Comprehensive evaluation on progressive development strategies in DENV surveillance and monitoring infection rate among vector population.

The elevated rise in dengue infection rate has been a health burden worldwide and it will continue to impact global health for years to come. Accumulated literature holds accountable the geographical expansion of the mosquito species transmitting the dengue virus DENV. The frequency of this viral disease outbreaks has increased rapidly in the recent years, owing to various geo-climatic and anthropological activities. Due to scarcity of any effective control measures, there has been a continuous traceable rise in mortality and morbidity rates. However, it has been reported that the spate of incidences is directly related to density of the virus infected vector (mosquito) population in a given region. In such a scenario, systems capable of detecting virus infected vector population would aid in estimating prediction of outbreak, as well as provide time to deploy suitable management strategies for vector control, and to break the vector-human transmission chain. This would also help in identifying areas, where much improvement is needed for vector management. To this context, we illustrate an exhaustive overview of both gold standards and as well as emerging advents for sensitive and specific mosquito population strategized viral detection technologies. We summarize the cutting-edge technologies and the challenges faced in pioneering to field application. Regardless the proven popularity of the gold standards for detection purpose, they offer certain limitations. Thus with the surge in the infection rate globally, approaches for development of newer advancements and technique upgradation to arrest the infection escalation and for early detection as a part of vector management should be prioritized.

Malaria and dengue fever in febrile children entering healthcare facilities in Mwanza, Tanzania.

Plasmodium spp. infections and cases of malaria are a long-standing public health problem for children living in middle- and low-income countries. Dengue virus causes an emerging under-recognized disease burden. A cross sectional study was conducted between March 2020 and December 2021 to determine the status of malaria and dengue fever, and the associated factors in children living in Mwanza, Tanzania. Clinical features were recorded; blood samples were analyzed using dengue NS1 rapid diagnostics test (NS1-RDT), malaria rapid diagnostic test (MRDT) and PCR and microscopy for malaria parasites. Descriptive analysis was based on infection status; odds ratio and confidence interval were used to determine the factors associated with dengue fever and malaria. The prevalence of malaria in the 436 children included in the final analysis was 15.6%, 8.5%, and 12.1% as determined by MRDT, blood smear examination and PCR, respectively. The prevalence of dengue fever determined by the NS1-RDT was 7.8%. Body rash, muscle and joint/bone pain were associated with a positive rapid dengue test result. Retro-orbital pain characterized Plasmodium spp. and dengue virus co-infections. Clinical signs and symptoms could not readily differentiate between malaria and dengue fever patients or patients co-infected with both causative agents underscoring the urgent need for the accurate laboratory diagnostics. Additional large-scale studies are required to assess the epidemiological burden of acute febrile illness in developing countries and to produce data that will guide empirical treatment.

Dengue in Pune city, India (2017-2019): a comprehensive analysis.

Objectives: To understand the dynamics of dengue disease with special reference to (1) age (2) primary/secondary infections (3) serostatus and (4) serotypes examined during three consecutive years. Methods: During 3 dengue seasons (2017-19), NS1/IgM ELISAs were used for dengue diagnosis in one of the 15 administrative wards of Pune City, India. Predefined symptoms were recorded at the time of diagnosis/hospitalization. IgG-capture ELISA (Panbio) was used to differentiate primary/secondary infections. DENV serotypes were determined for 260 viral RNA-positive patients. Results: During the 3 years, 3,014/6,786 (44.4%, 41.4-49.9%) suspected cases were diagnosed as dengue. Use of either NS1 or IgM would have missed 25.5% or 43% of the confirmed dengue cases, respectively. Notably, a higher proportion of secondary dengue cases remained mild while a substantial proportion of primary infections developed warning signs. The symptoms among Dengue/non-dengue patients and primary/secondary infections varied and influenced by age and serostatus. The number and proportion of dengue serotypes varied yearly. A remarkable decline in dengue cases was observed during the COVID-19 pandemic years. Conclusion: A substantial proportion of primary and secondary dengue patients progress to warning signs/severity or mild infection respectively, underscoring the possible role of non-ADE mechanisms in causing severe dengue that requires hospitalization. Both NS1 and IgM should be used for efficient diagnosis.

Detection of dengue virus serotype 4 in Panama after 23 years without circulation.

