Seroepidemiology of Chikungunya Virus Among Febrile Patients in Eight Health Facilities in Central and Northern Mozambique,2015

Abstract

Background:

The burden and spread of chikungunya virus (CHIKV) are rapidly increasing worldwide, but the epidemiology in Mozambique is barely known. The aim of this study was to determine the seroepidemiology of CHIKV in central and northern Mozambique.

Methods:

A cross-sectional study was conducted between March 2015 and May 2016 in eight health facilities situated in central and northern Mozambique to recruit 392 patients with undifferentiated febrile illness from outpatient clinics. Serum samples from each participant were screened using commercially available enzyme-linked immunosorbent assay for detection of anti-CHIK IgM and IgG antibodies. A subset of study samples (n = 37) was further tested by the plaque reduction neutralization assay (PRNT).

Results:

The median age of participants was 23 years (IQR: 7–34), and 45.7% were female. The frequency of participants with seropositivity for IgM and IgG anti-CHIKV antibodies was 1.5% (6/392) and 28.6% (112/392), respectively. Patients with seropositivity for IgM anti-CHIKV were significantly younger. Frequency of patients with seropositivity for IgG anti-CHIKV increased with age. Frequency of patients with seropositivity for IgM anti-CHIKV was higher in Tete province, but most patients with seropositivity for IgG anti-CHIKV infection were from Cabo Delgado and Sofala provinces.

Conclusions:

Our data demonstrate serological evidence of CHIKV in central and northern Mozambique, expanding the limited evidence of the virus in the country. We recommend that CHIKV should be considered in the differential diagnosis of febrile illness throughout the country.

Introduction

Chikungunya fever (CHIK) is an arbovirus caused by chikungunya virus (CHIKV), an alphavirus of the Togaviridae family. The virus is primarily transmitted to humans by the bite of infected Aedes aegypti and Aedes albopictus (Staples et al. 2009, Kucharz and Cebula-Byrska 2012, Lo Presti et al. 2014). The most common symptomatology is fever accompanied by rash, headache, myalgia, and arthralgia (Ng Ching and Hapuarachchi 2010, Burt et al. 2012, Rougeron et al. 2015). CHIKV is emerging as a global threat because of its rapid spread worldwide. The virus was first isolated in 1952/3 during an outbreak in villages in southern Tanzania, which also affected few villages in northern Mozambique (Lo Presti et al. 2014, Gudo et al. 2016).

In recent years, the number of countries reporting CHIKV for the first time increased significantly (Doudou et al. 2015, Rana and Lunia 2015, Luksic et al. 2017) and autochthonous transmission of the virus had been reported in temperate countries in Europe (Liumbruno et al. 2008, Delisle et al. 2015, Tanay 2016). Of concern, CHIKV has no specific treatment or vaccine and reports of cases of severe diseases are increasing (Her et al. 2009, Caglioti et al. 2013, Crosby et al. 2016, Karen et al. 2017).

Main drivers of rapid spread of CHIKV worldwide include the following: (i) changes in vector distribution pattern and its abundance due to climate change, (ii) increased contact between people and vector, due to the progressive invasion of the wild environment, (iii) rapid and unplanned urbanization, and (iv) increase of international trade (Chevillon et al. 2008, Her et al. 2009, Meason and Paterson 2014, Weaver 2014, Rana and Lunia 2015).

In Mozambique, data on CHIKV epidemiology are scarce and the disease is heavily neglected. Consequently, CHIKV is not considered in the differential diagnosis of fever in the country. The few studies on CHIKV in the country are very old or were conducted in Maputo city, which is situated in the southern part of the country (Samo et al. 2016). The risk of CHIKV in central and northern Mozambique is considered high, considering that cases of CHIKV have been reported in recent years in Tanzania, a country that shares the border with Mozambique in the north (Ganu and Ganu 2006, Kajeguka et al. 2016). Also, a large entomological survey conducted in 2016 in 32 districts in Mozambique found that the highest container index of A. aegypti was found in the center and north of the country (MISAU 2017).

The aim of the study was to investigate the frequency of CHIKV in febrile patients attending outpatient clinics in eight health facilities situated in five provinces in central and northern Mozambique.

