Outbreak of Chikungunya virus in a vulnerable population of Sergipe, Brazil - A molecular and serological survey

Abstract

Background

Chikungunya virus (CHIKV) is a re-emerging arbovirus that is causing outbreaks in several countries of the Americas. The virus was introduced in Brazil in 2014, and since then, several Brazilian states have notified autochthonous cases.

Objectives

Provide additional evidence on a CHIKV outbreak and an outline of the laboratory and clinical profile of symptomatic patients in Sergipe, Brazil.

Study design

In February 2016, we collected 142 serum samples from symptomatic patients for arboviruses in Sergipe, Brazil. All samples were submitted to qRT-PCR for the emerging arboviruses circulating in Brazil - ZIKV, CHIKV, and DENV - and later submitted to the immunoenzymatic assay. RNA positive samples were randomly selected and sequenced for characterization of the genotype involved in the outbreak.

Results

Our study had 75.35% (107/142) positivity for CHIKV infection, with all age groups and genera being equally infected. The virus was identified in 11 of the 13 cities studied in that state, including the ECSA genotype. Importantly, fever was the only statistically significant symptoms for CHIKV infection (p < 0.05), while asthenia was significantly associated with symptomatic patients that were CHIKV-negative (p < 0.05).

Conclusions

Our findings support the importance of fever as a clinical marker and contribute to molecular and serological surveillance data, which may help in the understanding of CHIKV circulation, emergence and clinical description.

1. Background

Chikungunya virus (CHIKV: Togaviridae: Alphavirus) is a mosquito-borne alphavirus mainly transmitted in urban and peri-urban areas by Aedes mosquitoes [1,2]. It was first described during a “dengue-like” outbreak in Tanzania between 1952-1953 [3]. In 2005, CHIKV caused a remarkable outbreak in La Réunion, a Madagascar island, with more than a third of its population testing positive for the infection [4]. The virus then spread across Asia, Europe, Pacific islands and the Americas, causing large outbreaks [2,5–8].

CHIKV genome is made of a single-stranded, positive-sense RNA of approximately 12 kb, which contains two open reading frames (ORFs) encoding for non-structural and structural proteins, respectively, all of which are arranged as a 5’-cap-nsP1-nsP2-nsP3-nsP4-(junction-region)-C-E3-E2-6K-E1-poly(A)-3’ [9]. CHIKV is classified into three genotypes based on its genetic diversity and the initial geographical distribution of its lineages that include West African, Asian, and East-Central-South-Africa (ECSA) genotypes [1].

CHIKV causes a self-limiting disease characterized by fever, asthenia, arthralgia, myalgia, headache and rash [1]. The acute phase of infection lasts one week and is characterized by the onset of fever and high viral loads. After the end of viremia, IgM becomes detectable, followed by severe myalgias and arthralgias [1]. The symptoms disappear over a period of time ranging from days to years [10,11].

The first autochthonous transmission of CHIKV in Brazil was described on 13 of September 2014, in the north region of Brazil caused by Asian genotype, and seven days later, autochthonous cases of ECSA genotype was confirmed in the Northeast of Brazil [12]. In the following years, the ECSA genotype continued to be identified in the Northeast, as well as in the Southeast region of Brazil [12–14]. In Brazil, CHIKV was associated with severe and fatal cases [15,16], posing not only a major threat to public health but also, a serious economic burden due to the prolonged disability and high medical costs imposed. Moreover, this situation is made worse by the lack of available treatments and/or vaccines against this virus.

2. Objectives

The present study aimed to provide additional evidence on a CHIKV outbreak and an outline of the laboratory and clinical profile of symptomatic patients during an outbreak in the state of Sergipe, Brazil. This state is located in northeastern Brazil, a region considered endemic for dengue virus (DENV) and zika virus (ZIKV) in previous years [17].

3. Study design

3.1. Study location

The state of Sergipe is located in the Northeast Region and is surrounded by the Atlantic Ocean to the east and adjoins the states of Bahia to the west and south, and Alagoas to the north (Figure 1). It is the smallest State in Brazil, occupying a total area of 21 915,116 km², and has an estimated population of 2,068,017 inhabitants. The climate is characterized as tropical in the coastal region, and also as semi-arid and tropical savannah in the western region of the state.

Figure 1. Map of the state of Sergipe and its localization in Brazil with the origin location of all patients.

(A) Map with the location of the state of Sergipe in the Brazilian territory. (B) The state of Sergipe is located in the Northeast Region and is bounded by the Atlantic Ocean to the east and the states of Bahia to the west and south, and Alagoas to the north.

3.2. Ethical Statement

The samples analyzed in this study were collected during an ongoing project for arbovirus research in Sergipe, Brazil approved by the Ethics Committee of the Department of Microbiology of the Institute of Biomedical Sciences of the University of São Paulo (Protocol 1284/CEPSH - CAAE: 54937216.5.0000.5467). All participating subjects were asked to sign an informed consent and were subjected to a questionnaire that asked about clinical symptoms and socio-demographic information after their acceptance to participate in the study.