Panama is a country with endemic Dengue virus (DENV) transmission since its reintroduction in 1993. The four serotypes have circulated in the country and the region of the Americas, however, DENV-4 confirmed autochthonous cases have not been identified since 2000, despite its circulation in neighboring countries. Here, we report DENV-4 detection in Panama in the last four-month period of 2023 with co-circulation of the other serotypes, this was associated with a peak of dengue cases during the dry season even though most dengue outbreaks are described in the rainy season. Complete genomes of DENV-4 allowed us to determine that cases were caused by DENV-4 genotype IIb, the same genotype as 23 years ago, with high similarity to DENV-4 sequences circulating in Nicaragua and El Salvador during 2023. This report shows the importance of maintaining serotype and genotype surveillance for early detection of new variants circulating in the country.

The TIRS trial: Enrollment procedures and baseline characterization of a pediatric cohort to quantify the epidemiologic impact of targeted indoor residual spraying on Aedes-borne viruses in Merida, Mexico.

Aedes mosquito-borne viruses (ABVs) place a substantial strain on public health resources in the Americas. Vector control of Aedes mosquitoes is an important public health strategy to decrease or prevent spread of ABVs. The ongoing Targeted Indoor Residual Spraying (TIRS) trial is an NIH-sponsored clinical trial to study the efficacy of a novel, proactive vector control technique to prevent dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) infections in the endemic city of Merida, Yucatan, Mexico. The primary outcome of the trial is laboratory-confirmed ABV infections in neighborhood clusters. Despite the difficulties caused by the COVID-19 pandemic, by early 2021 the TIRS trial completed enrollment of 4,792 children aged 2-15 years in 50 neighborhood clusters which were allocated to control or intervention arms via a covariate-constrained randomization algorithm. Here, we describe the makeup and ABV seroprevalence of participants and mosquito population characteristics in both arms before TIRS administration. Baseline surveys showed similar distribution of age, sex, and socio-economic factors between the arms. Serum samples from 1,399 children were tested by commercially available ELISAs for presence of anti-ABV antibodies. We found that 45.1% of children were seropositive for one or more flaviviruses and 24.0% were seropositive for CHIKV. Of the flavivirus-positive participants, most were positive for ZIKV-neutralizing antibodies by focus reduction neutralization testing which indicated a higher proportion of participants with previous ZIKV than DENV infections within the cohort. Both study arms had statistically similar seroprevalence for all viruses tested, similar socio-demographic compositions, similar levels of Ae. aegypti infestation, and similar observed mosquito susceptibility to insecticides. These findings describe a population with a high rate of previous exposure to ZIKV and lower titers of neutralizing antibodies against DENV serotypes, suggesting susceptibility to future outbreaks of flaviviruses is possible, but proactive vector control may mitigate these risks.

Circulating serotypes and genotypes of dengue virus during the 2023 outbreak in Eastern Nepal.

Dengue virus (DENV) is one of the most significant mosquito-borne diseases in Nepal. In 2023, DENV outbreaks began in Eastern Nepal, near the border with India, and rapidly spread nationwide. The study aims to describe the outbreak's epidemiological pattern, laboratory characteristics, DENV serotypes, and genotypes. A hospital-based cross-sectional study was conducted in four hospitals in Jhapa, Eastern Nepal, in 2023. Acute serum samples were obtained from dengue suspected patients within 7 days of illness and subjected to virus isolation, conventional and real-time polymerase chain reaction (RT-PCR), and phylogenetic analysis. Out of 60 samples, 42 (70 %), 11 (18.3 %) and 7 (11.7 %) were primary, secondary and non-dengue infection, respectively. Among 53 dengue confirmed patients, 46 (86.7 %) were positive for NS1 and 12 (22.6 %) were positive for both NS1 and IgM. Out of 42 dengue isolates, a new clade of the cosmopolitan genotype of DENV-2 was the most prevalent (28, 66.7 %), followed by genotype III of DENV-3 (11, 26.2 %) and genotype V of DENV-1 (3, 7.1 %). Genotype III of DENV-3 was first introduced in 2022-2023 in Nepal. Phylogenetic analysis of the E gene revealed the DENV-2 isolates from Nepal had 98 % homologous nucleotide similarity with the strains from India and Bangladesh. To our knowledge, this is the first report of circulating serotypes and genotypes of DENV in Jhapa. Integrating molecular findings into the dengue control plan can enhance surveillance efforts, monitor disease trends, and implement proactive measures to reduce the burden of dengue and prevent fatalities in future outbreaks.

Diagnostic Accuracy of Five Molecular Assays for the Detection of Dengue Virus.

