Malaria surveillance from both ends: concurrent detection of Plasmodium falciparum in saliva and excreta harvested from Anopheles mosquitoes

Background: Malaria is the most important vector-borne disease in the world. Epidemiological and ecological studies of malaria traditionally utilize detection of Plasmodium sporozoites in whole mosquitoes or salivary glands by microscopy or serological or molecular assays. However, these methods are labor-intensive, and can over- or underestimate mosquito transmission potential. To overcome these limitations, alternative sample types have been evaluated for the study of malaria. It was recently shown that Plasmodium could be detected in saliva expectorated on honey-soaked cards by Anopheles stephensi, providing a better estimate of transmission risk. We evaluated whether excretion of Plasmodium falciparum nucleic acid by An. stephensi correlates with expectoration of parasites in saliva, thus providing an additional sample type for estimating transmission potential. Mosquitoes were exposed to infectious blood meals containing cultured gametocytes, and excreta collected at different time points post-exposure. Saliva was collected on honey-soaked filter paper cards, and salivary glands were dissected and examined microscopically for sporozoites. Excreta and saliva samples were tested by real time polymerase chain reaction (RT-rtPCR). Results: Plasmodium falciparum RNA was detected in mosquito excreta as early as four days after ingesting a bloodmeal containing gametocytes. Once sporogony (the development of sporozoites) occurred, P. falciparum RNA was detected concurrently in both excreta and saliva samples. In the majority of cases, no difference was observed between the Ct values obtained from matched excreta and saliva samples, suggesting that both samples provide equally sensitive results. A positive association was observed between the molecular detection of the parasites in both samples and the proportion of mosquitoes with sporozoites in their salivary glands from each container. No distinguishable parasites were observed when excreta samples were stained and microscopically analyzed. Conclusions: Mosquito saliva and excreta are easily collected and are promising for surveillance of malaria-causing parasites, especially in low transmission settings or in places where arboviruses co-circulate

Nucleic Acid Preservation Card Surveillance Is Effective for Monitoring Arbovirus Transmission on Crocodile Farms and Provides a One Health Benefit to Northern Australia

The Kunjin strain of West Nile virus (WNVKUN) is a mosquito-transmitted flavivirus that can infect farmed saltwater crocodiles in Australia and cause skin lesions that devalue the hides of harvested animals. We implemented a surveillance system using honey-baited nucleic acid preservation cards to monitor WNVKUN and another endemic flavivirus pathogen, Murray Valley encephalitis virus (MVEV), on crocodile farms in northern Australia. The traps were set between February 2018 and July 2020 on three crocodile farms in Darwin (Northern Territory) and one in Cairns (North Queensland) at fortnightly intervals with reduced trapping during the winter months. WNVKUN RNA was detected on all three crocodile farms near Darwin, predominantly between March and May of each year. Two of the NT crocodile farms also yielded the detection of MVE viral RNA sporadically spread between April and November in 2018 and 2020. In contrast, no viral RNA was detected on crocodile farms in Cairns during the entire trapping period. The detection of WNVKUN and MVEV transmission by FTATM cards on farms in the Northern Territory generally correlated with the detection of their transmission to sentinel chicken flocks in nearby localities around Darwin as part of a separate public health surveillance program. While no isolates of WNVKUN or MVEV were obtained from mosquitoes collected on Darwin crocodile farms immediately following the FTATM card detections, we did isolate another flavivirus, Kokobera virus (KOKV), from Culex annulirostris mosquitoes. Our studies support the use of the FTATM card system as a sensitive and accurate method to monitor the transmission of WNVKUN and other arboviruses on crocodile farms to enable the timely implementation of mosquito control measures. Our detection of MVEV transmission and isolation of KOKV from mosquitoes also warrants further investigation of their potential role in causing diseases in crocodiles and highlights a “One Health” issue concerning arbovirus transmission to crocodile farm workers. In this context, the introduction of FTATM cards onto crocodile farms appears to provide an additional surveillance tool to detect arbovirus transmission in the Darwin region, allowing for a more timely intervention of vector control by relevant authorities

Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

The human prolyl-4-hydroxylase domain (PHD) proteins 1–3 are known as cellular oxygen sensors, acting via the degradation of hypoxia-inducible factor (HIF) α-subunits. PHD2 and PHD3 genes are inducible by HIFs themselves, suggesting a negative feedback loop that involves PHD abundance. To identify novel regulators of the PHD2 gene, an expression array of 704 transcription factors was screened by a method that allows distinguishing between HIF-dependent and HIF-independent promoter regulation. Among others, the E-twenty six transcription factor ETS translocation variant 4 (ETV4) was found to contribute to PHD2 gene expression particularly under hypoxic conditions. Mechanistically, complex formation between ETV4 and HIF-1/2α was observed by mammalian two-hybrid and fluorescence resonance energy transfer analysis. HIF-1α domain mapping, CITED2 overexpression and factor inhibiting HIF depletion experiments provided evidence for cooperation between HIF-1α and p300/CBP in ETV4 binding. Chromatin immunoprecipitation confirmed ETV4 and HIF-1α corecruitment to the PHD2 promoter. Of 608 hypoxically induced transcripts found by genome-wide expression profiling, 7.7% required ETV4 for efficient hypoxic induction, suggesting a broad role of ETV4 in hypoxic gene regulation. Endogenous ETV4 highly correlated with PHD2, HIF-1/2α and several established markers of tissue hypoxia in 282 human breast cancer tissue samples, corroborating a functional interplay between the ETV4 and HIF pathways

Illumina sequencing of clinical samples for virus detection in a public health laboratory

High-throughput sequencing (HTS) provides the opportunity, once a diagnostic result is obtained, to extract additional information from a virus-containing sample. Hence, it offers advantages over established quantitative amplification technology, such as quantitative PCR, particularly in a public health environment. At this early stage of its clinical application, there have been limited studies comparing HTS performance to that of the more established quantitative PCR technology for direct detection of viruses. In this pilot-scale study, we tested HTS with a range of viruses and sample types routinely encountered in a public health virology laboratory. In comparison with quantitative PCR, our HTS method was able to sensitively (92%) detect all viruses in any sample type with the exception of certain tissues. Moreover, sufficient nucleotide sequence information was obtained to enable genotyping of strains detected, thus providing additional useful epidemiological information. While HTS sensitivity may not yet match that of PCR, the added value through enhanced epidemiological data has considerable potential to enable real-time surveillance of circulating strains so as to facilitate rapid and appropriate response to outbreaks and virus zoonotic spillover events

Stability of West Nile Virus (Flaviviridae: Flavivirus) RNA in mosquito excreta

Arbovirus surveillance is crucial for the implementation of vector-borne disease control measures. Recently, it has been demonstrated that mosquitoes with a disseminated arbovirus infection excrete viral RNA, which can be detected by molecular methods. Thereby, mosquito excreta has been proposed as a sample type that could be utilized for arbovirus surveillance. In this study, we evaluated if West Nile virus (Kunjin strain, WNVKUN) RNA in Culex annulirostris Skuse (Diptera: Culicidae) excreta deposited on different substrates could be detected after storage for up to 2 wk at tropical conditions of high heat and humidity. No significant drop in relative quantity of WNVKUN RNA (determined by comparison of Ct values) in excreta deposited on Flinders Associate Technologies (FTA) cards was observed over 14 d, suggesting that RNA was stable for that time. There was no significant difference in relative quantity of WNVKUN RNA in excreta deposited on FTA cards or polycarbonate substrates after 24 h. However, after 7 and 14 d, there was a significant decline in the relative quantity of viral RNA in the excreta stored on polycarbonate substrates. For incorporation in arbovirus surveillance programs, we recommend the use of polycarbonate substrates for excreta collection in mosquito traps deployed overnight, and the integration of FTA cards in traps serviced weekly or fortnightly. Polycarbonate substrates facilitate the collection of the majority of excreta from a trap, and while FTA cards offer limited area coverage, they enable preservation of viral RNA in tropical conditions for extended periods of time

Mosquito excreta: A sample type with many potential applications for the investigation of Ross River virus and West Nile virus ecology.

