INTRODUCTION

Several required conditions are associated with the emergence of a new arboviral disease. Both introduction and dissemination must occur simultaneously to support the maintenance of complex cycles involving vectors, hosts, and human infection. In Brazil, the current climate changes associated with deforestation and socioeconomic conditions of the population represent critical aspects that are favorable to the emergence and reemergence of arboviruses transmitted by Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus mosquitoes.¹

Although a significant number of Zika (ZIKV), Dengue (DENV), and Chikungunya (CHIKV) infections is asymptomatic, these viruses are responsible for moderate and severe syndromes. The acute febrile syndrome caused by DENV has been a significant public health challenge worldwide for decades, with a dramatic increase in cases in recent years, and it has been responsible for about 5,486 deaths until 39^th epidemiological week of 2024 in Brazil.² Additionally, in recent years, two new emerging arboviruses were introduced in Brazil, ZIKV and CHIKV, both of which cause acute febrile syndromes and a severe impairment for human health. CHIKV is responsible for severe cases of polyarthralgia, and ZIKV, for congenital zika syndrome (CZS), leading to neuronal abnormalities, delayed neuro-psychomotor development in newborns, and Guillain-Barré syndrome (GBS) in adults, representing a public health emergency of international concern according to the World Health Organization.^3-4

The clinical symptoms associated with these arboviral infections are quite similar, especially in mild cases. Gastrointestinal, musculoskeletal, hematological, ophthalmological disorders, fever, itching, and headache are nonspecific symptoms of arboviral diseases, which hinder the differential clinical diagnosis and the correct notification of suspected cases. Therefore, effective notification associated with accurate diagnosis and the correct use of the Brazilian Health Information System are paramount and represent an impacting factor for public health.⁵ The work aimed to describe the epidemiological and laboratorial profile of arboviruses of suspected cases reported in southeastern Mato Grosso (MT) during the emergence of ZIKV in Brazil.

METHODS

Study population

A retrospective and experimental study of suspected cases of arboviruses DENV, ZIKV and CHIKV fever reported in the municipality of Rondonópolis from August 2015 to August 2016 was carried out. During this period, Brazil experienced the emergence and dissemination of ZIKV.

Suspected cases of arboviruses assisted by the municipality’s Primary Care Health Units were referred to the Central Laboratory of Rondonópolis, and clinical serum samples were obtained. The collected samples underwent molecular and serological analysis at the Universidade Federal de Rondonópolis Laboratory of Basic and Applied Virology. The study included patients up to five days after the onset of symptoms during the introduction of ZIKV in Brazil (2015-2016). Samples with more than five days of the onset of symptoms, insufficient volume for laboratorial analysis and inadequate storage conditions were excluded. Epidemiological data were obtained from July 2019 to November 2019 by notifiable forms, according to the Notifiable Diseases Information System (In Portuguese, Sistema de Informação de Agravos de Notificação - SINAN) of the Ministry of Health Brazil.

Demographic and clinical information analysis

The descriptive study was performed using sociodemographic and epidemiological data from 197 SINAN notifications; of these, 179 samples were included in laboratory analysis. Data were tabulated and analyzed using Microsoft Excel^® (Washington, USA). The spatial distribution of patients’ household was achieved from the open-source Geographic Information System (QGIS v3.18) software.

Laboratory analysis

Molecular investigation was performed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) in serum samples. The RNA was extracted and purified from 150μL of serum using silica columns (NucleoSpin^® RNA Virus, Macherrey-Nagel, GER) according to the manufacturer’s instructions. The one-step real-time ZDC Multiplex RT-PCR Assay^® (BioRad, USA) was performed for RNA detection of DENV, ZIKV, and CHIKV viruses, following the manufacturer’s instructions, on a CFX96 Touch^TM Thermal Cycler (BioRad, USA).

Serological assessment was performed by enzyme-linked immunosorbent assay (ELISA) to detect immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies anti-ZIKV according to the manufacturer’s instructions (Euroimmun AG, GER).

The interpretation of serological results was conducted carefully, considering the possibility of cross-reaction among arboviruses. In this regard, the molecular and serological diagnosis employed in this study present specific features, as shown on Table 1.⁶

The study was conducted obeying the ethical aspects that regulate scientific research involving human, according to Resolution 466/12 of the Brazilian National Health Council, and was approved by the Universidade Federal de Mato Grosso Research Ethics Committee, under CAAE 10767319.8.0000.8088.

