Kathryn B. Anderson

Determinants of Inapparent and Symptomatic Dengue Infection in a Prospective Study of Primary School Children in Kamphaeng Phet, Thailand

Background Dengue viruses are a major cause of morbidity in tropical and subtropical regions of the world. Inapparent dengue is an important component of the overall burden of dengue infection. It provides a source of infection for mosquito transmission during the course of an epidemic, yet by definition is undetected by health care providers. Previous studies of inapparent or subclinical infection have reported varying ratios of symptomatic to inapparent dengue infection. Methodology/Principal Findings In a prospective study of school children in Northern Thailand, we describe the spatial and temporal variation of the symptomatic to inapparent (S:I) dengue illness ratio. Our findings indicate that there is a wide fluctuation in this ratio between and among schools in a given year and within schools over several dengue seasons. The most important determinants of this S:I ratio for a given school were the incidence of dengue infection in a given year and the incidence of infection in the preceding year. We found no association between the S:I ratio and age in our population. Conclusions/Significance Our findings point to an important aspect of virus-host interactions at either a population or individual level possibly due to an effect of heterotypic cross-reactive immunity to reduce dengue disease severity. These findings have important implications for future dengue vaccines.

A Shorter Time Interval Between First and Second Dengue Infections Is Associated With Protection From Clinical Illness in a School-based Cohort in Thailand

BACKGROUND Despite the strong association between secondary dengue virus (DENV) infections and dengue hemorrhagic fever (DHF), the majority of secondary infections are subclinical or mild. The determinants of clinical severity remain unclear, though studies indicate a titer-dependent and time-dependent role of cross-protective anti-DENV antibodies. METHODS Data from 2 sequential prospective cohort studies were analyzed for subclinical and symptomatic DENV infections in schoolchildren in Kamphaeng Phet, Thailand (1998-2002 and 2004-2007). Children experiencing ≥ 1 DENV infection were selected as the population for analysis (contributing 2169 person-years of follow-up). RESULTS In total, 1696 children had ≥ 1 DENV infection detected during their enrollment; 268 experienced 2 or more infections. A shorter time interval between infections was associated with subclinical infection in children seronegative for DENV at enrollment, for whom a second-detected DENV infection is more likely to reflect a true second infection (average of 2.6 years between infections for DHF, 1.9 for DF, and 1.6 for subclinical infections). CONCLUSIONS These findings support a pathogenesis model where cross-reactive antibodies wane from higher-titer, protective levels to lower-titer, detrimental levels. This is one of the first studies of human subjects to suggest a window of cross-protection following DENV infection since Sabins challenge studies in the 1940s.

Preexisting Japanese Encephalitis Virus Neutralizing Antibodies and Increased Symptomatic Dengue Illness in a School-Based Cohort in Thailand

Background Dengue viruses (DENVs) and Japanese encephalitis virus (JEV) have significant cross-reactivity in serological assays; the clinical implications of this remain undefined. An improved understanding of whether and how JEV immunity modulates the clinical outcome of DENV infection is important as large-scale DENV vaccine trials will commence in areas where JEV is co-endemic and/or JEV immunization is routine. Methods and Findings The association between preexisting JEV neutralizing antibodies (NAbs) and the clinical severity of DENV infection was evaluated in a prospective school-based cohort in Thailand that captured asymptomatic, non-hospitalized, and hospitalized DENV infections. Covariates considered included age, baseline DENV antibody status, school of attendance, epidemic year, and infecting DENV serotype. 942 children experienced at least one DENV infection between 1998 and 2002, out of 3,687 children who were enrolled for at least one full year. In crude analysis, the presence of JEV NAbs was associated with an increased occurrence of symptomatic versus asymptomatic infection (odds ratio [OR] = 1.55, 95% CI: 1.08–2.23) but not hospitalized illness or dengue hemorrhagic fever (DHF). The association was strongest in children with negative DENV serology (DENV-naive) (OR = 2.75, 95% CI: 1.12–6.72), for whom the presence of JEV NAbs was also associated with a symptomatic illness of longer duration (5.4 days for JEV NAb+ versus 2.6 days for JEV NAb-, p = 0.048). JEV NAbs were associated with increased DHF in younger children with multitypic DENV NAb profiles (OR = 4.05, 95% CI: 1.18 to 13.87). Among those with JEV NAbs, the association with symptomatic illness did not vary by antibody titer. Interpretation The prior existence of JEV NAbs was associated with an increased probability of symptomatic as compared to asymptomatic DENV illness. These findings are in contrast to previous studies suggesting an attenuating effect of heterologous flavivirus immunity on DENV disease severity.

