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Keywords:

  • salivary biomarker;
  • Aedes bites;
  • dengue risk;
  • spatial distribution;
  • residential neighbourhoods;
  • Vientiane

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Objective

Using human IgG antibody response to the Aedes Nterm-34 kDa salivary peptide as an indicator of human exposure to Aedes bites in surveying exposed populations from areas at risk of dengue virus (DENV) transmission in urban settings of Vientiane city, Lao PDR.

Methods

Enzyme-linked immunosorbent assay tests were performed to measure the IgG response to Nterm-34 kDa peptide in blood samples collected within a flavivirus seroprevalence survey carried out in 2006 including 3558 randomly selected individuals. The level of IgG response to the Nterm-34 kDa peptide in individuals was analysed in relation to the level of urbanisation of the individual's residence, areas that presented significant differences in the prevalence of recent DENV infection.

Results

No differences were observed in the anti-Nterm-34 kDa IgG level between DENV-positive and DENV-negative individuals. However, the level of specific IgG response was higher among individuals living in slightly urbanised neighbourhoods than among those in more highly urbanised areas (P < 0.0001). Interestingly, a similar pattern had already been observed concerning the prevalence of recent DENV infection in the same populations.

Conclusion

The results of this retrospective study indicate that the evaluation of human IgG response to the Aedes Nterm-34 kDa salivary peptide could be a useful indicator to identify places with risk of dengue virus transmission in urban endemic areas.

Objectif

Utiliser la réponse de l'anticorps IgG humaine au peptide salivaire Nterm-34 kDa de l’Aedes comme un indicateur de l'exposition humaine aux piqûres de l’Aedes dans la surveillance des populations exposées dans les zones à risque de transmission du virus de la dengue (DENV) en milieu urbain de la ville de Vientiane, au Laos.

Méthodes

Des tests ELISA ont été effectués pour mesurer la réponse IgG au peptide Nterm-34 kDa dans des échantillons de sang prélevés dans une étude de séroprévalence du flavivirus réalisée en 2006, sur 3558 personnes choisies aléatoirement. Le niveau de réponse IgG contre le peptide Nterm-34 kDa chez les individus a été analysé en relation avec le niveau d'urbanisation du lieu de résidence des individus, les zones qui présentaient des différences significatives dans la prévalence de l'infection DENV récente.

Résultats

Aucune différence n'a été observée dans le niveau d'IgG anti-Nterm-34 kDa entre les individus positifs et négatifs pour le DENV. Cependant, le niveau de la réponse IgG spécifique était plus élevé chez les personnes vivant dans des quartiers un peu urbanisés que chez ceux vivant dans des zones plus fortement urbanisées (P < 0,0001). De façon intéressante, une tendance similaire avait déjà été observée dans la prévalence de l'infection DENV récente dans les mêmes populations.

Conclusion

Les résultats de cette étude rétrospective indiquent que l’évaluation de la réponse IgG humaine au peptide salivaire Nterm-34 kDa d’Aedes pourrait être un indicateur utile pour identifier les zones à risque de transmission du virus de la dengue dans les zones endémiques urbaines.

Objetivo

Utilizando la respuesta de anticuerpos IgG humanos al péptido salivar Nterm-34 kDa de Aedes como un indicador de la exposición humana a picaduras de Aedes, estudiar las poblaciones expuestas pertenecientes a áreas en riesgo de transmisión del virus del dengue (VDEN) en zonas urbanas de la ciudad de Vientiane, Laos.

Métodos

Se realizaron pruebas de ELISA para medir la respuesta de IgG al péptido Nterm-34 kDa en muestras de sangre recogidas como parte de un estudio de seroprevalencia de flavivirus llevado a cabo en el 2006, incluyéndose 3558 individuos escogidos al azar. Se analizó el nivel de respuesta de IgG al péptido Nterm-34 kDa con relación al nivel de urbanización de la residencia del individuo, áreas que presentaron diferencias significativas en la prevalencia de infecciones recientes por VDEN.

