D. Safronetz. Laboratory of Virology, Division of Intramural Research, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA. Tel.: +1 406 375 7425; Fax: +1 406 375 9620; E-mail: email@example.com
Lassa virus (LASV) is endemic in Sierra Leone, Guinea and Liberia (known as the Mano River region) and Nigeria and Lassa fever cases from these countries are being reported annually. Recent investigations have found evidence for an expanded endemicity zone between the two known Lassa endemic regions indicating that LASV is more widely distributed throughout the Tropical Wooded Savanna ecozone in West Africa.
• Lassa fever is a considerable public health problem in Sierra Leone/Guinea/Liberia (also known as the Mano River region) and Nigeria.
• There is evidence for an expanded endemicity zone between the two known endemic regions (e.g. southern Mali, Burkina Faso, Côte d’Ivoire, Ghana) which shares a similar ecozone - Tropical Wooded Savanna.
• Future ecological and epidemiological studies need to address the prevalence of Lassa virus in these predicted risk areas to prepare for proper public health measures.
Lassa fever was first documented in 1969 when a small outbreak of a mysterious disease claimed the lives of two missionary nurses serving in the town of Lassa, Nigeria. Following the initial identification of Lassa virus (LASV), and its subsequent association with Mastomys natalensis, cases of Lassa fever were being diagnosed annually in two geographically separate regions in West Africa; Nigeria and Sierra Leone/Guinea/Liberia (otherwise referred to as the Mano River region) (Buckley and Casals, 1970; Frame et al., 1970; Monath et al., 1974; reviewed in Khan et al., 2008) (Fig. 1). Currently, an estimated 300 000–500 000 cases of Lassa fever are diagnosed annually in the four endemic countries (Ogbu et al., 2007). In addition, at least 29 imported cases of Lassa fever have been documented worldwide (one third of which were fatal), with an average of one imported case diagnosed each year since 2000 (E-alert, 2006; Macher and Wolfe, 2006; Atkin et al., 2009; Kitching et al., 2009; Amorosa et al., 2010). Based on these findings, LASV is arguably the most prevalent Biosafety Level (BSL) four agent and a prominent threat to human health worldwide.
One of the enduring questions regarding Lassa fever is why cases are not recognized in the countries between the two regions of endemicity (Fig. 1). Throughout the 1970s and 80s, extensive clinical and ecological research on LASV was conducted in West Africa, mostly notable in Sierra Leone and Nigeria (reviewed in Khan et al., 2008). Interestingly, in one of the earliest serological surveys for LASV in humans, John Frame retrospectively identified potential cases of Lassa fever in missionaries stationed in Upper Volta (now Burkina Faso), Mali, Côte d’Ivoire, and Central African Republic/Zaire (now Democratic Republic of Congo), as well as Nigeria, Liberia and Guinea (Frame, 1975). Although the study by Frame could not definitively prove the cases originated from the countries of residence, the results suggested the potential of a wider region of endemicity. However, despite these early findings, to date little work has been done in countries like Mali, Côte d’Ivoire, Burkina Faso, Ghana, Togo or Benin in an attempt to evaluate the presence of LASV.
Evidence of a wider area of endemicity
Not unexpectedly, the majority of imported cases of Lassa fever have originated from Nigeria, Sierra Leone, Liberia or Guinea (Macher and Wolfe, 2006). However, to-date, at least three imported cases have been identified from outside these four countries, suggesting a wider distribution of LASV and challenging the current dogma of LASV endemicity. In 1980, a non-fatal case of Lassa fever was diagnosed in The Netherlands in an aid worker stationed in Burkina Faso (Macher and Wolfe, 2006). In 2000, Gunther et al. (2000) reported the characterization of a unique LASV strain (AV) which was isolated from a fatal case of Lassa fever. The patient, a young woman from Germany, was travelling in West Africa through Ghana, Côte d’Ivoire and Burkina Faso. Nearly a decade later a similar scenario unfolded in the United Kingdom (Atkin et al., 2009). In February 2009, a previously healthy British man was medically evacuated from Mali to London for treatment. Upon arrival the patient was alert, but rapidly deteriorated and succumbed to infection within 24 h. A post-mortem diagnosis of Lassa fever was made through amplification of LASV specific fragments of multiple genomic targets. Nucleotide sequence analysis confirmed the diagnosis and revealed that the LASV strain in the Malian case was phylogenetically most closely related to strain AV, from the German case. Unlike the young woman from Germany, the British man had only travelled in Mali and resided in Soromba, a small village in the southern region near the border of Côte d’Ivoire, where he had been working with an aid agency to build a bridge.
More compelling evidence of an expanded distribution of LASV can be found in recent cases of Lassa fever in Ghana. In late October 2011, the first case of Lassa fever was diagnosed in a young man who resided in the Amansie West district within the Ashanti region of southern Ghana (World Health Organization, 2011a). He had no travel history outside the area during the incubation period suggesting that the infection was locally acquired, possibly in surrounding forests where he routinely hunted rodents. Lassa fever was confirmed by reverse transcription polymerase chain reaction (RT-PCR) on a blood sample collected on the day of death. Within 3 weeks of the index case, two additional cases of Lassa fever were confirmed in patients from geographical distinct areas in southern Ghana. The second case also occurred in the Amansie West district, whereas the third occurred in the West Akim district (World Health Organization, 2011b). All three cases were fatal.
