Evidence of Low-Level Dengue Virus Circulation in the South Region of Cameroon in 2018

Introduction

Dengue fever is an arthropod-borne viral disease caused by any of four known dengue virus (DENV) serotypes and is transmitted primarily by female Aedes aegypti mosquitoes in Africa and by Aedes albopictus, a secondary dengue vector in Asia, which has spread to North America and >25 countries in Europe. Although the majority of DENV infections are mild or asymptomatic, ∼25% of infected individuals experience severe febrile disease. Dengue fever is primarily found in tropical and subtropical regions around the world and is considered an emerging disease. However, despite the high number of global dengue fever cases, there are still knowledge gaps surrounding DENV prevalence in Africa. In Cameroon evidence for DENV circulation has been sporadic, but is increasing (Kuniholm et al. 2006, Tchuandom et al. 2018). To date, DENV serotypes 1 and 2 have been detected in Cameroon using serology or quantitative PCR (qPCR) (Fokam et al. 2010, Demanou et al. 2014, Monamele and Demanou 2018, Nemg Simo et al. 2018, Yousseu et al. 2018). The goal of this study, which was undertaken as part of the global One Health project PREDICT on zoonotic disease surveillance, was to screen human blood for flavivirus RNA using broad range conventional PCR. Samples were collected from individuals in urban, peri-urban, and rural sites in South Cameroon, which is considered to be a hot spot for zoonotic disease transmission.

Materials and Methods

The sampling for this study was carried out in two district hospitals in the South Region of Cameroon (Meyomessala and Sangmelima District Hospitals) and in surrounding communities in the Dja-et-Lobo Department. Hospital surveillance of syndromic patients and community sampling of healthy individuals were implemented in collaboration with staff at the district hospitals who had been trained in the protocols for sample collection and processing, biosafety, and research ethics. The inclusion criteria for syndromic patients were a fever of at least 38°C and symptoms related to emerging infectious diseases, such as hemorrhage, encephalitis, diarrhea, and vomiting. Healthy community members were included as a method to uncover background infections or those that had not yet progressed to febrile disease. The limit for the inclusion of children was 20% in each group, whereas there was no selection for gender. Participation in this study was voluntary and written informed consent was obtained from adult participants or from parents or guardians of children, and child assent obtained from children aged between 12 and 17 years old. To ensure anonymity of the data, a unique code was assigned to each participant, and all information collected was stored in compliance with national and international rules of confidentiality. Participants underwent a quantitative interview and biological sample collection, which involved drawing whole blood, of which 1 mL was stored in Trizol (Invitrogen, Waltham, MA) and 1 mL in universal viral transport medium (BD, Franklin Lakes, NJ) and frozen at −80°C. Samples were transferred to the Military Health Research Center (CRESAR) in Yaoundé in a liquid nitrogen dry shipper.

At CRESAR total RNA was extracted from the whole blood specimens in Trizol and cDNA generated, whereas the universal viral transport medium was kept as a backup for potential viral growth experiments. All extracted samples were tested with a broad range PCR for flaviviruses capable of detecting all four DENV serotypes (Moureau et al. 2007), as well as a Cytochrome B PCR to verify successful extraction of genetic material; both were run as conventional PCRs. Bands of the corresponding size for flavivirus amplicons were sent for cloning and sequencing (Macrogen, South Korea), and sequences were compared with the GenBank database using the BLASTn tool.

Results

Between September 2017 and September 2018, 649 individuals were enrolled from district hospitals in Meyomessala (216) and Sangmelima (218), and from the Sangmelima community (215); 649 questionnaires were collected, and 629 of these individuals also agreed to provide whole blood samples. Patients in the hospitals were primarily diagnosed with malaria, typhoid, and/or HIV, with 22% of patients from Sangmelima and 8% from Meyomessala having more than one of these infections concurrently (Fig. 1). None of the enrolled patients was diagnosed with dengue fever or any other flavivirus infection at the hospital level. All laboratory-processed samples passed the Cytochrome B quality test, and two were also found to be positive for flavivirus RNA, accounting for an overall 0.3% positive rate, or 0.5% in the wet season only. The sequencing of the amplified products revealed sequences identical with DENV-1 isolate Gabon 2012 (MG877557) in both cases. The two patients who were found to be infected with DENV were both men living in Meyomessala. The first patient, a 21-year-old student reporting chills, fever, headache, and loss of appetite, was seen in the Sangmelima District Hospital in May 2018, and was diagnosed with malaria through microscopy. The second patient, a 60-year-old construction worker reporting abdominal pain, fever, headache, chills, and loss of appetite, was seen at the Meyomessala District Hospital in August 2018, and was diagnosed with malaria through microscopy and typhoid fever through Widal test.

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FIG. 1.

Map of Cameroon showing sampling locations (red and yellow dots) with key findings as well as sites of previous dengue detection (black and gray squares and hexagons). Color images are available online.

Discussion

The DENV prevalence observed in this study in South Cameroon is significantly lower than in other PCR-based studies conducted in Cameroon, which detected DENV-1 RNA in between 7% and 11% of febrile patients (Nemg Simo et al. 2018, Yousseu et al., 2018). However, these previous studies used a different assay (qPCR) and were conducted in different geographic areas, with further differences in study duration (much shorter) and population compared with this study. The degree of urbanization and associated socioeconomic factors certainly influence the risk for dengue transmission, as does the vicinity to the sea. The city of Douala, for example, is a large urban center and the samples from the coastal city Kribi were collected after a suspected dengue fever outbreak. The low prevalence and months in between the cases do not indicate a classical dengue fever outbreak in our case, but might rather point toward endemic circulation with either fewer susceptible individuals due to previous infections, limited vector presence (rural environment), or a local nonhuman reservoir (sylvatic cycle) (Fagbami et al. 2018). The sequence identity with an isolate from neighboring Gabon suggests a local circulation of that strain.

None of the DENV positive patients, nor any of the other enrolled individuals, was diagnosed with dengue fever on the hospital level. Considering that both DENV positive patients were also diagnosed with malaria at the same time, and one was also diagnosed with typhoid (for a total of three simultaneous coinfections), that observation does not seem surprising, as symptoms are overlapping. With DENV screening being primarily limited to research studies, and routine testing not readily available, coupled with a high prevalence of both malaria and typhoid, dengue fever is thus likely underdiagnosed in Cameroon, especially as it appears that typhoid is dramatically overdiagnosed (Nsutebu et al. 2003). The conventional, relatively easy, and inexpensive broad range reverse transcription (RT)-PCR proved successful in detecting DENV in whole blood samples.

Overall, the results from this study support previous findings, which indicate at least a low-level circulation of DENV in Cameroon. They also indicate that dengue fever is likely underdiagnosed and point toward a need for rapid tests being readily available to clinicians.