Resultados

No se observaron diferencias entre el nivel de IgG anti-Nterm-34 kDa entre individuos positivos y negativos para VDEN. Sin embargo, el nivel de respuesta IgG específica era mayor entre individuos viviendo en barrios relativamente urbanizados, que entre aquellos en áreas muy urbanizadas (P < 0.0001). Se observó un patrón similar con respecto a la prevalencia de infecciones recientes de VDEN entre la misma población.

Conclusión

Los resultados de este estudio retrospectivo indican que la evaluación de la respuesta de IgG humana al péptido salivar Nterm-34 kDa de Aedes podría ser un indicador útil para identificar áreas con riesgo de transmisión del virus del dengue en áreas endémicas urbanas.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Dengue fever (DF) has become the most rapidly expanding arthropod-borne disease worldwide. Almost half of the world's population is now at risk of infection, especially in urban areas of developing countries (Guzman et al. 2010). Dengue fever presents a real risk of emerging in developed countries where many cases have now been reported. One example confirming this threat is the autochthonous sporadic outbreak reported in 2010 in southeastern France (Gould et al. 2010). Adequate strategies must be urgently developed to control this disease. Dengue virus (DENV) is transmitted by the bite of infected Aedes mosquitoes, mainly of two species: Aedes aegypti and Aedes albopictus. As no drugs and vaccines are available, the reduction and/or interruption of human-vector contacts remain the key strategy to prevent transmission.

The potential evaluation of human-vector contact is commonly based on the entomological monitoring of immature stages (larvae and/or pupae) of Aedes mosquitoes in container habitats and the calculation of House, Container and Breteau indices (Focks 2004). Nevertheless, these indices are a poor proxy for measuring adult mosquito abundance and are not accurate for assessing transmission risks (Focks 2004). Adult mosquito abundance is considered to be the most appropriate way to assess transmission risk (Focks 2004). However, collection of adult mosquitoes is a fastidious task, especially for Aedes mosquitoes, which bite both day and night. The sampling method may raise ethical concerns, especially for human landing collection, because no vaccines and treatments are available against arbovirus diseases. Given these limitations, the identification of new complementary indicators for monitoring dengue transmission risk is a research priority and has been declared as such by WHO (Farrar et al. 2007). For this purpose, the study of human immune response to Aedes mosquito salivary proteins is a promising approach. Indeed, as observed for numerous arthropods, antibody (Ab) responses to salivary antigens could be a pertinent biomarker for measuring the level of human exposure to Aedes bites (Remoue et al. 2007; Fontaine et al. 2011; Doucoure et al. 2012a,b).

We have found the human IgG Ab response to the Aedes aegypti Nterm-34 kDa salivary peptide to be a specific candidate biomarker of human exposure to Aedes bites (Elanga et al. 2012). The main objective of this study was to apply this salivary biomarker for evaluating human exposure to Aedes vector bites in urban settings of Vientiane, the capital of Laos. We explored relationships between the human IgG Ab response to this peptide, the level of urbanisation of residential communities and the spatial distribution of recent dengue infection.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

The IgG response to the Nterm-34 kDa peptide was assessed in human blood samples collected in February and March 2006 (dry season), within urban neighbourhoods of Vientiane, where Ae. aegypti and Ae. albopictus are the major DENV vectors (Vallée et al. 2009). As previously described by Vallée et al. (2007, 2009), neighbourhoods were classified by level of urbanisation using 13 human and environmental indicators: the proportion of built-up area; density of the population; changes in the built-up surface area between 1981 and 1999; the proportion of public infrastructure buildings; the proportion of trade buildings; the number of markets nearby; distance to the city centre via the road network; the average distance of every building to the road network; access to running water, electricity and toilets; the proportion of concrete houses; and the proportion of the population involved in agricultural activities. These indicators are derived from the 1999 aerial photographs processed in the Atlas Infographique de Vientiane and the 1995 census from the Lao National Statistical Center (Rossi et al. 2003).

