Nguyet Minh Nguyen

Host and viral features of human dengue cases shape the population of infected and infectious Aedes aegypti mosquitoes

Dengue is the most prevalent arboviral disease of humans. The host and virus variables associated with dengue virus (DENV) transmission from symptomatic dengue cases (n = 208) to Aedes aegypti mosquitoes during 407 independent exposure events was defined. The 50% mosquito infectious dose for each of DENV-1–4 ranged from 6.29 to 7.52 log10 RNA copies/mL of plasma. Increasing day of illness, declining viremia, and rising antibody titers were independently associated with reduced risk of DENV transmission. High early DENV plasma viremia levels in patients were a marker of the duration of human infectiousness, and blood meals containing high concentrations of DENV were positively associated with the prevalence of infectious mosquitoes 14 d after blood feeding. Ambulatory dengue cases had lower viremia levels compared with hospitalized dengue cases but nonetheless at levels predicted to be infectious to mosquitoes. These data define serotype-specific viremia levels that vaccines or drugs must inhibit to prevent DENV transmission.

A Randomized, Double-Blind Placebo Controlled Trial of Balapiravir, a Polymerase Inhibitor, in Adult Dengue Patients

Background. Dengue is the most common arboviral infection of humans. There are currently no specific treatments for dengue. Balapiravir is a prodrug of a nucleoside analogue (called R1479) and an inhibitor of hepatitis C virus replication in vivo. Methods. We conducted in vitro experiments to determine the potency of balapiravir against dengue viruses and then an exploratory, dose-escalating, randomized placebo-controlled trial in adult male patients with dengue with <48 hours of fever. Results. The clinical and laboratory adverse event profile in patients receiving balapiravir at doses of 1500 mg (n = 10) or 3000 mg (n = 22) orally for 5 days was similar to that of patients receiving placebo (n = 32), indicating balapiravir was well tolerated. However, twice daily assessment of viremia and daily assessment of NS1 antigenemia indicated balapiravir did not measurably alter the kinetics of these virological markers, nor did it reduce the fever clearance time. The kinetics of plasma cytokine concentrations and the whole blood transcriptional profile were also not attenuated by balapiravir treatment. Conclusions. Although this trial, the first of its kind in dengue, does not support balapiravir as a candidate drug, it does establish a framework for antiviral treatment trials in dengue and provides the field with a clinically evaluated benchmark molecule. Clinical Trials Registration. NCT01096576.

Tracking Dengue Virus Intra-host Genetic Diversity during Human-to-Mosquito Transmission.

Dengue virus (DENV) infection of an individual human or mosquito host produces a dynamic population of closely-related sequences. This intra-host genetic diversity is thought to offer an advantage for arboviruses to adapt as they cycle between two very different host species, but it remains poorly characterized. To track changes in viral intra-host genetic diversity during horizontal transmission, we infected Aedes aegypti mosquitoes by allowing them to feed on DENV2-infected patients. We then performed whole-genome deep-sequencing of human- and matched mosquito-derived DENV samples on the Illumina platform and used a sensitive variant-caller to detect single nucleotide variants (SNVs) within each sample. >90% of SNVs were lost upon transition from human to mosquito, as well as from mosquito abdomen to salivary glands. Levels of viral diversity were maintained, however, by the regeneration of new SNVs at each stage of transmission. We further show that SNVs maintained across transmission stages were transmitted as a unit of two at maximum, suggesting the presence of numerous variant genomes carrying only one or two SNVs each. We also present evidence for differences in selection pressures between human and mosquito hosts, particularly on the structural and NS1 genes. This analysis provides insights into how population drops during transmission shape RNA virus genetic diversity, has direct implications for virus evolution, and illustrates the value of high-coverage, whole-genome next-generation sequencing for understanding viral intra-host genetic diversity.