RESULTS

This study included 197 patients with acute febrile syndrome and clinical suspicion of DENV, ZIKV, or CHIKV infections. According to the 197 patients’ SINAN forms, 124 (63%) were female; of these, eight were pregnant, four in the first trimester and the other half in the third trimester of pregnancy. The mean age was 32 years, and the youngest patient was two months old, whereas the oldest was 70 years old. The most frequent age group was 19-59 years. Considering ethnic identity, 28% of patients self-declared as white, and three cases were reported as indigenous. The educational level was missing in 57% of notification forms. On the other hand, for 17 patients (8.6%), this variable was inapplicable according to age (Table 2).

With respect to patients’ residence areas, 114 (57.9%) reported living in urban areas and three patients lived in indigenous territory of Rondonópolis. Only four notifications presented patients belonging to other municipalities, which are included to the same health region (southeast), according to the division of Mato Grosso Regions of Health.⁷

In our study, the included cases were distributed in 79 (33%) neighborhoods in the municipality of Rondonópolis. Geoprocessing analysis showed a high density of notified cases in three urban neighborhoods of the city, in the central and north, as shown in Figure 1.

According to clinical presentations data, only 46 (23.3%) forms had some information. Musculoskeletal disorders such as arthralgia, joint swelling, and myalgia were reported for 45 (22.8%) patients, who presented at least one of these symptoms, representing the main complaints. Fever (13.7%), headache (11.7%), itching (10%), and symptoms related to hematological disorders, such as rash and petechiae (12.6%), were also notified. Other described symptoms were ophthalmological (6.1%) and gastrointestinal (5.6%) manifestations (Table 3).

Experimental analyses were performed with a total of 179 serum samples (Table 4). Molecular assay revealed three (1.7%) patients infected with DENV. Additionally, 16 (8.9%) patients had positive results for ZIKV, and CHIKV RNA was not detected in any clinical serum samples. Therefore, a total of 19 patients tested positive in molecular assays performed by the study, of which 18 (94.7%) were women. Regarding the ZIKV serology, IgM was detected in 19 (10.6%) samples and IgG in 100 (55.8%).

DISCUSSION

In this retrospective study, we assessed clinical, epidemiological, and laboratorial data of DENV, ZIKV, and CHIKV suspected cases in a Brazilian municipality located in southeastern MT, Midwest region of Brazil. Such geographic area is endemic for DENV, with the concomitant occurrence of ZIKV and CHIKV. Our study reveals unprecedented data for arboviruses in this geographic region during the introduction of ZIKV and CHIKV in Brazil, 2015-2016. Molecular arbovirus investigation was performed during the same period, in Cuiabá, the state capital of MT, which is a distant region.⁸

Rondonópolis has approximately 236,000 inhabitants, and is the reference point to 18 other cities belonging to the Southeast Health region. During the period 2015–16, the city reported 8,597 notifications of DENV, 535 of ZIKV, and 70 of CHIKV fever, with a high incidence of DENV and ZIKV fever during the same period. In 2015–16, ZIKV and CHIKV fever cases were reported in about half of all Brazilian municipalities, while probable DENV cases were reported in all of them. Therefore, Brazil and consequently MT registered an expressive number of DENV, ZIKV, and CHIKV notifications due to the introduction and expansion of the recently emerging arboviruses associated with the persistence and endemicity of DENV. And the increased number of notifications occurred mainly during rainy seasons, according to SINAN notifications.^7,9-13 Our data supported this observation, with the highest number of cases (61%) detected by molecular tests reported between December 2015 and April 2016, with poor impact in reducing cases through educational campaigns for vector control implemented by Brazilian health authorities.

Following the introduction of ZIKV and the beginning of the outbreak in 2015 in Brazil, accurate laboratorial diagnostics were not available for MT, as in most parts of the country. The first positive confirmed results were reported only in February 2016 by the epidemiological surveillance program of the Department of Health of Mato Grosso. On the other hand, ZIKV was detected in a human sample collected in the municipality of Tapurah, northern MT, in August 2015.⁸ The first positive cases in pregnant women were also confirmed in February 2016 from Primavera do Leste, a municipality belonging to the same health region of Rondonópolis, reinforcing viral circulation in this area.¹³ However, the first suspected case of CZS in this region probably occurred in June 2015, which would confirm the circulation of ZIKV in MT in the middle of that year.⁴ In the same way, the first laboratory-confirmed cases of CHIKV fever were reported only in March 2016.⁷

In this regard, adequate filling of notification forms and protocols available by Brazilian health authorities is essential for implementing epidemiological surveillance programs, especially considering the introduction of new arboviruses in DENV endemic areas, as observed in the CHIKV and ZIKV outbreak cases in recent years.⁵ Therefore, the high number of variables missing in different fields of the notification forms assessed in our study highlight the need for proper filling of notification forms for suspected cases, as clinical diagnosis was essential for introducing these arboviruses in Brazil, mainly due to the lack of serological or molecular diagnoses.