3.3. Patients

This was an observational cross-sectional study. It was conducted with sera samples obtained from 142 patients with symptoms of acute disease presenting an “arbovirus-like” infection. Samples were collected in February 2016 in Sergipe state, Brazil. All patients were attended at basic health units in Sergipe. All samples were kept in storage at −80 °C until processing.

3.4. Molecular characterization

Nucleic acid extraction was performed using the QIAamp Viral RNA Mini Kit (Qiagen; Valencia, CA) and carried out according to the manufacturer’s instructions. Molecular detection of DENV, CHIKV, and ZIKV was performed with the use of the Quantitative RT-PCR ReadyMix™ reagents (Sigma-Aldrich, St. Louis, MO, EUA). Primers/probes used for these viruses were previously described for DENV [18], ZIKV [19] and for CHIKV [20]. qRT-PCR reactions consisted of a step of reverse transcription at 44 °C for 30 minutes of the enzyme activation at 94 °C for 2 minutes, and 40 cycles at 94 °C for 15 seconds and 60 °C for 1 minute for hybridization and extension with the use of ABI7500 equipment (Thermo Fisher Scientific, Waltham, MA, USA).

3.5. Serological characterization

Sera samples were evaluated with a commercial semi-quantitative ELISA kit (enzyme-linked immunosorbent assay), which detects anti-CHIKV IgM and IgG antibodies. All procedures were carried out according to the manufacturer’s instructions (Euroimmun, Lubeck, Germany). Briefly, sera were diluted 1:101 in sample buffer and incubated at 37 °C for 60 min in a microplate well; a calibrator, positive and negative controls, provided by the manufacturer, was used. The optical density (O.D.) was measured in an Epoch Microplate Spectrophotometer (BioTek, Vermont, USA) and the results were calculated according to the manufacturer's instructions. Values < 0.8 were regarded as negative, ≥ 0.8 to < 1.1 as borderline, and ≥ 1.1 as positive.

3.6. Phylogenetic characterization

To determine circulating CHIKV genotypes, positive samples were subjected to a reverse transcription reaction with High-Capacity cDNA Reverse Transcription Kits (Applied Biosystem, Foster City, CA, USA) following the manufacturer's recommendations. The Invitrogen protocol for PCR amplification was executed with minor modifications. Briefly, reactions were conducted in 22 µL total containing 2 µL CHIKV cDNA, 1 µL each (10 µM) forward and reverse primers previously described [21], 0.3 µL (5 U/µL) Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA, USA), 2.5 µL 10 x PCR Buffer (reaching a final concentration of 1.13 x per reaction), 1 µL (50 mM) MgSO₄, 1 µL (10 mM) dNTPs, and 13.2 µL nuclease-free water. Amplification was carried out as follows: 94 °C for 3 min, followed by 40 cycles at 94 °C for 15 s, 56 °C for 30 s, and 68 °C for 4 min, followed by a final extension at 68 °C for 10 min.

Sequencing reactions were performed with the BigDye terminator v.3.1 cycle sequencing kit (Applied Biosystems) using specific primer pairs in the Genetic analyzer automated sequencer “Applied Biosystems PRISM” 3130xl (Applied Biosystems). Chromatograms were analyzed with CodonCode Aligner 3.7.1 (Sequence Assembly and Alignment Software — CodonCode Corporation) with a Phred quality score of 20 as the cut-off for trimming of low-quality sequences. Sequences were aligned using Clustal X2 [22] and the alignment was manually edited using AliView [23]. Viral phylogenies based on the partial E1-3’NTR region were estimated using Maximum Likelihood (ML) considering the nucleotide substitution model general time-reversible with gamma-distributed rate variation (GTR+G) and 10,000 replicates implemented in FastTree 2 [24]. The final tree was then visualized and plotted using FigTree v.1.4.2 (http://tree.bio.ed.ac.uk).

3.7. Data analysis

The available information from the patient's medical records was analyzed together with the molecular and serological data. Statistical analysis was performed using the online contingency table analysis from the GraphPad Prism website assuming a two-tailed Fisher’s exact test. Statistical significance was assessed, considering 95% confidence intervals, and p values below 0.05.

4. Results

The CHIKV prevalence in the samples was 75.35% (107/142) considering the results of qRT-PCR and/or IgM-ELISA. 64.1% (91/142) of the patients were positive for viral RNA, with low dispersion and CT (cycle threshold) values at the beginning of infection, with an increase in both parameters in the cases with longer periods after the onset of symptoms (Figure 2). Moreover, 11.3% (16/142) were only positive for IgM in the whole cohort (Table 1). There were no statistically significant differences between the ages and sexes of the CHIKV positive individuals, but the majority of samples were of female patients, 61.7% (66/107), and in general patients were between 15 to 30 years old, 44.9% (48/107) (Table 2). We found CHIKV circulation in eleven out of thirteen cities in Sergipe, shown in Table 3.Symptomatic patients had statistically significant (p < 0.05) association with both fever and asthenia. Importantly, fever associated with CHIKV-positive cases, while asthenia associated with CHIKV-negative cases. However, reported symptoms such as joint pain, hemorrhagic signs, headache, rash, signs of meningoencephalitis, itching, retro-orbital pain, abdominal pain, myalgia, and vomiting had no significant association with acute CHIKV infection.