Background and Objectives: The steady spread of dengue virus (DENV) poses a profound public health threat worldwide. Reverse transcription real-time polymerase chain reaction (RT2-PCR) has been increasingly recognized as a reference method for the diagnosis of acute dengue infection. The goal of this study was to assess the diagnostic accuracy of five different RT2-PCR kits for the detection of DENV in a historically processed set of sera samples. Materials and Methods: In this retrospective study, 25 sera samples from routinely processed unique adult patients with a known DENV status (previously tested in both molecular and serological assays) were tested in parallel using four conventional (RealStar Dengue PCR Kit 3.0, Clonit'ngo Zika, Dengue & Chikungunya, BioPerfectus Zika Virus/Dengue Virus/Chikungunya Virus Real Time PCR Kit and Novaplex Tropical fever virus) and one sample-to-result (STANDARD M10 Arbovirus Panel) RT2-PCR assays. Additionally, an end-point dilution analysis was conducted in quintuplicate on six serial dilutions of an RNA preparation obtained from a culture-grown DENV serotype 1 strain for a total of 150 tests. Results: The overall accuracy of the evaluated tests ranged from 84% to 100%. In particular, the sensitivity of three conventional RT2-PCR assays (RealStar, Clonit'ngo and Novaplex) was 100% (95% CI: 79.6-100%), while it was lower (73.3%; 95% CI: 48.1-89.1%) for the BioPerfectus kit. The sample-to-result STANDARD M10 panel performed comparatively well, showing a sensitivity of 92.9% (95% CI: 68.5-98.7%). No false positive results were registered in any assay. The end-point dilution analysis suggested that the RealStar kit had the lowest limit of detection. Conclusions: Available RT2-PCR kits for the detection of DENV are highly specific and generally sensitive and, therefore, their implementation in diagnostic pathways is advisable.

Peri-domestic entomological surveillance using private traps allows detection of dengue virus in Aedes albopictus during an autochthonous transmission event in mainland France, late summer 2023.

While locally-acquired dengue virus (DENV) human infections occur in mainland France since 2010, data to identify the mosquito species involved and to trace the virus are frequently lacking. Supported by a local network gathering public health agencies and research laboratories, we analysed, in late summer 2023, mosquitoes from privately-owned traps within a French urban neighbourhood affected by a dengue cluster. The cluster, in Auvergne-Rhône-Alpes, comprised three cases, including two autochthonous ones. Upon return from a recent visit to the French Caribbean Islands, the third case had consulted healthcare because of dengue-compatible symptoms, but dengue had not been recognised. For the two autochthonous cases, DENV-specific antibodies in serum or a positive quantitative PCR for DENV confirmed DENV infection. The third case had anti-flavivirus IgMs. No DENV genetic sequences were obtained from affected individuals but Aedes albopictus mosquitoes trapped less than 200 m from the autochthonous cases' residence contained DENV. Genetic data from the mosquito-derived DENV linked the cluster to the 2023-2024 dengue outbreak in the French Caribbean Islands. This study highlights the importance of raising mosquito-borne disease awareness among healthcare professionals. It demonstrates Ae. albopictus as a DENV vector in mainland France and the value of private mosquito traps for entomo-virological surveillance.

Re-Emergence of DENV-3 in French Guiana: Retrospective Analysis of Cases That Circulated in the French Territories of the Americas from the 2000s to the 2023-2024 Outbreak.

French Guiana experienced an unprecedented dengue epidemic during 2023-2024. Prior to the 2023-2024 outbreak in French Guiana, DENV-3 had not circulated in an epidemic manner since 2005. We therefore studied retrospectively the strains circulating in the French Territories of the Americas (FTA)-French Guiana, Guadeloupe, and Martinique-from the 2000s to the current epidemic. To this end, DENV-3 samples from the collection of the National Reference Center for Arboviruses in French Guiana (NRCA-FG) were selected and sequenced using next-generation sequencing (NGS) based on Oxford Nanopore Technologies, ONT. Phylogenetic analysis showed that (i) the 97 FTA sequences obtained all belonged to genotype III (GIII); (ii) between the 2000s and 2013, the regional circulation of the GIII American-I lineage was the source of the FTA cases through local extinctions and re-introductions; (iii) multiple introductions of lineages of Asian origin appear to be the source of the 2019-2021 epidemic in Martinique and the 2023-2024 epidemic in French Guiana. Genomic surveillance is a key factor in identifying circulating DENV genotypes, monitoring strain evolution, and identifying import events.