Based on all these criteria, three strata of nine neighbourhoods each were identified: the central zone, the first urbanised belt and the second urbanised belt. As described in previous studies (Vallée et al. 2007, 2009), in each neighbourhood, households were randomly selected from a national list (from the Lao National Statistical Center). For each selected households, only one adult (≥35 years) and one child (≥6 months and <6 years) were included in the study. A total of 3692 individuals were interviewed, and 3634 blood samples were obtained. After excluding 76 samples from the analysis (inadequate specimen sampling and/or registration), overall 1990 adults and 1568 children were included in a serological survey to evaluate the seroprevalence of DENV and Japanese encephalitis infections in Vientiane urban settings. The overall study received ethics approval from the Lao National Ethics Committee for Health Research in Lao PDR and from the Oxford University Tropical Research Ethics Committee.

This study only focused on recent DENV infections. For this purpose, from the database provided by Vallée et al. (2009), we selected 126 individuals who positively reacted to dengue virus (DENV) IgM Ab (DEN IgM MAC-ELISA was performed for serological assays) among the whole study population (corresponding to 3.5% of entire cohort; 99 adults and 27 children) as previously described (Vallée et al. 2009). Blood samples (dried blood spot on filter paper) were available for 114/126 individuals (89 adults and 25 children). Additionally, an equal number of individuals who were negative for recent DENV infection (i.e. negative anti-DENV IgM Ab response; IgM DENV-) was randomly selected and matched-paired with regard to the neighbourhoods, sex and age. Blood samples (= 10) from individuals living in Montpellier (France) and presenting no exposure to Ae. aegypti mosquitoes were used as negative controls to determine the specific IgG level threshold (cut-off of immune responders), as previously described (Elanga et al. 2012). Enzyme-linked immunosorbent assay (ELISA) was performed for quantitative detection of IgG Ab against Nterm-34 kDa peptide as previously described (Elanga et al. 2012). Briefly, ELISA tests were carried out on dried blood-spot eluates. Individual results were expressed as the ∆OD value: ∆OD = ODxODn, where ODx represents the mean of individual optical density (OD) in both antigen wells and ODn the individual OD in a blank well containing no Nterm-34 kDa antigen. Individuals with a specific IgG response higher than the cut-off were considered positive.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Overall 45.1% (103/228, 95% CI = 38.7–51.6) of individuals living in Vientiane were immune responders to Ae. aegypti Nterm-34 kDa peptide, whatever neighbourhood, age or DENV serological status. In both groups of individuals (positive and negative to DENV IgM), the level (median values) of the IgG response to Nterm-34 kDa peptide was higher than the level observed in the negative controls (< 0.05; Mann–Whitney U test; Figure 1). However, the median IgG level remained very low and below the cut-off for both groups (Figure 1). No significant difference in the level of specific IgG response was observed among Vientiane individuals according to DENV status (Figure 1).

image

Figure 1. IgG Ab response to Nterm-34 kDa salivary peptide from urban population of Vientiane municipality in 2006. The IgG specific response is presented for both individuals with a positive (IgMDENV+) or negative (IgMDENV−) serological DENV IgM Ab, status and in comparison with unexposed individuals. Black dots indicate an individual IgG response (∆OD) and bars represent the median value in each group. Dotted line represents the cut-off of the specific Ab response (∆OD > 0.250), and P-values were calculated using the Mann–Whitney U-test.

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According to the neighbourhoods' degree of urbanisation, the level of IgG Ab response to Nterm-34 kDa peptide was significantly different between the three strata of urbanisation initially defined (= 0.0003; Kruskal–Wallis test; Figure 2). Specific IgG response among individuals living in the second urbanised belt (the periphery with low urbanisation) was higher than those from the first urbanised belt (< 0.0001, Mann–Whitney test) or from the central highly urbanised area (< 0.0001, Mann–Whitney test). Nevertheless, no difference in the level of anti-Nterm-34 kDa IgG response was observed between the first urbanised belt and the central zone. The same spatial distribution was observed for the seroprevalence of recent DENV infections in the whole study population (Table 1). Indeed, the prevalence of anti-DENV IgM Ab was higher (< 0.01) in individuals living in the periphery (60/1324, 4.5% and 43/1203, 3.6% in the second and the first belt, respectively) than in those living in the central zone (23/1031, 2.2%).