Comparative Susceptibility of Aedes albopictus and Aedes aegypti to Dengue Virus Infection After Feeding on Blood of Viremic Humans: Implications for Public Health

Aedes albopictus is secondary to Aedes aegypti as a vector of dengue viruses (DENVs) in settings of endemicity, but it plays an important role in areas of dengue emergence. This study compared the susceptibility of these 2 species to DENV infection by performing 232 direct blood-feeding experiments on 118 viremic patients with dengue in Vietnam. Field-derived A. albopictus acquired DENV infections as readily as A. aegypti after blood feeding. Once infected, A. albopictus permitted higher concentrations of DENV RNA to accumulate in abdominal tissues, compared with A. aegypti. However, the odds of A. albopictus having infectious saliva were lower than the odds observed for A. aegypti (odds ratio, 0.70; 95% confidence interval, .52–.93). These results quantitate the susceptibility of A. albopictus to DENV infection and will assist parameterization of models for predicting disease risk in settings where A. albopictus is present.

Epidemiology and Virology of Acute Respiratory Infections During the First Year of Life: A Birth Cohort Study in Vietnam

Background: Understanding viral etiology and age-specific incidence of acute respiratory infections in infants can help identify risk groups and inform vaccine delivery, but community-based data is lacking from tropical settings. Methods: One thousand four hundred and seventy-eight infants in urban Ho Chi Minh City and 981 infants in a semi-rural district in southern Vietnam were enrolled at birth and followed to 1 year of age. Acute respiratory infection (ARI) episodes were identified through clinic-based illness surveillance, hospital admissions and self-reports. Nasopharyngeal swabs were collected from infants with respiratory symptoms and tested for 14 respiratory pathogens using multiplex reverse transcription-polymerase chain reaction. Results: Estimated incidence of ARI was 542 and 2691 per 1000 infant-years, and hospitalization rates for ARI were 81 and 138 per 1000 infant-years, in urban and semi-rural cohorts, respectively, from clinic- and hospital-based surveillance. However self-reported ARI episodes were just 1.5-fold higher in the semi-rural versus urban cohort, indicating that part of the urban–rural difference was explained by under-ascertainment in the urban cohort. Incidence was higher in infants ≥6 months of age than <6 months, but this was pathogen-specific. One or more viruses were detected in 53% (urban) and 64% (semi-rural) of samples from outpatients with ARI and in 78% and 66% of samples from hospitalized ARI patients, respectively. The most frequently detected viruses were rhinovirus, respiratory syncytial virus, influenza virus A and bocavirus. ARI-associated hospitalizations were associated with longer stays and more frequent ICU admission than other infections. Conclusions: ARI is a significant cause of morbidity in Vietnamese infants and influenza virus A is an under-appreciated cause of vaccine-preventable disease and hospitalizations in this tropical setting. Public health strategies to reduce infant ARI incidence and hospitalization rates are needed.

Field- and clinically derived estimates of Wolbachia-mediated blocking of dengue virus transmission potential in Aedes aegypti mosquitoes.

Significance In laboratory experiments, Wolbachia (wMel strain)-infected Aedes aegypti are refractory to disseminated arboviral infections. Yet previous characterizations of wMel-mediated blocking have not considered several biologically and ecologically important factors likely to influence the virus–mosquito interaction. After direct feeding on 141 viremic dengue patients, we demonstrate wMel lowers dengue virus (DENV) transmission potential and lengthens the extrinsic incubation period. Subsequently, using established field populations of wild-type and wMel-infected Ae. aegypti, we compared field- versus laboratory-rearing conditions on mosquito susceptibility to disseminated DENV infection. The magnitude of wMel-mediated virus blocking was even greater when mosquitoes developed under field conditions. These clinically and ecologically relevant findings support Wolbachia introgression into Ae. aegypti populations as a biocontrol method to reduce the transmission of DENV and other arboviruses. The wMel strain of Wolbachia can reduce the permissiveness of Aedes aegypti mosquitoes to disseminated arboviral infections. Here, we report that wMel-infected Ae. aegypti (Ho Chi Minh City background), when directly blood-fed on 141 viremic dengue patients, have lower dengue virus (DENV) transmission potential and have a longer extrinsic incubation period than their wild-type counterparts. The wMel-infected mosquitoes that are field-reared have even greater relative resistance to DENV infection when fed on patient-derived viremic blood meals. This is explained by an increased susceptibility of field-reared wild-type mosquitoes to infection than laboratory-reared counterparts. Collectively, these field- and clinically relevant findings support the continued careful field-testing of wMel introgression for the biocontrol of Ae. aegypti-born arboviruses.