Materials and Methods

Study design and settings

A cross-sectional study was conducted in eight health centers, one district hospital, one provincial hospital, and one central hospital, situated in five cities (Fig. 1) from central and northern Mozambique, namely Pemba city (Natite Health Center and Pemba Provincial Hospital), Lichinga city (Lichinga Health Center), Nampula city (Nampula Central Hospital), Tete city (Number 2 Health Center, Number 3 Health Center, and Moatize Health Center), and Caia district (Caia District Hospital). Mozambique is situated in the southeast coast of Africa.

Fig. 1.

Geographical location of study sites.

The climate in Mozambique is tropical with two distinct seasons, the rainy season from November to April and cold season from May to October. Although more than 50% of the population lives in rural areas, the country is undergoing rapid and unplanned urbanization, resulting in rapid expansion of poor slums where population density is high, sanitation is precarious, and breeding sites for mosquitoes are abundant.

Lichinga, Pemba, Nampula, and Tete cities are capital of Niassa, Cabo Delgado, Nampula, and Tete provinces. These cities comprise a small urban area, surrounded by a large suburban slum, with precarious and overcrowded dwellings and poor sanitation. Of these cities, Nampula is the most populous, with highest precipitation, Lichinga is the coldest city, and Tete is the warmest city with lowest precipitation and lowest humidity rate. Pemba city has the highest humidity. Caia is a rural village and is the capital of Caia District.

Participants and sample collection

A total of 392 patients with undifferentiated fever who attended the outpatient clinic at the study sites, between March 2015 and May 2016, were enrolled in this study. Individuals with psychiatric disease or with a readily identifiable focus of infection, such as otitis, sinusitis, purulent pharyngitis, cellulitis, urinary tract infection, dental abscess, septic arthritis, pneumonia, or pelvic inflammatory disease, were excluded.

A total volume of 12 mL of blood was requested from each participant and collected into a 6-mL K3EDTA tube and into a 6-mL serum separation tube (both from BD Vacutainer). Serum samples were aliquoted and kept at −20°C until shipment to the Virus Isolation Laboratory (VIL) at the National Institute of Health in Maputo. On receipt at VIL, all serum samples were stored at −80°C until testing.

Ethics statement

The protocol of this study was approved by Mozambique's National Bioethics Committee for Health (ref#: IRB00002657). Written and informed consent was obtained from each eligible individual before enrollment.

Case definition

A case of CHIKV seropositivity was defined as the presence of positive results for anti-CHIK IgM or IgG antibodies using enzyme-linked immunosorbent assay (ELISA). Participants with negative results for anti-CHIK IgM and IgG antibodies were labeled as negative for CHIK infection.

Questionnaire

A questionnaire was used to collect clinical, epidemiological, and demographic information from all patients.

Laboratory testing

All serum samples (n = 392) were tested using ELISA at VIL, in Maputo, Mozambique, and a subset of these samples (n = 37) was randomly selected among ELISA-positive and ELISA-negative samples shipped and tested by neutralization assay at the Institute of Virology, University of Bonn Medical Centre, Bonn, Germany, using a VRP neutralization assay.

Enzyme-linked immunosorbent assay

For serological screening, all serum samples were tested using commercial ELISA kits for the detection of anti-CHIKV IgM and IgG antibodies (EUROIMMUN AG, Lübeck, Germany), following the instructions provided by the manufacturer.

Polymerase chain reaction

RNA was extracted using the QIAamp RNA Viral Mini Kit (QIAGEN, Inc., CA) following the manufacturer's instruction. For amplification of CHIKV RNA, a real-time reverse-transcriptase polymerase chain reaction assay (RT-PCR) was carried out using the CHIKV real-time RT-PCR diagnostic panel and protocol developed and kindly provided by Karolinska Institutet, Sweden. For this PCR, we used the following sequence of primers and probes: F: 5′-TCGCATCTAGCTATAAAACTAATAGAGCAG-3′ and R: 5′-CTGTCCGACATCATCCTCCTTG-3′ and probe 5′-CGACTCAACCATCCTG −3′ (FAM-MGB).