Predominance of Dengue Virus Serotype-1/Genotype-I in Eastern and Southeastern Ethiopia.

We determined the dengue virus (DENV) serotypes and genotypes in archived serum samples that were collected during the 2014-2016 and 2021 dengue outbreaks in Dire Dawa City and the Somali region in Ethiopia. DENV serotype 1 (DENV-1) was predominant followed by DENV serotype 2 (DENV-2). Thirteen of the DENV-1 strains were assigned to Genotype-I, while the remaining two were found to be Genotype-III. All three DENV-2 strains were assigned the Cosmopolitan Genotype. The DENV strains responsible for the outbreaks are genetically closely related to the DENV strains that circulated in neighboring and Asian countries. The findings also showed continued local transmission of a monophyletic lineage and a co-circulation of DENV-1 and DENV-2 during the outbreaks. There is a need to strengthen DENV genomic surveillance capacity for the early detection of circulating serotypes, and prevent devastating consequences of future outbreaks due to the co-circulation of different serotypes.

Detection of Dengue Virus 1 and Mammalian Orthoreovirus 3, with Novel Reassortments, in a South African Family Returning from Thailand, 2017.

In July 2017, a family of three members, a 46-year-old male, a 45-year-old female and their 8-year-old daughter, returned to South Africa from Thailand. They presented symptoms consistent with mosquito-borne diseases, including fever, headache, severe body aches and nausea. Mosquito bites in all family members suggested recent exposure to arthropod-borne viruses. Dengue virus 1 (Genus Orthoflavivirus) was isolated (isolate no. SA397) from the serum of the 45-year-old female via intracerebral injection in neonatal mice and subsequent passage in VeroE6 cells. Phylogenetic analysis of this strain indicated close genetic identity with cosmopolitan genotype 1 DENV1 strains from Southeast Asia, assigned to major lineage K, minor lineage 1 (DENV1I_K.1), such as GZ8H (99.92%) collected in November 2018 from China, and DV1I-TM19-74 isolate (99.72%) identified in Bangkok, Thailand, in 2019. Serum samples from the 46-year-old male yielded a virus isolate that could not be confirmed as DENV1, prompting unbiased metagenomic sequencing for virus identification and characterization. Illumina sequencing identified multiple segments of a mammalian orthoreovirus (MRV), designated as Human/SA395/SA/2017. Genomic and phylogenetic analyses classified Human/SA395/SA/2017 as MRV-3 and assigned a tentative genotype, MRV-3d, based on the S1 segment. Genomic analyses suggested that Human/SA395/SA/2017 may have originated from reassortments of segments among swine, bat, and human MRVs. The closest identity of the viral attachment protein σ1 (S1) was related to a human isolate identified from Tahiti, French Polynesia, in 1960. This indicates ongoing circulation and co-circulation of Southeast Asian and Polynesian strains, but detailed knowledge is hampered by the limited availability of genomic surveillance. This case represents the rare concurrent detection of two distinct viruses with different transmission routes in the same family with similar clinical presentations. It highlights the complexity of diagnosing diseases with similar sequelae in travelers returning from tropical areas.

What Is Dengue?

This JAMA Patient Page describes the viral infection dengue and its signs and symptoms, diagnosis, treatment, and prevention measures.

Complete Blood Count Values Over Time in Young Children During the Dengue Virus Epidemic in the Dominican Republic From 2018 to 2020.

Background: Dengue fever (DF) is a mosquito-borne illness with substantial economic and societal impact. Understanding laboratory trends of hospitalized Dominican Republic (DR) pediatric patients could help develop screening procedures in low-resourced settings. We sought to describe laboratory findings over time in DR children with DF and DF severity from 2018 to 2020. Methods: Clinical information was obtained prospectively from recruited children with DF. Complete blood count (CBC) laboratory measures were assessed across Days 1-10 of fever. Participants were classified as DF-negative and DF-positive and grouped by severity. We assessed associations of DF severity with demographics, clinical characteristics, and peripheral blood studies. Using linear mixed-models, we assessed if hematologic values/trajectories differed by DF status/severity. Results: A total of 597 of 1101 with a DF clinical diagnosis were serologically evaluated, and 574 (471 DF-positive) met inclusion criteria. In DF, platelet count and hemoglobin were higher on earlier days of fever (p < = 0.0017). Eighty had severe DF. Severe DF risk was associated with thrombocytopenia, intraillness anemia, and leukocytosis, differing by fever day (p < = 0.001). Conclusions: In a pediatric hospitalized DR cohort, we found marked anemia in late stages of severe DF, unlike the typically seen hemoconcentration. These findings, paired with clinical symptom changes over time, may help guide risk-stratified screenings for resource-limited settings.