Naturally-Acquired Dengue Virus Infections Do Not Reduce Short-Term Survival of Infected Aedes aegypti from Ho Chi Minh City, Vietnam

Transmission of dengue virus (DENV) from mosquito to human is dependent upon the survival of the mosquito beyond the virus extrinsic incubation period. Previous studies report conflicting results of the effects of DENV on Aedes aegypti survival. Here, we describe the effect of DENV on the short-term survival (up to 12 d) of 4,321 Ae. aegypti mosquitoes blood-fed on 150 NS1-positive dengue patients hospitalized in the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam. Mosquito survival was not different between cohorts that fed upon blood from which 0% of mosquitoes became DENV infected (N = 88 feeds), or 100% became infected (N = 116 feeds). Subgroup analysis also did not reveal serotype-dependent differences in survival, nor a relationship between survival and human plasma viremia levels. These results suggest that DENV infection adds minimal cost to Ae. aegypti, an important finding when parameterizing the vector competence of this mosquito.

Physicians, Primary Caregivers and Topical Repellent: All Under-Utilised Resources in Stopping Dengue Virus Transmission in Affected Households

Background Primary health care facilities frequently manage dengue cases on an ambulatory basis for the duration of the patient’s illness. There is a great opportunity for specific messaging, aimed to reduce dengue virus (DENV) transmission in and around the home, to be directly targeted toward this high-risk ambulatory patient group, as part of an integrated approach to dengue management. The extent however, to which physicians understand, and can themselves effectively communicate strategies to stop focal DENV transmission around an ambulatory dengue case is unknown; the matter of patient comprehension and recollection then ensues. In addition, the effectiveness of N,N-diethyl-3-methylbenzamide (DEET)-based insect repellent in protecting dengue patients from Aedes aegypti mosquitoes’ bites has not been investigated. Methodology A knowledge, attitude and practice (KAP) survey, focusing on the mechanisms of DENV transmission and prevention, was performed using semi-structured questionnaires. This survey was targeted towards the patients and family members providing supportive care, and physicians routinely involved in dengue patient management in Southern Vietnam. An additional clinical observational study was conducted to measure the efficacy of a widely-used 13% DEET-based insect repellent to repel Ae. aegypti mosquitoes from the forearms of dengue cases and matched healthy controls. Principal Findings Among both the physician (n = 50) and patient (n = 49) groups there were several respondents lacking a coherent understanding of DENV transmission, leading to some inappropriate attitudes and inadequate acute preventive practices in the household. The application of insect repellent to protect patients and their relatives from mosquito bites was frequently recommended by majority of physicians (78%) participating in the survey. Nevertheless, our tested topical application of 13% DEET conferred only ~1hr median protection time from Ae. aegypti landing. This is notably shorter than that advertised on the manufacturer’s label. No differences in landing time between febrile dengue cases or matched healthy controls (n = 19 experiments) were observed. Conclusion/Significance Our study identifies missed opportunities for primary care physicians to improve public health through communication of strategies that could prevent focal dengue transmission in and around a case household. We advocate better access to more efficient communication methods for physicians and auxilliary health workers, supporting to educate those at high risk of DENV transmission. Our empirical testing of a widely-available 13% DEET-based repellent was limited in its protective efficacy against Ae. aegypti mosquito bites, and therefore DENV transmission, suggesting more frequent application is necessary to be beneficial.

