The impact of large-scale deployment of Wolbachia mosquitoes on arboviral disease incidence in Rio de Janeiro and Niterói, Brazil: study protocol for a controlled interrupted time series analysis using routine disease surveillance data

Abbreviations

BG trap: BG-Sentinel trap; IBGE: Instituto Brasileiro de Geografia e Estatística (Brazilian Institute of Geography and Statistics); ITS: interrupted time series; MoH: Ministry of Health; PAHO: Pan American Health Organisation; PCR: polymerase chain reaction; qPCR: quantitative polymerase chain reaction; RCT: randomised controlled trial; SINAN: Sistema De Informação De Agravos De Notificação (Brazilian National Notifiable Diseases Information System); WHO: World Health Organisation

Background

The global incidence of dengue has increased dramatically in recent decades. Although cases are underreported, it is estimated that 390 million dengue virus infections occur every year, and of these 96 million have clinical manifestations of dengue or severe dengue. Globally, 3.9 billion people in 128 countries are at risk of infection¹. The primary vector of dengue is the Aedes aegypti mosquito, which is also capable of transmitting other arboviruses including chikungunya, Zika, yellow fever and Mayaro².

The first reported dengue outbreak in Brazil was in 1845, with subsequent outbreaks in 1880–1912 and 1916–1923². As a result of a coordinated effort from the Pan American Health Organization (PAHO) and the World Health Organization (WHO) to eradicate Ae. aegypti, Brazil was considered free of the mosquito in 1955, but the vector was reintroduced into the country two decades later³. In 1986, dengue virus serotype 1 (DENV1) was introduced to Rio de Janeiro and an estimated 1 million people were infected^3,4. Since then, dengue has become a major public health problem. From 1986 to 1993, outbreaks occurred approximately every 2–5 years, and from 1993 dengue became endemic with seasonal peaks in cases during the rainy season (December to May), but with ongoing transmission throughout the year^2,3. Between 2000 and 2007, more than 3 million dengue cases were reported (caused by DENV serotypes 1, 2 and 3) and in 2010, DENV4 re-emerged after 28 years of absence².

The recent introduction of Zika and chikungunya in dengue hyperendemic areas of Brazil has aggravated the situation. The overlapping clinical features, absence of serological assays for the Zika virus that can reliably distinguish between acute disease and past exposure, and the association of pregnancy-associated Zika virus infection with microcephaly and other neurologic complications represents a great challenge for public health that will require new strategies and innovations^5,6.

Between 2016 and 2017, 762 deaths were attributed to severe dengue in Brazil⁷. Additionally, in 2017, 127 deaths were confirmed to be caused by chikungunya. Yellow fever has spread from the North of Brazil to the Southeast over the last years, affecting humans and non-human primates. From July 2017 to April 2018, 1,266 cases of yellow fever including 415 deaths were confirmed in Brazil, with 223 cases and 73 deaths occurring in Rio de Janeiro State⁸. No autochthonous cases were reported in Rio de Janeiro city or Niterói, although some residents from those cities acquired yellow fever while traveling to other places in Brazil. A mass vaccination campaign against yellow fever began in 2016.

The costs of vector control, direct medical costs, and indirect costs related to dengue, Zika and chikungunya in Brazil were estimated to be 2.3 billion Brazilian reais ($650 million USD) in 2016⁹. In the absence of an effective vaccine for these arboviruses, disease prevention depends on vector control. Vector control guidelines in Brazil¹⁰ are focused on elimination or larvicide treatment of mosquito breeding sites and the control of adult mosquito populations with insecticides sprayed as ultra-low volume. The limited potential of these traditional vector control strategies to achieve large-scale and sustained reductions in dengue incidence is evidenced by the continuing public health burden of dengue throughout endemic areas where these measures are routinely employed, and the lack of robust efficacy data from well-designed trials to inform their optimal implementation¹¹.

The Wolbachia method (www.worldmosquito.org) is a novel, natural, and self-sustaining approach to reduce arboviral diseases transmitted by Ae. aegypti mosquitoes. The symbiotic Wolbachia bacterium is found naturally in over 60% of insect species, but not in Ae. aegypti, and is passed from one generation to the next through the insect’s eggs. Stable transinfection of Wolbachia into a local Ae. aegypti colony in the laboratory produces a lineage of Wolbachia-carrying mosquitoes which, upon release over several weeks, can achieve dissemination of Wolbachia into the local Ae. aegypti population through the processes of maternal inheritance and cytoplasmic incompatibility that give Wolbachia-carrying mosquitoes a reproductive advantage. The DENV-transmitting potential of Wolbachia infected mosquitoes is reduced by 66–75%^12,13, a phenotype which has been shown to persist in field mosquito populations up to five years after the end of releases. Mathematical modelling of this reduced transmissibility predicts a substantial and sustained reduction in dengue incidence in human populations where Wolbachia is established¹³. Laboratory data indicate a similar reduction in the competence of Wolbachia-carrying Ae. aegypti for transmitting other viruses including Zika, chikungunya, yellow fever and Mayaro^14–17.

