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Keywords:

  • Lassa virus;
  • Hantavirus;
  • seroprevalence;
  • Guinea

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objectives

To assess the public health relevance of Lassa arenavirus and hantavirus infections in a subpopulation of recently febrile patients.

Methods

In a human seroprevalence study, we enrolled 253 participants on the basis of reported high fever during the last 3 months. They represented roughly 20% of the population of Bantou and Tanganya villages. Comprehensive serological screening and confirmatory assays (enzyme-linked immunosorbent assay, immunofluorescence assay, Western blot analysis) with several Lassa virus and hantavirus antigens were used to ensure high specificity and broad detection capacity.

Results

We found a Lassa IgG prevalence of 40.3% (102/253) and a hantavirus IgG prevalence of 1.2% (3/253). The Lassa IgM prevalence reached 2.8% (7/253).

Conclusions

High Lassa virus seroprevalence in recently febrile patients indicates that Lassa fever is a significant public health problem in the region. Human hantavirus infections also occur in the region but their public health relevance remains to be determined.

Objectifs

Evaluer la relevance en santé publique des infections par les arénavirus Lassa et hantavirus dans une sous-population de patients récemment fébriles.

Méthodes

Dans une étude de séroprévalence humaine nous avons recruté 253 participants sur la base d'une forte fièvre rapportée au cours des 3 derniers mois. Ils représentaient environ 20% de la population des villages Bantou et Tanganya. Le dépistage sérologique complet et les tests de confirmation (ELISA, immunofluorescence, Western blot) avec plusieurs antigènes du virus de Lassa et hantavirus ont été utilisés pour assurer une spécificité élevée et une capacité de détection étendue.

Résultats

Nous avons trouvé une prévalence des IgG de Lassa de 40,3% (102/253) et une prévalence des IgG de hantavirus de 1,2% (3/253). La prévalence des IgM de Lassa atteignait 2,8% (7/253).

Conclusions

La séroprévalence élevée du virus de Lassa chez les patients récemment fébriles indique que la fièvre de Lassa est un important problème de santé publique dans la région. Les infections humaines à hantavirus surviennent aussi dans la région, mais leur relevance en santé publique reste à déterminer.

Objetivos

Evaluar la relevancia a nivel de salud pública de las infecciones por los virus de Lassa y hantavirus en una subpoblación de pacientes con fiebre reciente.

Métodos

En un estudio de seroprevalencia en humanos se incluyeron 253 participantes que reportaron haber tenido fiebre alta en los últimos 3 meses. Representaban aproximadamente un 20% de la población de los poblados de Bantou y Tanganya. Se realizaron pruebas serológicas exhaustivas y pruebas confirmatorias (ELISA, prueba de inmunofluorescencia, análisis de Western blot) con varios antígenos del virus de Lassa y hantavirus para asegurar una alta especificidad y una capacidad de detección amplia.

Resultados

Hemos encontrado una prevalencia de IgG de Lassa del 40.3% (102/253) e IgG de prevalencia de hantavirus del 1.2% (3/253). La prevalencia de IgM de Lassa alcanzó un 2.8% (7/253).

Conclusiones

Se ha encontrado una alta seroprevalencia del virus de Lassa en pacientes con historia reciente de fiebre, indicando que la fiebre de Lassa es un problema significativo de salud pública en la región. También había infecciones en humanos por hantavirus, pero su relevancia a nivel de salud pública continúa sin ser determinada.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Arenaviruses (genus Arenavirus, family Arenaviridae) and hantaviruses (genus Hantavirus, family Bunyaviridae) are zoonotic viruses that are spread by aerosolised excreta of rodents and, in the case of hantaviruses, also other small mammals. They cause chronic infections with no apparent harm in their natural hosts. Lassa virus, the most prominent arenavirus, causes Lassa fever in West Africa, where up to 300 000 clinical cases are estimated to occur annually (McCormick 1999). Other arenaviruses – such as Lujo, Junin, Machupo, Guanarito and Sabia viruses – are causative agents of haemorrhagic fevers in South Africa and America. The type species of the genus, Lymphocytic choriomeningitis virus, occasionally causes acute central nervous system disease and congenital malformations (Charrel & de Lamballerie 2003; Gunther & Lenz 2004; Paweska et al. 2009). Hantaviruses cause haemorrhagic fever with renal syndrome (HFRS) in Asia and Europe, and hantavirus cardiopulmonary syndrome in the Americas (Schmaljohn & Hjelle 1997; Kruger et al. 2001 Kruger et al. 2011; Peters & Khan 2002).