Figure 2. Threshold cycle according to days of symptoms in positive patients.

The initial phase of infection with low dispersion and CT (cycle threshold) values, and increase in both parameters, CT, and dispersion, during the final viremia. Boxplots represent the 75th percentile, median, 25th percentile and the whiskers extend to the highest and lowest value in the 1.5x interquartile range.

Serological results showed that 4.2% (6/142) patients were IgG positive, and all of them were also IgM positive (Table 1). No DENV or ZIKV genomic RNA was detected in all sera samples.

A phylogenetic tree, including sequences from Sergipe and representative sequences for all the known genotypes, indicated that the Sergipe sequences belonged to a monophyletic group, characterized as ECSA and were related to sequences isolated in previous years in Northeast region of Brazil between 2014 to 2016 (Figure 3).

Figure 3. Maximum likelihood phylogeny of CHIKV partial E1-3’NTR region sequences.

The dataset consist in 36 CHIKV sequences with: West African genotype (n = 5), Asian genotype (n = 7), ECSA genotype (n = 19) and the sequences generated in this study (n = 5). All sequences used are labeled in the format: genotype/accession number/country/year of isolation. Bootstrap scores are shown next to well-supported nodes and the phylogeny was midpoint rooted. The Brazilian sequences belonging to the ECSA genotype are highlighted in blue, and the sequences characterized in the present study from Sergipe-2016 are highlighted with the dot in black color.

5. Discussion

Here we present clinical, serological and molecular data showing a high prevalence of CHIKV genotype ECSA in symptomatic patients from a susceptible population spread over a considerable geographical region of Sergipe. CHIKV was first identified in Brazil in September 2014, with two distinct introductions, one in Oiapoque-Amapá in the North Region caused by the Asian genotype, and another in Feira de Santana-Bahia in the Northeast region of Brazil, caused by the ECSA genotype [12]. In 2015, the Northeast region of Brazil was hit by a large outbreak caused by the ZIKV associated with the co-circulation of the CHIKV and DENV [13], with cases of co-infections [25].

The State of Sergipe is located in a region with intense DENV [26] and ZIKV [17] circulation in previous years, due to the presence of Aedes aegypti, which is the main vector responsible for the circulation of these viruses in the urban settings [27,28]. In this study, we highlight a high positivity of CHIKV genomic RNA detection in symptomatic patients in the state of Sergipe during the summer of 2016. This was the first study that showed the circulation of the virus in susceptible population in Sergipe. Moreover, it revealed that the virus was widely distributed in that State, being identified in 11 of the 13 cities sampled.

It is noteworthy that CHIKV virus caused a major outbreak in Salvador, capital of the State of Bahia at the end of 2015-2016, and apparently, 16.7% of symptomatically infected patients were PCR positive [29]. Moreover, in a seroepidemiological study carried out in the district of Chapada-Bahia, IgM and IgG antibodies were detected in 20% of the sampled individuals, indicating that virus had already spread in that region [30].

The absence of association of infection with the age and gender was characteristic of emerging viruses spreading among an immune-naïve population [12]. Our findings agree with those of Cunha et al. [30], which reported fever among seroconverters in Brazil. Nevertheless, we further observed that fever was the symptom associated with the acute, initial phase of infection, which was also consistently reported by previous studies in other countries [32,33]. Recent studies suggest that during the beginning of the infection, there are differences in gene expression, of several pathways involved in the biosynthesis of molecules related to antiviral responses (e.g., immunological, inflammation, energy deprivation, and joint pain), correlating with symptoms shown at the acute phase of infection [33,34].

Taken together, symptoms that are collectively associated with CHIKV infections were present in the majority of our cases. Crucially, only fever was statistically associated with the infection, while symptoms such as joint pain (98/107), headaches (93/107) and myalgia (74/107) did not show statistical significance. We argue that another agent causing asthenia was co-circulating with CHIKV in these localities.

The phylogenetic characterization of the sequences from the state of Sergipe indicated that they were closely related, given the high bootstrap support, to other isolates characterized during previous years in the Northeast region of Brazil [12,25,28,35]. These results showed that the ECSA genotype is an important genotype associated with the emergence and persistence of the cases of Chikungunya fever in this region of Brazil. Specific and sensitive laboratorial diagnostic of different arboviruses is a goal to be achieved. Nevertheless, it is important to account for clinical signs and symptoms as relevant in helping the attending physician to choose among diagnostic tools [13,33].