Utilization of novel molecular multiplex methods for the detection and, epidemiological surveillance of dengue virus serotypes and chikungunya virus in Burkina Faso, West Africa.

BACKGROUND: Dengue virus (DENV) and Chikungunya virus (CHIKV) are major arboviruses that are transmitted to humans by Aedes aegypti (A. aegypti) and Aedes Albopictus (A. Albopictus) mosquitoes. In absence of specific antivirals and vaccine against these two viruses, prompt diagnosis of acute infections and robust surveillance for outbreak identification remain crucial. Therefore, rapid, robust, high-throughput, accessible, and low-cost assays are essential for endemic countries. This study evaluated our recently developed multiplex RT-PCR and RT-qPCR assays to screen for DENV1-4 and CHIKV circulation in Burkina Faso. METHODS AND RESULTS: This study, conducted between June to August 2023, enrolled patients with suspected arbovirus infection presenting at healthcare facilities in three Burkina Faso cities (Bobo-Dioulasso, Houndé, and Ouagadougou). Serum samples were collected and screened for DENV serotypes and CHIKV using our newly multiplex RT-PCR and RT-q PCR techniques recently developed. A total of 408 patients (age median = 33, range from 3 to 84 years) participated in this study. Of these, 13.7% (56/408) had DENV infection; DENV-1 was 32.1% (18/56) and DENV-3 was 67.9% (38/56). DENV-2, DENV-4 and CHIKV were not detected. CONCLUSIONS: This study demonstrates the effectiveness of our molecular methods for DENV detection and serotyping in Burkina Faso. The affordability of our methods makes them valuable for implementing widespread routine clinical diagnostics or arbovirus surveillance in resource-limited settings.

Simultaneous detection of arboviruses by a multiplex RT-qPCR assay in Tocantins, a northern state of Brazil.

In Brazil, Dengue, Zika and Chikungunya viruses constitute a major threat to the public health system. Simultaneous circulation of these arboviruses occurs in many regions of the world due to the expansion of transmission vectors. The infection by these arboviruses triggers similar symptoms during their acute phase. However, in some cases, severe symptoms may occur, leading to different types of disabilities and even death. In this context, considering the similarity of the symptoms, the problems caused by the infection of these arboviruses, and the increasing risk of coinfection in humans, the differential diagnosis of these infections is essential for clinical management and epidemiological investigation. Thus, this study aimed to identify, through diagnosis via Quantitative Polymerase Chain Reaction with Reverse Transcription, arbovirus coinfection in patients from the Tocantins state (Northern Brazil). A total of 495 samples were analyzed, three from which were determined to be a coinfection of Dengue and Chikungunya viruses. The data obtained here indicate the co-circulation and coinfection by Dengue and Chikungunya viruses in the Tocantins state. These results highlight the importance of monitoring the circulation of these arboviruses for the development of health actions that aim their prevention and combat, as well as their clinical and therapeutic management.

Field-deployable viral diagnostic tools for dengue virus based on Cas13a and Cas12a.

The diagnosis of dengue virus (DENV) has been challenging particularly in areas far from clinical laboratories. Early diagnosis of pathogens is a prerequisite for the timely treatment and pathogen control. An ideal diagnostic for viral infections should possess high sensitivity, specificity, and flexibility. In this study, we implemented dual amplification involving Cas13a and Cas12a, enabling sensitive and visually aided diagnostics for the dengue virus. Cas13a recognized the target RNA by crRNA and formed the assembly of the Cas13a/crRNA/RNA ternary complex, engaged in collateral cleavage of nearby crRNA of Cas12a. The Cas12a/crRNA/dsDNA activator ternary complex could not be assembled due to the absence of crRNA of Cas12a. Moreover, the probe, with 5' and 3' termini labeled with FAM and biotin, could not be separated. The probes labeled with FAM and biotin, combined the Anti-FAM and the Anti-Biotin Ab-coated gold nanoparticle, and conformed sandwich structure on the T-line. The red line on the paper strip caused by clumping of AuNPs on the T-line indicated the detection of dengue virus. This technique, utilizing an activated Cas13a system cleaving the crRNA of Cas12a, triggered a cascade that amplifies the virus signal, achieving a low detection limit of 190 fM with fluorescence. Moreover, even at 1 pM, the red color on the T-line was easily visible by naked eyes. The developed strategy, incorporating cascade enzymatic amplification, exhibited good sensitivity and may serve as a field-deployable diagnostic tool for dengue virus.