Recently, several arenaviruses and hantaviruses were found in Guinea, West Africa. Lassa virus was detected in Mastomys natalensis (Lecompte et al. 2006) and a novel arenavirus, Kodoko virus, was found in Mus minutoides (Lecompte et al. 2007). The first indigenous African hantaviruses were discovered in Guinea too; Sangassou virus in Hylomyscus simus (Klempa et al. 2006, 2012) and Tanganya virus in a shrew, Crocidura thereseae (Klempa et al. 2007).

The identification of the first African hantaviruses in 2006/07 raised the question of the relevance of hantaviruses as rodentborne pathogens additional to Lassa virus. To assess public health relevance of these two pathogens, we performed a human seroprevalence study in a subpopulation of recently febrile patients. Very recently, intensive serological search provided evidence of human hantavirus infections and HFRS-like clinical cases in Forest Guinea where Sangassou virus (SANGV) had been detected (Klempa et al. 2010). In the current seroepidemiological study, we focused on another area of Guinea: the savannah (Upper Guinea), a region where Lassa virus and Tanganya virus have been found.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Blood collection

For this serological investigation, 253 sera (9 ml each) were collected in May and October 2004 in the population of two rural villages of Upper Guinea, Bantou and Tanganya. They were located in the Faranah prefecture, where previous Lassa virus studies had been performed from 2003 to 2005 (Fichet-Calvet et al. 2007, 2008) and where, moreover, Tanganya hantavirus had been found (Klempa et al. 2007). Approval for the investigation was obtained from the National Ethics Commission of Guinea. All participants were self-recruited, after a public announcement. Sampling took place in health facility, where a doctor did consultancies after bleeding. The participants gave informed consent prior to enrolment and were enrolled on the basis of reported high fever during the last 3 months. They represented roughly 20% of the population; 132 of about 800 inhabitants of Bantou and 121 of about 500 inhabitants of Tanganya. The sample size was based on the formula n = (z2pq)/d2, with “z” corresponding to the confidence interval at 90% (1.64), “p” to the probability to be infected by Lassa in the area (0.35), “q” to the probability to not be infected by Lassa (0.65) and “d” to the precision of the result desired (0.04). The number of persons to be sampled in each village was then 87, but we increased it because of possible loss of samples between Africa and Europe, or during testing.

Serology

We used comprehensive serological screening and confirmatory assays with several Lassa virus and hantavirus antigens to ensure high specificity and broad detection capacity of our diagnostic approach.

All serum samples were tested for IgG antibodies against Lassa virus (Josiah and AV strains) by enzyme-linked immunosorbent assay (ELISA) and, in parallel, immunofluorescence assay (IFA), with serum dilutions of 1:10, 1:20 and 1:40. Reciprocal titres of 40 by ELISA and 20 by IFA were considered as positive (Wulff & Lange 1975; Emmerich et al. 2006). IgM antibodies against Lassa virus strain Bantou were tested by IFA with a serum dilution of 1:40. For IFA-positive samples, end-point titres (EPT) of IgM and IgG were determined.

All sera were screened for hantavirus IgG and IgM by ELISA in separate assays using recombinant nucleocapsid protein of three hantaviruses, Puumala virus (PUUV), Dobrava-Belgrade virus (DOBV) and SANGV, as antigens (Meisel et al. 2006; Klempa et al. 2010). If confirmed by Western blot analysis (WB) based on antigens of PUUV, DOBV, Hantaan virus (HTNV) and Seoul virus (SEOV) (recomBlot; Mikrogen, Martinsried, Germany), positive samples were further subjected to (i) an in-house immunofluorescence assay (IFA, using slides with SANGV-infected cells prepared as principally described before [Schilling et al. 2007]) and (ii) a commercial IFA kit (Hantavirus Mosaic 2; Euroimmun; Lübeck, Germany).

Molecular testing

All sera were screened for Lassa virus acute infection by RT-PCR targeted on GP gene (Olschlager et al. 2010). The extraction of RNA was performed by pooling the sera by 3 and using the QIAamp viral RNA kit (Qiagen, Hilden, Germany). The primers used for amplification were S36 (5′-ACCGGGGATCCTAGGCATTT-3′) and LVS339 (5′-GTTCTTTGCGCAGGA(AC)AGGGGCAT(GT)CAT-3′).