As for socio-demographic analyses, Female patients accounted for almost 63% of the total cases in our study, and eight of them were pregnant. A similar rate is commonly observed in other surveys, probably because women are more likely to seek healthcare compared to men, at least for arbovirus investigation. This phenomenon could also be explained by a greater risk of exposure to mosquito bites because of women domestic costumes. Other relevant fact about these data can be closely related to the protocol established by Brazilian authorities, which focused on women, mainly during pregnancy, because of the occurrence of CZS, especially during 2015–16. This fact may have motivated females to search for more healthcare, thus resulting in a greater number of positive cases, as observed in our study.^11,14 A larger number of notifications was observed for patients in the age group of 19–59 years, which represents the economically more active population. Although professional occupation was not assessed in the study, this profile represents important insight for the local economy, as arboviruses affect patient productivity, with a critical impact on daily work routines. Similar patterns were shown in other profile epidemiological studies conducted in Brazil.^11,15

It was observed that notifications were concentrated in urban areas with high population density and hence higher risk of infection. This fact reinforces and corroborates the urban features of DENV, ZIKV, and CHIKV, transmitted by anthropophilic mosquitoes to humans, which act as amplification hosts for these arboviruses critical to public health.¹⁶

Although only three notifications were reported in indigenous territory, these rural areas are close to urban areas of Rondonópolis and may represent a potential risk zone for future outbreaks, depending on ecological conditions. Therefore, these suspected cases highlight the importance of consider indigenous territory in the municipality epidemiological surveillance programs.

Epidemiological features and climatic and ecological conditions, such as rainfall periods associated with high temperatures, may predict arboviral epidemics. These features, accompanied by disorderly urban growth, overpopulation, and precarious sanitary conditions, combined with inefficient vector control programs, result in more mosquito breeding grounds and provide conducive conditions for increased proliferation of infectious disease vectors, such as arboviruses. In this regard, low socioeconomic status and less education levels are both important for individuals and consequently familiar risk to contract these febrile syndromes.^17,18 Therefore, due to the accelerated and disordered urbanization process of the municipality of Rondonópolis, the notable social, economic, and environmental differences among all urban neighborhoods can influence the high incidence of these diseases.^16,19

The three arboviral diseases investigated in this study are closely related and cause DENV-like clinical signs and symptoms. Moreover, about 80% of ZIKV cases are asymptomatic and may thus hinder differential clinical diagnosis, resulting in arbovirus misclassification. Therefore, the concomitant circulation of ZIKV, CHIKV, and other arboviruses in an area endemic to DENV, where laboratory tests are not always available for prompt confirmation, is a challenge for public health authorities and especially for health professionals, which in turn interfere with obtaining accurate diagnosis.^8,20-22

In this scenario, it is important that clinical assessment is followed by laboratory diagnosis, especially molecular assays, which provide a specific diagnosis since the onset of symptoms. Serological assays may present the possibility of cross-reaction and false positive results for arboviruses with the synthesis of cross-reactive antibodies in patients previously exposed to some heterologous flaviviruses, often leading to inconclusive serology diagnostics. Furthermore, serological assays do not determine which DENV serotype is indeed circulating, which is very relevant, mainly in epidemic situations.^11,6

It is important to point out that molecular and serological assays may be performed at different stages of the arboviral disease. Molecular assays may only be feasible during viremia stages, approximately five days after symptom onset. On the other hand, serology can be applicable after antibody synthesis, which is detectable at the end of the viremia stage.⁶ Thus, our results showed one positive DENV sample in the molecular assay with the presence of ZIKV IgM antibodies, probably due to cross-reactivity. In addition, 14 positive ZIKV IgM were negative by molecular test. Furthermore, IgM was not detected in other 12 from 16 ZIKV RT-qPCR positive samples, probably because antibody synthesis was not completed or under the ELISA detection limits. Therefore, only four samples reached concomitantly serological (IgM) and molecular positive diagnosis, which combined with the high number of IgG positive results reinforce the possibility of cross-reaction, since the virus had just introduced into the region, showing that serological diagnosis is neither adequate nor timely.