With releases now conducted in eight countries over the past eight years, the World Mosquito Program has demonstrated that Wolbachia can be successfully established and maintained in both small-scale and large-scale urban settings in multiple ecological environments^18–22.

A core objective of these releases has been to ensure strong community acceptance and government support for the approach, achieved through embedding community and stakeholder engagement within the project activities in each site. Observational evidence of the impact of Wolbachia releases on arboviral disease in pilot sites has been encouraging, with no evidence of local dengue transmission where Wolbachia has established at high levels. Following city-wide deployment in Townsville, Australia, there has been no confirmed local dengue transmission in Wolbachia-treated areas for four seasons since completion of releases, despite local transmission every year for the prior 13 years and ongoing importation of DENV infection in travelers²¹. A cluster randomized controlled trial (RCT) to generate a robust and quantitative estimate of the impact of Wolbachia on dengue incidence commenced in Yogyakarta, Indonesia in 2017, with reporting of results expected in 2021²³.

In Brazil, planned scale up of Wolbachia deployments from demonstration projects in Rio de Janeiro and Niterói municipalities to large-scale releases was accelerated by the declaration of Zika as a public health emergency by the WHO in early 2016, and the recommendation by WHO’s Vector Control Advisory Group in March 2016 that the Wolbachia method be evaluated in rigorously monitored pilot deployments under operational conditions, to build evidence of epidemiological effectiveness against Aedes-borne viruses²⁴. Given the imperative from stakeholders and funders to scale up deployment within a relative short time frame, and to retain sufficient flexibility to optimize methods for large-scale deployment in the varied micro-environments within Niterói and Rio de Janeiro, an RCT or other carefully controlled deployment was not considered feasible. Instead releases under operational conditions, and with pragmatic evaluation of disease impact using data routinely collected for public health purposes, was favoured.

Here we describe a protocol for evaluating the effect of large-scale non-randomized Wolbachia releases on the incidence of dengue, Zika and chikungunya in the municipalities of Niterói and Rio de Janeiro. The proposed strategy employs a controlled interrupted time series analysis of routinely notified suspected and laboratory-confirmed arboviral cases, together with monitoring of arbovirus activity with outbreak signals routinely used in public health disease surveillance. This methodology allows measurement of the impact of the intervention at the population level over time, accounting for the seasonal trends and inter-annual fluctuations often observed in dengue and other arboviral disease incidence.

Methods

Study setting and population

Rio de Janeiro and Niterói cities are located in the State of Rio de Janeiro, Brazil. Niterói has an area of 134 km² and a population of 487,562 in 2010. Rio de Janeiro is the second largest city in Brazil with 6,320,446 inhabitants in 2010 and an area of 1,200 km². The two cities sit on opposite sides of the Guanabara Bay and are linked by a long bridge, which transports a large commuter population between Niterói and Rio de Janeiro.

The cities are divided into health districts, for the purpose of planning and delivering care - ten in Rio de Janeiro and seven in Niterói. In Rio de Janeiro, Wolbachia deployments will be conducted in one of the administrative areas of the city (Figure 1; produced in ArcMap version 10.5, ESRI, CA), in an area of approximately 90 km² and with 886,551 inhabitants, 39% of whom live in slums. The total release area in Niterói is approximately 31 km² covering a population of 268,536 (Table 1). For the purposes of Wolbachia deployment, the Rio de Janeiro and Niterói intervention areas are divided into 4 and 3 release zones, respectively, which are aligned with neighbourhood administrative boundaries.

Figure 1.

Map of (a) Rio de Janeiro and (b) Niterói Wolbachia-treated and untreated areas (produced in ArcMap version 10.5, ESRI, CA).

Table 1. Sociodemographic characteristics of Wolbachia treated and untreated areas, Niterói and Rio de Janeiro (source: 2010 Brazil population census).

	Neighbourhoods	Population	Area (km2)	Population density
Niterói
Zone 1	4	23,747	9.2	2581
Zone 2	11	68,695	50.6	1357
Zone 3	13	178,891	12.6	14,197
Non-release control area	24	216,229	62.1	3481
Total	52	487,562	134.5	3624
Rio de Janeiro
Zone 1	10	107,130	11.8	9078
Zone 2	6	150,646	33.5	4496
Zone 3.1	8	408,036	32.6	12,516
Zone 3.2	4	220,739	12.0	18,394
Non-release control area	52	1,512,608	117.3	12,895
Non-release area	80	3,921,287	996.5	3935
Total	160	6,320,446	1203.7	5250

In Rio de Janeiro, two administrative areas adjacent to the release area have been designated a priori as a comparative control zone (Figure 1), based on comparable sociodemographic characteristics and synchronous historical dengue time series (Figure 2 and Table 1). In Niterói, the remaining untreated area of the city has been designated as the comparative control zone (Figure 1).

Figure 2.

Dengue incidence in Wolbachia-treated vs untreated areas of (a) Rio de Janeiro (2000–2017) and (b) Niterói (2007–2016) (source: Brazilian National Disease Surveillance System (SINAN)).