Table 1. Percentage of anti-DENV IgM antibody positive sera in the study population according to the level of urbanisation of their place of residence within Vientiane city, 2006
 Second urbanised zone (n = 1324)First urbanised zone (n = 1203)Central zone (n = 1031)P
N° (%)95% CIN° (%)95% CIN° (%)95% CI
Recent dengue virus infections60 (4.5)3.4–5.643 (3.5)2.5–4.623 (2.2)1.3–3.1<0.01
image

Figure 2. IgG Ab response to Nterm-34 kDa salivary peptide in the population studied according the level of urbanisation of their residential areas in Vientiane city, 2006. Black dots indicate individual IgG response (∆OD), and bars represent the median value in each group. Dotted line represents the cut-off of a specific Ab response (∆OD > 0.250). Statistically significant differences between the three groups (P = 0.0003, nonparametric Kruskal–Wallis test) and between the first and second urbanised belts (P < 0.0001, Mann–Whitney U-test) are indicated.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

We reported for the first time the presence of an IgG Ab response to the Aedes Nterm-34 kDa salivary peptide in Asian populations living in a dengue endemic area. Such specific Ab responses have already been detected in African children permanently exposed to the bites of Aedes mosquitoes (Elanga et al. 2012). This highlights the usefulness of this biomarker for the assessment of human exposure to Aedes bites, in all the geographical distribution areas of arbovirus vectors worldwide.

However, the design of our study did not allow discriminating the exposure to the bites of each of the two dengue vectors present in the study area. Indeed, even though the Nterm-34 kDa peptide has been identified in Ae. aegypti saliva, we cannot exclude a potential cross-reactivity with Ae. albopictus salivary proteins, as previously observed for whole saliva extracts (Doucoure et al. 2012b). The low level of IgG Ab response observed probably indicates a low exposure to Aedes bites during the dry season due to low mosquito densities, as previously observed in children in West Africa (Elanga et al. 2012). This emphasizes the sensitive potential of such salivary biomarker to detect human exposure to vector bites even in case of very low exposure, as previously shown for exposure to Anopheles bites (Remoue et al. 2006; Poinsignon et al. 2008, 2010). The higher level of the IgG Ab response to the Nterm-34 kDa peptide observed in the periphery than in the central zone suggests that the human exposure to Aedes bites may be higher in slightly urbanised neighbourhoods than in highly urbanised areas. This spatial differentiation can be explained by the presence of more favourable conditions for mosquito breeding and human – vector contact in the periphery than in the urbanised areas. Indeed, the central zone of Vientiane city (highly urbanised) has undergone remarkable urban transformation. In contrast, major changes characterised by poor water and unsatisfactory sanitary conditions due to heavy and rapid expansion associated with improper urbanisation of the city have occurred in the periphery (Vallée et al. 2009). This seems to indicate that, as observed today in Thailand (Chareonsook et al. 1999), slightly urbanised neighbourhoods (i.e. periphery) are potentially at higher risk of dengue virus transmission in Vientiane city than fully urbanised zones. This hypothesis seems to be strengthened by the results of the present study, as similar trends were observed between the spatial distribution of the prevalence of recent dengue infections and the human exposure to Aedes bites, estimated by the human IgG response to the Nterm-34 kDa salivary peptide.

We cannot exclude that inhabitants of Vientiane have been bitten and/or have become infected outside their residential neighbourhoods (i.e. work place, leisure, travel, etc.). However, our findings appear to indicate a relationship between the human IgG Ab response to Aedes Nterm-34 kDa salivary peptide, the level of urbanisation, and recent dengue virus infections, in the urban settings of Vientiane. Interestingly, the results of this retrospective study indicate a reliable usefulness of human IgG Ab response to Nterm-34 kDa salivary peptide, for predicting areas with a higher risk of dengue virus transmission in urban settings of developing countries. However, further studies in different epidemiologic settings are required for the validation of this biomarker as a new and complementary tool for evaluating dengue virus transmission risk.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
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