The amplification was carried out in 25 μL reaction mixtures containing 5 μL RNA template, nuclease-free water, SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase, 0.9 μM of each primer, and 0.2 μM probe. We used the following cycling parameters: reverse transcription at 50°C for 30 min, inactivation at 95°C for 2 min followed by 45 cycles of fluorescence detection at 95°C for 15 s, and annealing at 60°C for 1 min.

VRP neutralization assay

The VRP neutralization assay was performed on BHK-21 cells as described previously (Gläsker et al. 2013). VRPs at MOI 2,5 (calculated based on VRP RNA copies/mL) dilutions were incubated with serially diluted human serum for 1 h at 37°C before adding the mixture to a monolayer of BHK-21 cells in a 96-well plate (total volume per well 40 μL). After incubation for 1 h at 37°C, the inoculums were removed, cells were washed once with PBS, and medium was added. Supernatants for Gluc measurement were taken at 24 h. The luciferase reporter assay was performed using a Renilla Luciferase Assay System (Promega, Madison, WI) and measurement was performed automated in a Synergy 2 microplate reader (BioTek) using polystyrene microplates (Greiner Bio-one).

Neutralization potency was determined as percentage of measured Gluc activity compared to Gluc readout after VRP application without serum (Gläsker et al. 2013).

Statistical analysis

Data were analyzed using the statistical software package STATA 12.0 (College Station, TX: StataCorp, 2011). Study groups were compared using Fisher's exact test for categorical variables and Kruskal–Wallis for numerical variables (medians). A p-value <0.05 was considered statistically significant.

Results

Sociodemographic characteristics of the study participants are depicted in Table 1. The median age of study participants was 23 years (IQR: 7–34), and 45.7% were female. The majority of participants had a Mozambican citizenship (260/283; 91.9%). A total of 6 (1.5%) patients showed seropositivity for anti-IgM CHIKV infection and 112 (28.6%) were seropositive for anti-IgG CHIKV infection. Frequency of patients with seropositivity for anti-IgM CHIKV infection was higher in Tete (3/6; 50%) and for anti-IgG CHIKV infection was higher in Pemba city (50/112; 44.6%) and Caia district (37/112; 33.1%). Frequency of patients with anti-IgG CHIKV-positive results increased with age, peaking at age category of [25–49] years.

Table 1.