Interference and Facilitation Between Dengue Serotypes in a Tetravalent Live Dengue Virus Vaccine Candidate

BACKGROUND Live, multivalent vaccines have historically exhibited interference in humans; live dengue virus (DENV) vaccines have proven no exception. METHODS To characterize interactions between DENV serotypes in a tetravalent live-attenuated virus vaccine candidate, we analyzed data from a factorial clinical trial in which all combinations of high- and low-dose DENV serotypes were combined in 16 live-attenuated tetravalent vaccine formulations (N = 64) and administered to flavivirus-naive adult volunteers. Regression models considered the outcomes of reactogenicity and seroconversion, controlling for all serotype doses simultaneously. Additionally, results were compared against earlier evaluations of the same viruses administered as monovalent formulations. RESULTS DENV-1 was immunologically dominant in both monovalent and tetravalent formulations. In tetravalent formulations, DENV-1 and DENV-2 antagonized each other, with a high dose of one decreasing seroconversion to the other. However, high-dose DENV-1 significantly increased seroconversion against 3 or more serotypes, increasing seroconversion to DENV-1, DENV-3, and DENV-4. The highest reactogenicity occurred when DENV-1 was at high dose and all others were low; reactogenicity decreased with the incorporation of other high-dose serotypes. CONCLUSIONS Interference and facilitation occurred between serotypes in the live vaccine candidate evaluated. These analyses suggest that it may be possible to exploit facilitation to increase overall seroconversion.