A pregnant woman with acute cardiorespiratory failure: dengue myocarditis

In April, 2014, a 29-year-old Vietnamese woman, 31 weeks into her fourth pregnancy, presented to a clinic in Ho Chi Minh City with a 2 day history of fever, dry cough, chest pain, and shortness of breath. She had a history of partial thyroidectomy for hyperthyroidism in 2012, and had had three miscarriages. She took 150 μg thyroxine daily and was a non-smoker. She was initially treated for presumed pneumonia with imipenem. However, her respiratory function deteriorated overnight and she was transferred to the intensive care unit (ICU) at our hospital. On admission she was febrile (38°C), tachycardic (pulse 115 beats per min), and tachypnoeic (respiratory rate 28 breaths per min), with an SpO2 of 94% on 5 L oxygen. Heart sounds were normal but she had bibasal crackles on chest auscultation. Apart from a gravid uterus, abdominal examination was normal. Chest radiograph showed bilateral infi ltrates suggesting pulmonary oedema (appendix). Haemoglobin was 92 g/L, haematocrit 29·2%, total white cell count 8·5 × 109/L, (neutrophil count 7·53 × 109/L and lymphocyte count 0·4 × 10/L), with a platelet count of 134 × 109/L; INR was 1. Urea, electrolytes, liver transaminases, and procalcitonin were normal. Arterial blood gas analysis showed pH 7·49, PaO2 69 mm Hg, PaCO2 30·9 mm Hg, HCO3 23·7 mmol/L, and lactate 1·2 mmol/L. We started oseltamivir and continued imipenem and maintenance fl uids. Her respiratory function deteriorated further (respiratory rate 35 breaths per min, SpO2 85% on 15 L oxygen) so we started non-invasive ventilation with bilevel positive airway pressure (BiPAP) and added vancomycin and azithromycin. ECG showed inverted T waves in the inferior leads (appendix), and troponin I was raised (1·1 μg/L; normal <0·3 μg/L) peaking at 1·6 μg/L the next day, with pro-brain natriuretic peptide 1913 ng/L (normal ≤125 ng/L). Portable ultrasonography showed small bilateral pleural eff usions, but echocardiography was not available. NS1 dengue rapid test was positive and we decided to deliver the baby that day (illness day 4) in view of the likelihood of worsening thrombocytopenia and coagulopathy during the critical period of dengue. A baby girl weighing 2·22 kg was delivered by emergency caesarean section; she needed only supportive care until discharge from the neonatal unit 4 weeks later. The mother, who had been intubated during surgery, returned to ICU and needed a further 24 h of BiPAP. Her platelet count reached a nadir of 25× 109/L on illness day 6 but she had no signifi cant bleeding or shock. She had IgM seroconversion and real-time PCR confi rmed DENV-4. Throat swab was negative for infl uenza A and infl uenza B. Bronchoalveolar lavage and blood cultures were negative. Thyroid function was normal and autoimmune screen was negative. She was well enough to be discharged 8 days after admission, with a diagnosis of acute cardiac failure due to dengue myocarditis. At followup 4 weeks later, she had a normal ECG (appendix) and echocardiogram. Dengue is one of the fastest spreading viral infections and 2·5 billion people now live in endemic areas. Cardiac involvement in dengue can range from myocardial impairment and bradyarrhythmias to fulminant myocarditis. Dengue myocarditis can present at any time during the illness, unlike other severe manifestations that present during the critical phase around defervescence. Cardiac eff ects have rarely been reported in pregnant women with dengue, which might be due to underreporting because of limited diagnostic methods in endemic areas, or misdiagnosis. Dengue is associated with poor maternal and fetal outcomes, but the contribution of cardiac morbidity has not been defi ned. Pregnancy-related plasma volume expansion and raised capillary hydrostatic pressure could exacerbate the cardiovascular complications of dengue. As dengue incidence continues to increase, particularly in young adults, complications in pregnancy are likely to rise. This case highlights the need to consider dengue in patients living in or travelling from endemic areas, presenting with cardiopulmonary failure, particularly in pregnancy.