Clinical and Demographic Characteristics of Study Participants

Characteristics	All participants, n (%)	Anti-IgM CHIKV infection (%)	Anti-IgG CHIKV infection (%)	Negative CHIKV infection, n (%)	p-value ^a
Total	392 (100)	6/392 (1.5)	112/392 (28.6)	274/392 (69.9)
Sex	n = 352	n = 6	n = 104	n = 242
Male	191 (54.3)	2 (33.3)	64 (61.5)	125 (51,6)	0.173
Female	161 (45.7)	4 (66.7)	40 (38.5)	117 (48.3)	0.173
Age, median (IQR)	23 (7–34)	10 (3–25)	32 (25–40)	18 (0–29)	0.000^b
[0–5]	82 (20.9)	3 (50.0)	6 (5.3)	73 (27)	0.000
[6–15]	42 (10.7)	0 (0.0)	3 (2.7)	39 (14)
[16–24]	71 (18.1)	1 (16.7)	18 (16.1)	52 (19)
[25–49]	153 (39.0)	2 (33.3)	73 (65.2)	78 (28)
[>49]	33 (8.4)	0 (0.0)	12 (10.7)	21 (8)
NI	11 (2.8)	0 (0)	0 (0)	11 (4)
Nationality	n = 283	n = 6	n = 103	n = 174
Mozambique	260 (91.9)	6 (100.0)	102 (99.9)	152 (87.4)	0.002
Other	23 (8.1)	0 (0)	1 (0.1)	22 (12.6)	0.002
Academic level	n = 201	n = 5	n = 90	n = 106
None	31 (15.2)	4 (80.0)	15 (16.7)	12 (11.3)	0.001
Primary	77 (38.3)	0 (0)	34 (37.8)	43 (40.6)
Secondary	71 (35.3)	1 (20.0)	36 (40.0)	34 (32.1)
Higher level school	22 (11.0)	0 (0)	5 (5.4)	17 (16.0)
Study sites	N392	n = 6	n = 112	n = 274
Pemba city	166 (42.3)	1 (16.7)	50 (44.6)	115 (42.0)	0.000
Nampula city	110 (28.1)	0 (0)	8 (7.1)	102 (37.2)
Tete city	45 (11.5)	3 (50.0)	11 (9.8)	31 (11.3)
Lichinga city	25 (6.4)	1 (16.7)	6 (5.4)	18 (6.6)
Caia district	46 (11.7)	1 (16.7)	37 (33.1)	8 (2.9)
Clinical signs and symptoms
Fever	242 (62.5)	3 (50.0)	93 (83.0)	146 (54.1)	0.000
Headache	236 (60.2)	4 (66.7)	89 (79.5)	143 (52.1)	0.000
Myalgia	156 (40.5)	2 (33.3)	59 (52.7)	95 (35.1)	0.000
Arthralgia	135 (34.9)	2 (40.0)	51 (45.9)	82 (30.3)	0.014
Retro-orbital pain	53 (13.5)	1 (16.7)	21 (18.8)	31 (11.3)	0.149
Photophobia	33 (8.4)	1 (16.7)	9 (8.0)	23 (8.4)	0.719
Chills	187 (47.7)	2 (33.3)	66 (60.0)	119 (44.4)	0.017
Asthenia	46 (11.7)	0 (0.0)	9 (8.0)	37 (13.5)	0.212
Vomiting	51 (13.0)	1 (16.7)	16 (14.3)	34 (12.4)	0.852
Abdominal pain	52 (13.3)	1 (16.7)	12 (10.7)	39 (14.2)	0.632
Cough	20 (5.1)	2 (33.3)	9 (8.1)	9 (3.3)	0.001
Exanthema	15 (3.8)	0 (0)	1 (0.9)	14 (5.1)	0.130
Bruise	3 (0.8)	0 (0)	1 (0.9)	2 (0.7)	0.963
Petechiae	11 (2.8)	1 (16.7)	2 (1.8)	8 (2.9)	0.097
Epistaxis	9 (2.3)	1 (16.7)	2 (1.8)	6 (2.2)	0.059

Statistical significance was set at p < 0.05.

Pearson chi-squared test.

Mann–Whitney test.

The median age of the patients with seropositivity for anti-IgM CHIKV infection (median: 10 years; IQR: 3–25 years) was significantly lower, compared with those with seropositivity for anti-IgG CHIKV (median: 32 years; IQR: 25–40 years).

Common sign and symptoms were fever (242/387; 62.5%), headache (236/392; 60.2%), chills (187/392; 47.7%), myalgia (156/385; 40.5%), and arthralgia (135/387; 34.9%). Patients with seropositivity for anti-IgG CHIKV infection presented a slightly higher frequency of arthralgia (51/111; 45.9%), compared with patients with seropositivity for anti-IgM CHIKV infection (2/5; 40.0%) or with negative CHIKV infection (82/271; 30.3%). Moreover, fever (p = 0.000), headache (p = 0.000), myalgia (p = 0.000), and chills (p = 0.017) were more common in patients with seropositivity for anti-IgG CHIKV infection.

A subset of 37 samples was selected and shipped to the Institute of Virology, University of Bonn Medical Centre, Bonn in Germany, for assessing the level of agreement between ELISA and neutralization assay. As shown in Fig 2, there was a very high agreement between ELISA IgG-positive results (100%, n = 10) and ELISA IgM/IgG-positive results (88.2%, n = 17) and neutralization activity. Agreement was also high (100%) between ELISA-negative results and neutralization test. All ELISA-positive samples were negative when tested by real-time PCR.

Fig. 2.

Flowchart of patient recruitment and sample testing.

Discussion

No recent data on CHIKV in northern and central Mozambique are available. We found a frequency of patients with anti-IgM CHIKV- and anti-IgG CHIKV-positive results of 1.6% and 28.6%, respectively. Similar pattern was observed in a recent study conducted by our group in Maputo city, situated in southern Mozambique in 2013, and frequencies of IgM and IgG CHIKV antibodies were 4.3% and 21.2%, respectively (Samo et al. 2016).