The Emergence of Zika Virus: A Narrative Review

Key Summary Points Zika virus (ZIKV) was identified in 1947 and for decades caused only sporadic cases of mild human disease. The explosive nature of recent epidemics and links to GuillainBarr syndrome and microcephaly are concerning and remain poorly understood. There is evidence that maternal ZIKV infection at any stage of pregnancy may result in an increased risk for microcephaly, intrauterine growth restriction, and fetal death. The contribution of ZIKV infection to the total increase in microcephaly cases being observed, relative to other potential (unidentified) causes, remains unknown. There is evidence of sexual transmission of ZIKV by men, with virus detectable in semen by reverse transcriptase polymerase chain reaction for at least 2 months after infection. The relative importance of sexual transmission with regard to the overall burden of ZIKV transmission and risk for microcephaly is unknown. At present, no specific antiviral or vaccine is available for ZIKV, although vaccines are in development. Treatment is supportive. Pregnant women in unaffected areas are currently advised to postpone travel to ZIKV-endemic regions if possible and to avoid sex with male partners who have traveled to endemic regions. Providers should maintain a high level of suspicion for ZIKV infection in any patient presenting with rash and either a personal history of recent travel to an area with active ZIKV transmission or a history of travel in a sexual partner. Zika virus (ZIKV) was first isolated in 1947 from a sentinel rhesus monkey in the Zika forest near Entebbe, Uganda (1). It was one of many viruses discovered via the Rockefeller Foundations program on yellow fever in Africa and South America (2). In 1948, ZIKV was again isolated from the Zika forest, from Aedes africanus mosquitos (1). Serologic studies in people living near the Zika forest showed an estimated ZIKV seroprevalence of 6% (3). The first isolation of ZIKV from humans arose during investigation of an epidemic of jaundice in eastern Nigeria in 1952, when virus was isolated from a (nonjaundiced) child presenting with fever and headache (4). These symptoms were reproduced during experimental inoculation of a human volunteer in 1956, which resulted in a mild, self-limited febrile illness (5). The virus was next isolated in Uganda in 1962, from an individual with maculopapular rash, fever, and mild body pain (6). In the decade following initial discovery of the virus, sporadic febrile illnesses with seroconversion patterns suggestive of ZIKV infection were identified throughout Africa and Asia (711). Estimated seroprevalence was highest in Nigeria, at 48% to 56% (12, 13). Recent Epidemics The first recorded ZIKV epidemic occurred in 2007 on Yap Island, a small Micronesian island with approximately 10000 inhabitants. Physicians noted an outbreak of a mild dengue-like illness characterized by rash, fever, arthralgia, arthritis, and conjunctivitis (14). ZIKV was identified in 14% of serum samples from patients with acute illness. A limited serosurvey performed on the island found that 73% of Yap inhabitants older than 3 years of age had demonstrable IgM antibodies to ZIKV, indicating recent infection. Only 19% of these individuals reported a history of clinical illness consistent with suspected ZIKV infection, suggesting an asymptomatic-to-symptomatic ratio of 4 to 1. A second major ZIKV epidemic occurred in 2013 in French Polynesia, a chain of 67 islands with approximately 270000 inhabitants (15). Expanded surveillance led to an estimate of 28000 suspected cases during the epidemic, the largest on record at that time. This epidemic was also associated with the novel report of a case of GuillainBarr syndrome (GBS) that developed 1 week after a ZIKV-like illness (16). At the time, there were concurrent epidemics of dengue virus serotypes 1 and 3 and chikungunya in the Pacific Islands (17). Scientists postulated that a reported 20-fold increase in GBS cases in French Polynesia may have been linked to dengue virus infection, as had been reported previously (18); ZIKV infection; or some pathologic mechanism related to sequential infection with the 2 viruses (16). After the outbreak in French Polynesia, outbreaks of ZIKV occurred in New Caledonia, the Cook Islands, and Easter Island (19, 20). The largest ZIKV outbreak to date, by sheer numbers and geographic range, began in northeastern Brazil in May 2015 and is ongoing (21, 22). By October 2015, 14 states in Brazil and the department of Bolivar, Colombia, had confirmed autochthonous ZIKV transmission (23). By January 2016, the virus had spread to 20 countries or territories in the Americas and the Caribbean; regions with the highest burden of ZIKV transmission in Brazil, most notably Pernambuco State, were also reporting an increase in the number of infants born with microcephaly (24, 25). On 1 February 2016, the World Health Organization declared the reported clusters of microcephaly and other neurologic disorders a public health emergency of international concern (26), the fourth of its kind following swine flu (2009), the threatened resurgence of polio (2014), and Ebola (2014). As of March 2016, the epidemic had spread to 33 countries or territories, with transmission increasing in most countries (27). Early estimates from Brazil suggested a 20-fold increase in the number of microcephaly cases compared with previous years (25). Estimation of true differences in incidence by affected region and by year has been complicated by increased sensitivity of the current surveillance system at the cost of decreased specificity (28) and likely increased awareness and reporting; however, there appears to be a true increase in the occurrence of microcephaly in several countries in the Americas. Clear evidence now indicates that maternal ZIKV infection can cause fetal microcephaly. The virus has been isolated from the amniotic fluid of 2 pregnant women who had infants with microcephaly (29) and has been identified in brain tissue from 5 fetuses with microcephaly during autopsy (30, 31). All mothers reported a history of febrile illness with rash during their first trimester. It remains unclear whether other unidentified factors underlie the epidemic of microcephaly, including other known causes of microcephaly and possible surveillance artifacts. Seven countries in the Americas and the Caribbean have reported increases in the incidence of GBS concurrent with a rise in the number of ZIKV infections (27). In Brazil, a 19% increase in the incidence of GBS was reported for 2015 compared with previous years (32). Sixty-two percent of patients with GBS in Brazil reported a history of symptoms consistent with suspected ZIKV infection, and 7 patients with neurologic disease (including GBS) had laboratory-confirmed ZIKV infection. A retrospective study of GBS cases during the French Polynesia outbreak demonstrated that 100% of patients with GBS had antibodies to ZIKV compared with 56% of controls (33). To date, Europe, continental North America, and Australia have not reported mosquito-borne transmission of ZIKV (34). Transmission has been documented in Puerto Rico and the U.S. Virgin Islands and is expected to increase (35). Sexual transmission has been reported in the United States, Argentina, and Chile (27, 36, 37). Numerous imported cases of ZIKV infection in travelers returning from tropical countries to more temperate regions have also been reported, including 116 cases in the United States (38). Importation of ZIKV to susceptible countries across the globe raises concern for virus establishment in areas endemic for suitable mosquito vectors. A recent model that incorporated ecologic niche data for A aegypti and A albopictus, as well as regional travel patterns throughout the Americas, indicated that 60% of the U.S. population lives in areas conducive to at least seasonal, possibly year-round, ZIKV transmission (Figure 1) (39). Figure 1. Predicted risk for local Zika transmission, based on final destinations for travelers departing Brazil and incorporating ecological niche information for Aedes aegypti and A albopictus. Reproduced with permission from reference 39. The Zika virus is closely related to the Spondweni virus. There is a single serotype of ZIKV, and infection is thought to lead to lifelong immunity. The 2 lineages, Asian and African, are derived from a common ancestor in Uganda (40, 41). All 3 recent epidemics have been linked to expansion of the Asian lineage of ZIKV (Figure 2) (22, 41). It is not known whether strains of ZIKV are associated with an increased risk for neurologic disease or whether mutations have facilitated the explosive spread of ZIKV in recent years. Figure 2. Lineages of Zika virus isolates based on envelope gene sequences. Reproduced with permission from reference 41. Transmission The Zika virus is spread through the bite of mosquitoes, and multiple mosquito species are capable vectors. The virus was first isolated from A africanus in Uganda (1, 42). Aedes hensilli was the primary vector in the Yap epidemic (43), and A aegypti is likely a dominant vector in Asia and the Americas (4446). Multiple species are suggested as possible vectors, including a long list of Aedes species, Mansonia uniformis, Culex perfuscis, and Anopheles coustani (47, 48). Besides nonhuman primates and humans, ZIKV has been isolated from small mammals, reptiles, birds, and livestock (49). The previously sporadic occurrence of human infections suggests that humans were once accidental hosts in a sylvatic transmission cycle involving primates, possibly other animals, and forest mosquitos. Recent outbreaks indicate that the evolving epidemiology of ZIKV may now more closely mirror that of yellow fever virus, involving a sylvatic cycle as well as an efficient urban cycle involving human hosts and A aegypti mosquitoes. There is evidence of sexual transmission of ZIKV. In the first report in 2008, a U.S. scientist working in S