BACKGROUND:Emerging and re-emerging arthropod-borne viruses (arboviruses) cause human and animal disease globally. Field and laboratory investigation of mosquito-borne arboviruses requires analysis of mosquito samples, either individually, in pools, or a body component, or secretion such as saliva. We assessed the applicability of mosquito excreta as a sample type that could be utilized during studies of Ross River and West Nile viruses, which could be applied to the study of other arboviruses. METHODOLOGY/PRINCIPAL FINDINGS:Mosquitoes were fed separate blood meals spiked with Ross River virus and West Nile virus. Excreta was collected daily by swabbing the bottom of containers containing batches and individual mosquitoes at different time points. The samples were analyzed by real-time RT-PCR or cell culture enzyme immunoassay. Viral RNA in excreta from batches of mosquitoes was detected continuously from day 2 to day 15 post feeding. Viral RNA was detected in excreta from at least one individual mosquito at all timepoints, with 64% and 27% of samples positive for RRV and WNV, respectively. Excretion of viral RNA was correlated with viral dissemination in the mosquito. The proportion of positive excreta samples was higher than the proportion of positive saliva samples, suggesting that excreta offers an attractive sample for analysis and could be used as an indicator of potential transmission. Importantly, only low levels of infectious virus were detected by cell culture, suggesting a relatively low risk to personnel handling mosquito excreta. CONCLUSIONS/SIGNIFICANCE:Mosquito excreta is easily collected and provides a simple and efficient method for assessing viral dissemination, with applications ranging from vector competence experiments to complementing sugar-based arbovirus surveillance in the field, or potentially as a sample system for virus discovery

Detection of specific ZIKV IgM in travelers using a multiplexed flavivirus microsphere immunoassay

Zika virus (ZIKV) has spread widely in the Pacific and recently throughout the Americas. Unless detected by RT-PCR, confirming an acute ZIKV infection can be challenging. We developed and validated a multiplexed flavivirus immunoglobulin M (IgM) microsphere immunoassay (flaviMIA) which can differentiate ZIKV-specific IgM from that due to other flavivirus infections in humans. The flaviMIA bound 12 inactivated flavivirus antigens, including those from ZIKV and yellow fever virus (YFV), to distinct anti-flavivirus antibody coupled beads. These beads were used to interrogate sera from patients with suspected ZIKV infection following travel to relevant countries. FlaviMIA results were validated by comparison to the ZIKV plaque reduction neutralization test (PRNT). The results highlight the complexity of serological ZIKV diagnosis, particularly in patients previously exposed to or vaccinated against other flaviviruses. We confirmed 99 patients with ZIKV infection by a combination of RT-PCR and serology. Importantly, ZIKV antibodies could be discriminated from those ascribed to other flavivirus infections. Serological results were sometimes confounded by the presence of pre-existing antibodies attributed to previous flavivirus infection or vaccination. Where RT-PCR results were negative, testing of appropriately timed paired sera was necessary to demonstrate seroconversion or differentiation of recent from past infection with or exposure to ZIKV

A case of Japanese encephalitis with a fatal outcome in an Australian who traveled from Bali in 2019

A severe case of Japanese encephalitis virus (JEV) infection, resulting in fatality, occurred in an unvaccinated Australian male traveler from Bali, Indonesia, in 2019. During hospitalisation in Australia, patient cerebrospinal fluid (CSF) yielded JEV-specific IgM antibodies and RNA, and an isolate of the virus. Ongoing transmission of JEV in Bali underscores this pathogen as a public health risk and the importance of appropriate health, vaccination and mosquito avoidance advice to prospective travelers to the region