 and 14 post exposure (pe). The virus titer of the midgut and salivary glands of each mosquito were determined using a tissue culture infectious dose(50) (TCID(50)) assay. High midgut infection and salivary gland dissemination rates were observed. By day 5 after the infectious blood meal, ZIKV was found in the salivary glands of more than half of the mosquitoes tested (62%); and by day 10, all mosquitoes were potentially infective. CONCLUSIONS/SIGNIFICANCE: This study showed that Singapore's urban Ae. aegypti are susceptible and are potentially capable of transmitting ZIKV. The virus could be established in Singapore should it be introduced. Nevertheless, Singapore's current dengue control strategy is applicable to control ZIKV.

Evaluation of the Dengue NS1 Ag Strip® for detection of dengue virus antigen in Aedes aegypti (Diptera: Culicidae).

Dengue fever is currently one of the most important mosquito-borne diseases that affect humans. With neither vaccines nor treatment available, prevention of the disease relies heavily on surveillance and control of mosquito vectors. In the present study, we have evaluated and showed the potential use of the Dengue NS1 Ag Strip(®) for the detection of dengue virus (DENV) in Aedes aegypti. Initial results showed that the sensitivity of the test kit in detecting DENV in wild-caught mosquitoes is comparable to that of real-time reverse transcriptase-polymerase chain reaction. The detection of naturally infected Ae. aegypti with the NS1 rapid test kit in our dengue cluster investigation further illustrates its potential use for surveillance of DENV in wild mosquito populations. The kit can easily be used in a simple field station, and minimal training is required. The results can be obtained in less than an hour. Employment of the kit in the field could help guide mosquito control operations in the prioritization of resources in controlling the transmission of DENV. In this study the potential of the kit for field surveillance of infected dengue vectors, which are epidemiologically important, has been demonstrated.

Entomological investigation and control of a chikungunya cluster in Singapore.

In August 2008, a team from the National Environmental Agency conducted an entomological investigation of a chikungunya cluster in Singapore, with the primary aim of identifying the vector responsible for the outbreak and to assess the vector control operation. A total of 173 adult mosquitoes were caught using both the sweep-net method and the BG Sentinel Traps in and around the affected workers' quarters. Of these, 120 (69.4%) were Aedes albopictus and the rest were Culex quinquefasciatus. More than 2700 Ae. albopictus larvae were also collected from 33 breeding habitats detected. No Aedes aegypti was found. During the preintervention period, 6 (8.4%) out of 71 adult female Ae. albopictus were found positive for the chikungunya virus (CHIKV). Vector control measures resulted in a 90% reduction of adult Ae. albopictus caught by BG Sentinel Traps. Postintervention surveillance revealed the presence of CHIKV-positive mosquitoes. These findings led to continued intensive vector control operation in the affected area that further reduced vector population and interrupted the transmission of the disease. The E1 gene sequence of the CHIKV was identical to those of CHIKV isolated from human chikungunya cases working in the affected area, and contained the A226V mutation. The incrimination of Ae. albopictus as a major vector involved in the transmission of A226V CHIKV had led to the revision of chikungunya control strategy in Singapore. This study suggests the benefit of a vector control program that includes the evaluation of control measures in conjunction to virological surveillance in vector population.