New viruses recently discovered in the Pantanal biome in MT are indicators of the possibility of future interactions and compromise of human health.²³ Interestingly, it was observed that only 2% (4) of patients were submitted to some laboratorial diagnostic test by public health services in Rondonópolis during clinical assessment, and all were negative for acute infection by serological analysis. However, about 10.6% (19) of 179 sampled patients had positive RNA detection in our molecular investigation (IgM). Similar rates have been observed in related surveys.²⁴

In this context, the negative cases may have been probably caused by other pathogens not investigated during clinical follow-up. Considering the total number of reported notifications during the studied period, it is possible to infer that epidemiological and clinical criteria alone are insufficient for accurate diagnoses. Furthermore, accurate laboratory diagnosis may have implications for patient management and clinical evolution, such as effective treatment, and this highlights the importance of molecular assays for early detection and definitive confirmation of febrile syndrome etiologies.²⁵

It is important to address the limitations of this study, which were related to the quality of data obtained from patients’ SINAN forms, with high level of incomplete information. In addition to this, there is the possibility of other infectious etiologies since the survey was designed only for DENV, ZIKV, and CHIKV molecular detection and for ZIKV serological investigation. Moreover, the samples were collected between 2015–16, and molecular analyses were performed only in 2019–20, indicating the possibility of viral RNA degradation.

In conclusion, our study provides singular local research concerning clinical and laboratorial assessment of suspected arboviruses infected patients during the first outbreak of ZIKV and CHIKV in Brazil, in a region that has shown a high incidence of DENV cases, representing a relevant contribution to understanding the concomitant occurrence of these acute febrile syndromes.

Acknowledgments

The authors are grateful to the Municipal Health and Urbanism Departments of Rondonópolis, Mato Grosso State, Brazil.

REFERENCES

1. Jones R, Kulkarni MA, Davidson TMV, et al. Arbovirus vectors of epidemiological concern in the Americas: A scoping review of entomological studies on Zika, dengue and chikungunya virus vectors. PLoS One. 2020;15(2):e0220753. https://doi.org/10.1371/journal.pone.0220753

2. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde e Ambiente. Painel de Monitoramento das Arboviroses [internet] 2024 [citado 2024 out 04]. Disponível em: https://www.gov.br/saude/pt-br/assuntos/saude-de-a-a-z/a/aedes-aegypti/monitoramento-das-arboviroses

3. Souza WM, Lima STS, Mello LMS, et al. Spatiotemporal dynamics and recurrence of chikungunya virus in Brazil: an epidemiological study. Lancet Microbe. 2023 May;4(5):e319-e329. https://doi.org/10.1016/S2666-5247(23)00033-2

4. Roma JHF, Alves RC, Silva VSD, et al. Descriptive study of suspected congenital Zika syndrome cases during the 2015-2016 epidemic in Brazil. Rev Soc Bras Med Trop. 2019;52:e20190105. https://doi.org/10.1590/0037-8682-0105-2019

5. Carabali M, Jaramillo-Ramirez GI, Rivera VA, et al. Assessing the reporting of Dengue, Chikungunya and Zika to the National Surveillance System in Colombia from 2014-2017: A Capture-recapture analysis accounting for misclassification of arboviral diagnostics. PLoS Negl Trop Dis. 2021 Feb 4;15(2):e0009014. https://doi.org/10.1371/journal.pntd.0007721

6. Mota ML, Marinho RSS, Duro RLS, et al. Serological and molecular epidemiology of the Dengue, Zika and Chikungunya viruses in a risk area in Brazil. BMC Infect Dis. 2021 Jul 24;21(1):704. https://doi.org/10.1186/s12879-021-06401-3

7. Monitoramento dos casos de Dengue, Febre de Chikungunya e Febre pelo vírus Zika. Boletim Epidemiológico Nº 9 SE 11/2016. [internet]. Cuiabá: Secretaria de Estado de Saúde do Mato Grosso; 2016. Disponível em: http://200.252.205.48/suvsa/arquivos/526/boletins-epidemiologicos?page=3.

8. Costa MCS, Siqueira-Maia LM, Souza VC, Gonzaga AM, Azevedo VC, Martins LR, et al. Arbovirus investigation in patients from Mato Grosso during Zika and Chikungunya virus introdution in Brazil, 2015-2016. Acta Trop. 2019;190:395-402. https://doi.org/10.1016/j.actatropica.2018.12.019

9. Monitoramento dos casos de Dengue, Febre de Chikungunya e Febre pelo vírus Zika. Boletim Epidemiológico Nº 35 SE 50/2016. [internet]. Cuiabá: Secretaria de Estado de Saúde do Mato Grosso; 2016. Disponível em: http://200.252.205.48/suvsa/arquivos/526/boletins-epidemiologicos?page=2

10. Monitoramento dos casos de Dengue, Febre de Chikungunya e Febre pelo vírus Zika. Boletim Epidemiológico Nº 45 SE 52/2017. [internet]. Cuiabá: Secretaria de Estado de Saúde do Mato Grosso; 2017. Disponível em: http://200.252.205.48/suvsa/arquivos/526/boletins-epidemiologicos?page=1