Factors Influencing Dengue Virus Isolation by C6/36 Cell Culture and Mosquito Inoculation of Nested PCR-Positive Clinical Samples

Dengue viral isolation is necessary for definitive diagnosis, pathogenesis and evolutionary research, vaccine candidates, and diagnostic materials. Using standardized techniques, we analyzed isolation rates of 1,544 randomly selected polymerase chain reaction (PCR)-positive samples, representing all four dengue serotypes, from patients with serologically confirmed dengue infections and evaluated whether clinical and laboratory results could be predictive of isolation using standard and mosquito isolation techniques. Viruses were isolated from 62.5% of the samples by direct application to C6/36 cells and increased to 79.4% when amplifying C6/36 negative samples by intrathorasic inoculation in Toxyrhynchites splendens mosquitoes. High viremia, measured by reverse transcriptase (RT)-PCR, was a strong predictor for viral isolation by either method. Isolation was most successful in samples collected early in the disease, had low antibody levels, temperatures greater than 38°C, and had a final clinical diagnosis of dengue fever. Dengue serotypes also played a role in the success of viral isolation.

Chikungunya: acute fever, rash and debilitating arthralgias in a returning traveler from Haiti.

The following case report details a case of chikungunya fever in a returning traveler from Haiti. The report highlights the clinical presentation and natural history of the disease, and emphasizes that chikungunya has become established in the western hemisphere, with a resultant need for heightened provider awareness.

The dynamic role of dengue cross-reactive immunity: changing the approach to defining vaccine safety and efficacy

Dengue virus infections cause a substantial public health burden in tropical and subtropical regions. A single dengue vaccine has been approved by regulatory authorities in 19 countries, but concerns regarding vaccine safety in people who are dengue naive at the time of immunisation has introduced uncertainty into the vaccines future. As other dengue vaccines complete or enter large-scale efficacy trials, we argue that foundational work by Sabin, historic epidemiological observations of dengue outbreaks, and prospective cohort studies in Asia and the Americas indicate that modifications must be made to the methods of assessing dengue vaccines. In this Personal View, we review and relate previous data that supports a dynamic role of cross-protective dengue immunity to the goals and challenges of measuring and interpreting dengue vaccine immunogenicity, efficacy, and safety in clinical trials. We suggest that for partly protective vaccines, temporary cross-protective immunity could lead to overestimation of vaccine safety and efficacy in the early years following vaccination. We recommend that assessment of dengue vaccines should span several years, involve active surveillance to clinically characterise incident infections and regular blood draws to define kinetic changes in immunological profiles, and include sample sizes that are large enough to support detailed analyses of vaccine trial subgroups, such as individuals who are dengue naive.