Data analysis

The association of several variables such as the village (Bantou, Tanganya), the month of collection (May vs. October 2004), the sex and the age (considered as continuous variable) with Lassa IgG serological status was analysed by multiple logistic regression, using a binary factor (infected = 1, noninfected = 0) as the dependent variable and the independent variables described above. The level of significance was chosen for P = 0.05, and the analysis was performed with the software Statview (SAS Institute, Inc., Cary, NC).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Lassa virus

The overall Lassa IgG prevalence was 40.3% (102/253) by both methods (ELISA, IFA). The seroprevalences seemed similar in Bantou (42%; 55/132) and in Tanganya (39%; 47/121), in May 2004 (41%; 84/207) and in October 2004 (39%; 18/46), in females (39%; 45/116) and in males (42%; 57/137). This is confirmed by multivariate logistic regression in which the village (OR = 0.89, P = 0.67), the month of collection (OR = 0.89, P = 0.77) and the sex (OR = 1.03, P = 0.92) have no influence on the observed seroprevalences. The distribution by age shows the lowest seroprevalences during childhood and old age (20-30%, Figure 1) and the highest ones between 20 and 80 years (40–50%, Figure 1), although seroprevalences did not differ statistically by age (OR = 1.01, P = 0.09). Five persons, two females (40 and 50 years old) and two males (70 and 90 years old) in May, and one male (40 years old) in October had reciprocal IgG end-point titres (EPT) >1 280 as determined by IFA. Additionally, seven persons were IgM-positive; five in Bantou and two in Tanganya. The detailed EPT of the IgM-positive sera are summarised in Table 1. None of these sera were found to be RT-PCR-positive.

Table 1. Reciprocal end-point titres for the anti-Lassa virus IgM-positive persons
SampleSexAgeVillageDate of samplingIgM IFA BANTOUIgG IFA JOSIAHIgG IFA AVIgG IFA BANTOU
H33M71BantouMay 2004640160320160
H49F17BantouMay 20042560512051205120
H56M60BantouMay 20041280320320320
H140M75BantouMay 20041608016080
H165M36BantouMay 20042560512051205120
H182M40TanganyaMay 2004640negnegneg
H309M40TanganyaOct 200410240512051205120
image

Figure 1. Lassa IgG prevalence (n = 102 seropositives) in two villages located in Upper Guinea, Bantou and Tanganya, by age and by month of collection. Numbers above each bar show the absolute numbers of IgG-positives, numbers below the diagram the numbers of investigated persons.

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Hantavirus

Of 253 tested sera, 32 (12.2%) were initially found to be reactive in screening ELISA. However, finally only three of them (1.2%) were confirmed to be positive also in both WB and IFA. Antibody titres observed by IFA in positive serum samples were rather low (Table 2, Figure 2). One of the three IgG-positive sera (H3) showed weak reactivity also in IgM ELISA (data not shown); moreover, one serum (H186) was found to be hantavirus- and Lassa virus antibody-positive (cp. Table 2).

Table 2. Results of hantavirus confirmatory assays of finally positively scored serum samples
SampleSexAgeVillageIgG Western blotaIgG IFAb
PUUV+HTNVPUUVHTNVDOBVSEOVSFVSANGVcomb.
  1. ELISA-positive sera that were confirmed by western blot and IFA are shown.

  2. a

    +++, ++, +, +−, − rating of densities according to the manufacturer (Mikrogen); PUUV, Puumala virus; HTNV, Hantaan virus; DOBV, Dobrava-Belgrade virus; SEOV, Seoul virus; SFV, Sandfly fever virus.

  3. b

    Reciprocal end-point titres are given. -, negative; ND, not done; comb., antigen combination, Hantavirus Mosaic 2 IFA kit (Euroimmun); SANGV, Sangassou virus.

  4. c

    serum showed Lassa IgG antibodies, too (strains Josiah and AV).

H 3M75Bantou+++++++++40
H 139M65Bantou+++++ND100
H 186cM54Tanganya+++−+−ND100
image

Figure 2. Scheme and final results of the IgG screening algorithm employed in the study. In every box (besides the summarising one on the bottom of the figure), the first line indicates the serodiagnostic method, the second line the origin of used antigens and the third line the obtained results and final seroprevalence value. The last box shows the summing-up of the investigation. ELISA, Enzyme-linked immunosorbent assay; WB, Western blot; IFA, immunofluorescence assay; comb, Hantavirus Mosaic 2 IFA kit (Euroimmun); DOBV, Dobrava-Belgrade virus; PUUV, Puumala virus; SANGV, Sangassou virus; HTNV, Hantaan virus; SEOV, Seoul virus; LASV, Lassa virus.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In the Faranah region of Upper Guinea, the human IgG prevalence for Lassa virus is 40%, which is similar to the seroepidemiological data collected 13 years earlier in Gbetaya (43% seroprevalence) and Sangoyah (42% seroprevalence), two villages near those in our study (Lukashevich et al. 1993). The two other villages included in the previous study and showing lower seroprevalences, Tindo (33%) and Kamaraya (25%), are located along the main road but not in deep savannah. Epidemiologically, this seems to have an impact because the villages along the road are mainly infested by the black rat (Rattus rattus) and the domestic mouse (Mus musculus), which are not reservoirs of Lassa virus.