Discussion

The municipalities of Rio de Janeiro and Niterói in southeastern Brazil have been affected by dengue for more than 30 years, with epidemics occurring every 2 to 5 years. In recent years, outbreaks of the other Aedes-borne diseases chikungunya and Zika have presented further public health challenges, and since July 2017 at least 221 human cases of yellow fever have been confirmed in Rio de Janeiro State, including in urban areas⁸. Vector control strategies, based on elimination of mosquito breeding sites and use of insecticides to reduce adult populations, have not been effective in preventing dengue outbreaks²⁸. The Wolbachia method is a novel and self-sustaining approach for the biological control of arboviral diseases. The signature feature of Wolbachia is to reduce the arbovirus-transmission potential of Wolbachia-infected mosquitoes^12,14,16,17. The World Mosquito Program²⁹ is deploying Wolbachia-infected Ae. aegypti mosquitoes in Brazil with the purpose of achieving a large-scale and sustained reduction in arboviral disease burden in two cities where these diseases are public health priorities.

In March 2016, the WHO convened a Vector Control Advisory Group to review new and existing vector control tools for use in the response to the Zika virus outbreak. Based on the available evidence that Wolbachia reduces the Zika, dengue and chikungunya transmission potential of Ae. aegypti mosquitoes and field data showing long-term establishment of Wolbachia in mosquito populations in a range of environmental settings, the WHO recommended carefully monitored pilot implementation of the Wolbachia method in affected countries²⁴.

While RCTs are still considered the gold standard, they are not always feasible or agreeable to the community and government. The controlled ITS analysis is a quasi-experimental design that is commonly used to evaluate population-level public health intervention^27,30,31 and is a pragmatic alternative design where an RCT is considered infeasible, particularly in the presence of a well-matched untreated control area^32,33. Given the public health emergency posed by the Zika epidemic at the time of this study’s inception and the need to scale up Wolbachia deployment in Rio de Janeiro and Niterói within a relative short time frame, an RCT or other carefully controlled deployment was not considered feasible. The controlled ITS is appropriate for the pragmatic evaluation of large-scale Wolbachia deployments given the long and reliable time series of dengue mandatory reporting data from both Rio de Janeiro and Niterói that allows for a longitudinal assessment of dengue trends before and after the Wolbachia intervention. Assessment of an impact of the intervention on chikungunya and Zika may be more difficult given their shorter time series.

Notifiable disease surveillance data can be limited by a lack of specificity in case definitions and inconsistent reporting practices, which may influence our ability to detect a true intervention effect on arboviral disease incidence. A subset of notified dengue cases are supported by laboratory diagnostic results, but these have several limitations: i) laboratory testing occurs infrequently (<15% of notified cases), particularly during outbreaks, ii) the cross-reactivity of IgM serology between dengue and Zika limits the utility of serological data since 2015, and iii) the restricted use of PCR in only certain patient populations limits the generalisability of PCR-positivity rates to all notified cases. We therefore base our analyses on all notified cases (suspected and confirmed). Benefits of using these routinely collected data include the availability of a long time series, reduced costs for data collection and timely acquisition of data.

Human mobility also presents a challenge to this study as individuals are likely to spend time in both Wolbachia-treated and untreated areas, making it difficult to determine the place of illness acquisition among notified cases of arboviral disease. Travel between areas means that the true Wolbachia exposure status of individuals resident in Wolbachia-treated and untreated areas becomes more similar, thereby diluting the observed intervention effect towards the null.

The introduction of Zika and chikungunya viruses brought new challenges to health surveillance and a greater willingness for better vector control in affected regions. With the re-emergence of yellow fever in Brazil, there is even more potential public health benefit if the Wolbachia intervention successfully reduces the vector competence of Ae. aegypti mosquitoes in the field and reduces arboviral disease incidence. No specific treatment for dengue, chikungunya or Zika currently exists. Although a vaccine against dengue (Sanofi Dengvaxia^®) was licensed in 2015, it was recently found to enhance the severity of subsequent dengue infection in individuals who were seronegative at the time of vaccination. As a result, a serology test prior to the administration of the vaccine is required to confirm previous dengue infection, increasing costs and decreasing feasibility in high-burden areas³⁴.

Releases of Wolbachia are completed or underway in eight countries, with no evidence of local transmission of dengue, Zika or chikungunya in places where Wolbachia is established at high levels²¹. The implementation of the Wolbachia intervention is complex and has not been done on a large scale in very densely populated urban areas in the Americas before. The implementation of the project was preceded by careful work to engage the community and gain public acceptance for the intervention, even in the most difficult contexts of poverty and urban violence present in both Rio de Janeiro and Niterói. Engagement, entomological monitoring, and public health impact assessment activities were developed in close partnership with local governments. If the current project is successful, this model can be expanded to the rest of the country and the Americas.

Ethics approval and consent to participate

The study was approved by the Brazilian Institutional Review Board (Conep) (CAAE: 59175616.2.0000.0008). The study uses pre-existing non-identifiable disease surveillance data, which does not require individuals’ consent.

Data availability

No data is associated with this article.