Resurgence of Dengue Virus Serotype 2: Findings from the 2023 Bangladesh Outbreak.

Bangladesh experienced the largest and deadliest dengue outbreak in 2023, after the virus had reappeared in the country 2 decades earlier. A total of 1,705 people died in Bangladesh, representing the highest case fatality rate (0.5%) due to dengue in the world for that year. The severity of dengue infection is to some extent related to the emergence of new circulating serotypes. To identify the possible predominant serotype in 2023, the reverse transcription polymerase chain reaction-based identification technique was used on stored serum samples of suspected dengue patients during the period between July and December 2023. The overall result of molecular serotyping showed that dengue virus (DENV-2) reappeared as the predominant serotype (74.1%), followed by a moderate number of samples with DENV-1 (19.8%) and DENV-3 (6.1%), in 2023. However, DENV-1 was found to be dominant in a few rural areas of Cox's Bazar districts. During the 2019 outbreak, DENV-3 was the dominant serotype, which seemed to be replaced by the DENV-2 serotype; this may have impacted the increased case fatality in 2023.

Aptamer-associated colorimetric reverse transcription loop-mediated isothermal amplification assay for detection of dengue virus.

Current diagnostic methods for dengue, such as serological tests, have limitations in terms of cross-reactivity with other viruses. To address this issue, we explored the potential of combining the loop-mediated isothermal amplification (LAMP) technique with the affinity of aptamers to develop point-of-care testing. In this study, we utilized 60 serum samples. An aptamer capable of binding to the dengue virus was employed as a platform for capturing genetic material, and its performance was compared to a commercial kit. Dengue virus was detected through RT-PCR and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP), allowing visual observation of the results without the need for equipment. In the context of the aptamer LAMP assay, our analysis revealed the detection of the dengue virus in 38 out of 60 samples, with 95% sensitivity and 100% specificity compared to RT-PCR and/or APTA-RT-PCR. Importantly, we observed no cross-reaction when assessing samples positive for the zika virus, underscoring the assay's selectivity. This innovative aptameric capture of the viral RNA in combination with the RT-LAMP (APTA-RT-LAMP) method has the potential to offer valuable molecular insights into neglected infectious diseases in a simpler and faster manner. IMPORTANCE: Dengue is a neglected tropical disease of significant epidemiological importance in tropical and subtropical countries. Current diagnostics for this infection present challenges, such as cross-reactivity in serological tests. Finding ways to enhance the diagnosis of this disease is crucial, given the absence of specific treatments. An accurate, simple, and effective diagnosis contributes to the improved management of infected individuals. In this context, our work combines molecular biology techniques, such as isothermal loop amplification, with aptamers to detect the dengue virus in biological samples. Our method produces colorimetric results based on a color change, with outcomes available in less than 2 hours. Moreover, it requires simpler equipment compared to molecular PCR tests.

Neuro-Ophthalmic Dengue Infection: A Case Report with a Multiple Body Site Sampling Strategy and Review of Laboratory Data.

Dengue neurological disease is an uncommon yet severe complication of dengue infection. It can manifest as encephalitis, encephalopathy, neuro-ophthalmic complications, or neuromuscular disorders. Severe infection can result in viral shedding across multiple body sites. We describe a case of severe neuro-ophthalmic dengue infection in an otherwise healthy returned traveller, presenting with prolonged multiple-body-site viral detections by PCR. The dengue virus (DENV) dynamics and serological response support a direct DENV neuropathogenicity. A retrospective review of the laboratory data at the Victorian Infectious Diseases Reference Laboratory (VIDRL) suggests that blood is the most frequent sample type with DENV detection (92% of all DENV-positive samples). Genotype variation is seen across different sample types. The similarity of CSF and nasopharyngeal DENV subtypes (genotype 1 and 3) suggests a possible correlation between nasopharyngeal replication and neurological complications. The case presented highlights the direct neuropathogenicity of DENV early in the course of infection, and a potential correlation between nasopharyngeal replication and neurological disease.