In our study in May 2004, the Lassa IgG seroprevalence values did not significantly differ in the age classes from 20–29 to 70–79 years. This finding suggests that most of the infections occur already during young age (until age 20). A possible decline in antibody prevalence with age might be compensated by few primary and secondary infections in adults, as evidenced by 7 IgM-positive adults in our study. The IgG seroprevalence of 30 – 50% in the savannah region differs mainly from those observed in forest Guinea, where the IgG prevalences were 5 – 30% (ter Meulen et al. 1996; Kerneis et al. 2009).

To determine Lassa fever incidence, we also analysed IgM seropositivity, which indicates recent infection. Lassa virus IgM usually peaks at 10–30 days after onset of fever and then decreases after 80 days (Wulff & Johnson 1979; Johnson et al. 1987). In this study, the Lassa IgM seropositivity reached 2.8%. To our knowledge, this is the first evaluation of Lassa IgM seropositivity of the rural population in a population-based study.

For hantavirus infections, the observed IgG seroprevalence of 1.2% is identical to that found in the forest of Guinea (Klempa et al. 2010), but much lower than that reported earlier from Gabon (8%; Dupont et al. 1987) and Central African Republic (4%; Gonzalez et al. 1988). The latter values probably are overestimates of antibody prevalence because no confirmatory tests were performed. On the one hand, a battery of different serological assays seems to be necessary to avoid false-positive results that might be caused by unspecific reactivity of sera from African people. On the other hand, it needs to be stated that a battery of assays might considerably decrease the sensitivity. However, in this very first assessment in the area, our priority was to clearly confirm whether human hantavirus infections occur. We therefore focused on specificity rather than sensitivity. Nevertheless, a seroprevalence of 1–2% is typical for hantavirus-endemic countries in other parts of the world, such as Germany (Zöller et al. 1995).

TANGV is, at the moment, the only hantavirus known to be present in the investigated area. Whether the hantavirus antibodies originate from infection with TANGV or another so far unknown hantavirus remains unclear. None of the sera exhibited measurable neutralising antibodies against PUUV, HTNV or SANGV (data not shown). Unfortunately, neutralisation tests with TANGV could not be performed as we failed to isolate TGNV on cell culture. These findings indicate that the observed weak reactivity in IFA (but also ELISA and Western blot) is due to the absence of autochthonous antigens in the tests formats and represents cross-reactivity to the heterologous hantavirus antigens used in the assays. The pathogen(s) causative of the human infections were most likely not represented by the panel of used antigens and viruses, and should be novel, more distantly related hantavirus(es).

The reservoir of Lassa virus, M. natalensis, is living in close contact with humans (Fichet-Calvet et al. 2007). This seems not to be the case for hantavirus reservoirs. The reservoir of TANGV, C. thereseae, is living in the savannah, forest and farms, but without invading the houses (Fichet-Calvet et al. 2010). The remarkable difference in seroprevalence rates for Lassa virus and hantaviruses suggests their dissimilar epidemiological characteristics. Most likely, a difference in frequencies for rodent/shrew – human contact could explain this phenomenon as the viruses are transmitted by different reservoir hosts. In agreement with the previous study from Forest Guinea (Klempa et al. 2010), the low hantavirus-specific seroprevalence suggest that these viruses are probably not transmitted (peri)domestically in villages. Other parameters such as differences in virus prevalence in rodents, virus shedding efficiency, virus stability and infectivity might contribute to the contrasting seroprevalence rates of the two viruses, too.

Despite the limitation in terms of the sample size, our study confirms that the South of Upper Guinea is a highly endemic zone for Lassa fever, as previously indicated by the report of four acute cases in Faranah's regional hospital in 1996–1999 (Bausch et al. 2001). This indicates that preventive measures focused on rodent control need to be considered by local public health authorities. Moreover, human hantavirus infections also occur in the region. Their public health relevance still needs to be determined. Further field studies leading to identification and isolation of local hantaviruses will allow to improve diagnostics and to assess their clinical relevance.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We appreciated the financial support by European Commission (grant INCO-DEV ICA4-CT2002-10050), Deutsche Forschungsgemeinschaft (grant KR1293/9-1/13-1)and a Marie Curie fellowship (PIEF-GA-2009-235164 to EFC). We are grateful to Amadou Doré, Fodé Kourouma, Barré Soropogui, Oumar Sylla, Thomas Strecker and Beate Becker-Ziaja for their technical help.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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