Model-based assessment of public health impact and cost-effectiveness of dengue vaccination following screening for prior exposure

The tetravalent dengue vaccine CYD-TDV (Dengvaxia®) is the first licensed vaccine against dengue, but recent findings indicate an elevated risk of severe disease among vaccinees without prior dengue virus (DENV) exposure. The World Health Organization currently recommends CYD-TDV only for individuals with serological confirmation of past DENV exposure. Our objective was to evaluate the potential health impact and cost-effectiveness of vaccination following serological screening. To do so, we used an agent-based model to simulate DENV transmission with and without vaccination over a 10-year timeframe. Across a range of values for the proportion of vaccinees with prior DENV exposure, we projected the proportion of symptomatic and hospitalized cases averted as a function of the sensitivity and specificity of serological screening. Scenarios about the cost-effectiveness of screening and vaccination were chosen to be representative of Brazil and the Philippines. We found that public health impact depended primarily on sensitivity in high-transmission settings and on specificity in low-transmission settings. Cost-effectiveness could be achievable from the perspective of a public payer provided that sensitivity and the value of a disability-adjusted life-year were both high, but only in high-transmission settings. Requirements for reducing relative risk and achieving cost-effectiveness from an individual perspective were more restricted, due to the fact that those who test negative pay for screening but receive no benefit. Our results predict that cost-effectiveness could be achieved only in high-transmission areas of dengue-endemic countries with a relatively high per capita GDP, such as Panamá (13,680 USD), Brazil (8,649 USD), México (8,201 USD), or Thailand (5,807 USD). In conclusion, vaccination with CYD-TDV following serological screening could have a positive impact in some high-transmission settings, provided that screening is highly specific (to minimize individual harm), at least moderately sensitive (to maximize population benefit), and sufficiently inexpensive (depending on the setting). AUTHOR SUMMARY Among several viral diseases transmitted by Aedes aegypti mosquitoes, dengue imposes the greatest and most persistent burden on global health. Efforts to curb its spread would benefit greatly from the availability of an effective vaccine. Currently, the only licensed dengue vaccine, known as CYD-TDV or by the brand name Dengvaxia®, is only recommended for use in people who are known to have been exposed to dengue virus in the past. Because symptoms of dengue can range from severe to mild to imperceptible, using clinical history alone to assess whether a person was previously exposed is unreliable. Instead, serological assays, which measure a person’s immune response to dengue virus, are necessary to confirm whether a person was previously exposed. Because serological assays can be subject to substantial error, we used a simulation model to assess how impactful CYD-TDV vaccination would be under different scenarios about the accuracy of a serological assay and the intensity of transmission in a given area. We found that the health impact and cost-effectiveness of CYD-TDV vaccination depended on the accuracy of the serological assay, its cost, and the setting in which it is deployed.

Clinical and laboratory predictors of influenza infection among individuals with influenza-like illness presenting to an urban Thai hospital over a five-year period

Early diagnosis of influenza infection maximizes the effectiveness of antiviral medicines. Here, we assess the ability for clinical characteristics and rapid influenza tests to predict PCR-confirmed influenza infection in a sentinel, cross-sectional study for influenza-like illness (ILI) in Thailand. Participants meeting criteria for acute ILI (fever > 38°C and cough or sore throat) were recruited from inpatient and outpatient departments in Bangkok, Thailand, from 2009–2014. The primary endpoint for the study was the occurrence of virologically-confirmed influenza infection (based upon detection of viral RNA by RT-PCR) among individuals presenting for care with ILI. Nasal and throat swabs were tested by rapid influenza test (QuickVue) and by RT-PCR. Vaccine effectiveness (VE) was calculated using the case test-negative method. Classification and Regression Tree (CART) analysis was used to predict influenza RT-PCR positivity based upon symptoms reported. We enrolled 4572 individuals with ILI; 32.7% had detectable influenza RNA by RT-PCR. Influenza cases were attributable to influenza B (38.6%), A(H1N1)pdm09 (35.1%), and A(H3N2) (26.3%) viruses. VE was highest against influenza A(H1N1)pdm09 virus and among adults. The most important symptoms for predicting influenza PCR-positivity among patients with ILI were cough, runny nose, chills, and body aches. The accuracy of the CART predictive model was 72.8%, with an NPV of 78.1% and a PPV of 59.7%. During epidemic periods, PPV improved to 68.5%. The PPV of the QuickVue assay relative to RT-PCR was 93.0% overall, with peak performance during epidemic periods and in the absence of oseltamivir treatment. Clinical criteria demonstrated poor predictive capability outside of epidemic periods while rapid tests were reasonably accurate and may provide an acceptable alternative to RT-PCR testing in resource-limited areas.