11. Farias PCS, Pastor AF, Gonçales JP, et al. Epidemiological profile of arboviruses in two different scenarios: dengue circulation vs. dengue, chikungunya and Zika co-circulation. BMC Infect Dis. 2023 Mar 22;23(1):177. https://doi.org/10.1186/s12879-023-08139-6

12. Man OM, Fuller TL, Rosser JI, et al. Re-emergence of arbovirus diseases in the State of Rio de Janeiro, Brazil: The role of simultaneous viral circulation between 2014 and 2019. One Health. 2022 Aug 10;15:100427. https://doi.org/10.1016/j.onehlt.2022.100427

13. Monitoramento dos casos de Dengue, Febre de Chikungunya e Febre pelo vírus Zika. Boletim Epidemiológico Nº 5 SE 07/2016. [internet]. Cuiabá: Secretaria de Estado de Saúde do Mato Grosso; 2016. Disponível em: http://200.252.205.48/suvsa/arquivos/526/boletins-epidemiologicos?page=3

14. França GV, Schuler-Faccini L, Oliveira WK, et al. Congenital Zika virus syndrome in Brazil: a case series of the first 1501 livebirths with complete investigation. Lancet. 2016;388(10047):891-7. https://doi.org/10.1016/S0140-6736(16)30902-3

15. Almeida JFPD, Alves WA. Descriptive profile of the occurrence of arboviruses in Governador Valadares, Minas Gerais, Brazil. J Health Biol Sci. 2020;8(1):1-7. https://doi.org/10.12662/2317-3076jhbs.v8i1.3226.p1-7.2020

16. Silva GB, Andrade CR, Salgado JVV, et al. Profile of hospitalization and death records associated to dengue and severe dengue in Minas Gerais between 2000 and 2015 from the Brazilian Public Health System perspective. PLoS One. 2023;58(1):54-62. https://doi.org/10.1371/journal.pntd.0011197

17. Renard A, Lombardini FP, Zapata MP, et al. Interaction of Human Behavioral Factors Shapes the Transmission of Arboviruses by Aedesand Culex Mosquitoes. Pathogens. 2023 Dec 6;12(12):1421. https://doi.org/10.3390/pathogens12121421

18. Power GM, Vaughan AM, Qiao L, et al. Socioeconomic risk markers of arthropod-borne virus (arbovirus) infections: a systematic literature review and meta-analysis. BMJ Glob Health. 2022 Apr;7(4):e007735. http://dx.doi.org/10. 1136/bmjgh-2021-007735

19. Cruz L, Guimarães AGF, Souza EM, et al. Influence of climatic variables on the Aedes aegypti and Culex quinquefasciatus populations in Mato Grosso, Brazil. Rev Soc Bras Med Trop. 2020;53:e20190185. https://doi.org/10.1590/0037-8682-0185-2019

20. Pavon JAR, Neves NADS, Silva LCF, et al. Neurological infection by chikungunya and a triple Arbovirus co-infection in Mato Grosso, Central Western Brazil during 2019. J Clin Virol. 2022 Jan;146:105056. https://doi.org/10.1016/j.jcv.2021.105056

21. Dias HG, Lima RC, Barbosa LS, et al. Retrospective molecular investigation of Mayaro and Oropouche viruses at the human-animal interface in West-central Brazil, 2016-2018. PLoS One. 2022 Nov 17;17(11):e0277612. https://doi.org/10.1371/journal.pone.0277612

22. Oliveira JF, Rodrigues MS, Skalinski LM, et al. Interdependence between confirmed and discarded cases of dengue, chikungunya and Zika viruses in Brazil: A multivariate time-series analysis. PLoS One. 2020;15(2):e0228347. https://doi.org/10.1371/journal.pone.0228347

23. Maia LMS, Pinto AZL, Carvalho MS, et al. Novel Viruses in Mosquitoes from Brazilian Pantanal. Viruses. 2019;11(10). https://doi.org/10.3390/v11100957

24. Frota CC, Correia FGS, Alves Vasconcelos LR, et al. Positivity of dengue, chikungunya, and Zika infections in women in Northeast Brazil post-Zika epidemic. Pathog Glob Health. 2023 Jul;117(5):485-492. https://doi.org/10.1080/20477724.2022.2142187

25. Fahsbender E, Costa AC, Gill DE, et al. Plasma virome of 781 Brazilians with unexplained symptoms of arbovirus infection include a novel parvovirus and densovirus. PLoS One. 2020;15(3):e0229993. https://doi.org/10.1371/journal.pone.0229993