This suggests that CHIKV also circulates in central and northern Mozambique. These findings are not surprising considering the following aspects: (i) several cases of CHIKV had been reported in Tanzania, which shares borders with Mozambique in the north (Chipwaza et al. 2014, Kajeguka et al. 2016), (ii) a recent entomological survey conducted in 32 districts in Mozambique found that A. aegypti was abundant in northern and central of the country (MISAU 2017), (iii) a recent case of severe CHIKV infection that occurred in northern Mozambique was recently reported by our group (Aly et al. 2017), and (iv) an old study conducted in nonfebrile individuals more than 50 years ago found serological evidence of CHIKV in several places in Mozambique, including in central and northern Mozambique (Kokernot et al. 1960).

Data of this study also showed that frequency of seropositivity for anti-IgG CHIKV infection increased with age, suggesting that the virus is circulating in these places for several years or decades, and probably caused unsuspected outbreaks in the past years.

Arthralgia is considered the most important symptom of CHIK, which in certain patients can become debilitating (Kucharz and Cebula-Byrska 2012). Our data showed that frequency of arthralgia was higher in patients with seropositivity for anti-IgG CHIKV, when compared to other groups. Unfortunately, we did not collect information on the duration of arthralgia to ascertain if these patients had chronic arthralgia or not. In addition, due to the transversal design of our study, the elapsed time since the last exposure to CHIKV was unknown. We recommend that longitudinal studies should be conducted to determine the chronic implication sequelae of CHIK in Mozambique.

A subset of samples was tested by neutralization assay. Our result showed a high level of agreement between ELISA IgG-positive results and neutralization results.

Lack of genomic RNA in all ELISA IgM-positive samples is not surprising, as previous studies from Mozambique and other countries in sub-Saharan Africa found similar results (Samo et al. 2015). This might be explained by the poor cold chain in Mozambique, which resulted in degradation of RNA, as well as due to the transient viremia of chikungunya (Caglioti et al. 2013).

We acknowledge few limitations of our study, such as the fact that the size of the group of IgM-positive patients was small and for this reason statistical comparison with other groups should be interpreted with caution. We also acknowledge that testing only a subset and not all serum samples for neutralization was a limitation. However, neutralization was performed not to determine the final result of each patient, but to validate the performance of ELISA.

Conclusion

Data from this study suggest that CHIKV is prevalent in northern and central Mozambique. Interventions are needed to increase clinical and public health awareness and prevent and mitigate the impact of CHIKV in Mozambique.

Footnotes

Acknowledgments

We thank all participants for accepting to participate in this study and all health professionals from Natite Health Center, Caia District Hospital, Pemba Provincial Hospital, Nampula Central Hospital, N4 Health Center, Number 3 Health Center, Moatize Health Center, and Lichinga city Health Center for their support during patient recruitment.

We also thank all staff from the Virus Isolation Laboratory, Instituto Nacional de Saúde, Maputo, Mozambique.

This study was funded by the European Foundation Initiative into Neglected Tropical Disease (grant # 91 488). This work was also supported, in part, by the U.S. Centers for Disease Control and Prevention through a cooperative agreement number 5NU14GH001237-03-00. The views expressed in this written publication do not necessarily reflect the official policies of the U.S. Department of Health and Human Services.

Authors' Contributions

V.S.A., A.F.M., S.A., V.M., F.M., I.C., I.S.C., J.O., and E.S.G. participated in the study design and patient recruitment. V.S.A., A.F.M., J.W., S.A., V.M., F.M., I.C., I.C., J.O., and B.M.K. participated in sample testing. All coauthors participated in data analysis and writing of the article.

Author Disclosure Statement

No competing financial interests exist.

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Seroepidemiology of Chikungunya Virus Among Febrile Patients in Eight Health Facilities in Central and Northern Mozambique,2015–2016

Abstract

Background:

Methods:

Results:

Conclusions:

Introduction

Materials and Methods

Study design and settings

Participants and sample collection

Ethics statement

Case definition

Questionnaire

Laboratory testing

Enzyme-linked immunosorbent assay

Polymerase chain reaction

VRP neutralization assay

Statistical analysis

Results

Discussion

Conclusion

Footnotes

Acknowledgments

Authors' Contributions

Author Disclosure Statement

References