Although the geographical origin of the AV strain of LASV remains unresolved, it is clear that it originated from an area where Lassa fever had not been previously reported (Gunther et al., 2000). The lack of travel history for the cases of Lassa fever in Mali and Ghana strongly supports circulation of LASV in rodents within these areas. Recent field studies confirmed this notion in southern Mali by demonstrating the presence of LASV infected rodents and epizootic transmission of virus in the village of Soromba (Safronetz et al., 2010). The identification of LASV and Lassa fever cases in these areas should not be surprising. Ecologically, much of southern Mali and Burkina Faso, as well as northern Côte d’Ivoire, Ghana, Togo and Benin share the same characteristics as the LASV endemic regions of Sierra Leone, Liberia, Guinea and Nigeria; and all are considered part of the Tropical Wooded Savanna ecozone (Fig. 1). The environmental factors (relative humidity, for example) within these areas may favour the circulation of LASV as well as M. natalensis (Fichet-Calvet and Rogers, 2009). In the area of southern Mali where LASV circulation was recently identified, rodent diversity was found to be extremely low with only M. natalensis captured, compared with other areas within Mali where captures included at least three species of Mastomys, as well as Praomys and several other species of small mammals (Safronetz et al., 2010; Safronetz, D., Sogoba, N., and Feldmann, H., unpublished results). Previously rodent diversity has been hypothesized to be inversely correlated with the prevalence of rodent-borne pathogens, suggesting that at least in southern Mali, conditions are conducive to LASV circulation (Mills, 2005). Finally genetic evidence proposes that Nigerian strains of LASV are ancestral to those from the Mano river region, suggesting that at some point LASV may have moved through the connecting countries (Bowen et al., 2000). In general M. natalensis are known to be peridomestic rodents and are thought to be relatively sedentary. Since LASV appears to have no adverse effects on the rodent reservoirs, and because it is believed to be transmitted both vertically and horizontally, it is likely that at the very least focal hotspots of LASV still exist in these connecting countries.
Infection with LASV results in a spectrum of symptoms which often precludes reliable clinical diagnosis, especially in areas where local physicians are not familiar with Lassa fever (McCormick et al., 1987; Richmond and Baglole, 2003; Gunther and Lenz, 2004). The incubation period for LASV is up to 21 days, after which the individuals may experience a gradual onset of mild, nondescript symptoms including low-grade fever, headache, malaise and general weakness. The majority of LASV infections (approximately 80%) are subclinical or do not progress beyond mild illness. After 4–7 days of mild illness, 20% of infect individuals experience more severe symptoms requiring medical attention. These can include haemorrhage, persistent vomiting, hypotension, oedema (commonly in the face and neck), hypovolemic shock and respiratory distress. Death generally occurs between 10–14 days post-symptom onset and results from multi-organ failure.
The remote locations where Lassa fever typically occur, in combination with the risk of secondary transmission of LASV and accidental exposure during autopsies has hampered clinical and pathological investigations into cases of Lassa fever. Although the pathogenesis of Lassa fever remains poorly understood there is epidemiological and experimental evidence suggesting that differences in virulence exists among LASV isolates (Jahrling et al., 1985; Gunther and Lenz, 2004). Estimates vary, however, it is commonly accepted that approximately 80% of LASV infections occurring within the endemic region are mild or asymptomatic. Additional experimental and epidemiological evidence is needed; however, it seems possible that the strains of LASV circulating in countries between the two endemic zones of Nigeria and Sierra Leone/Liberia/Guinea might be less pathogenic and therefore result in increased numbers of sub-clinical infections. In support of this hypothesis, there is evidence that LASV strain AV is less pathogenic in the highly sensitive cynomolgus macaque model of Lassa fever, with one of three animals surviving a challenge dose of 103 LASV particles (Baize et al., 2009). Although small animal numbers were used in the study, the challenge dose utilized has previously been shown to be 100% lethal in this model when more traditional LASV strains (i.e., Josiah) are used (Hensley et al., 2011). These potential differences could partially explain why Lassa fever has largely gone undiagnosed in countries like Mali, Côte d’Ivoire, Ghana, Togo and Benin (Fig. 1).
Rapid identification of Lassa fever cases is imperative for both patient management and outcome (Fisher-Hoch et al., 1995); however, the list of differential diagnosis is extensive and includes other prominent infectious diseases such as malaria, trypanosomiasis, Typhoid fever, yellow fever, Influenza, Measles and other viral hemorrhagic fever syndromes (Khan et al., 2008). Laboratory diagnosis of Lassa fever relies on detection of viral antigen or genetic material, or virus specific antibodies using methods including indirect fluorescent antibody (IFA) tests, enzyme-linked immunosorbent assays (ELISA), and conventional and real-time, RT-PCR; however, each assay has inherent disadvantages associated with it (Strong et al., 2006).
There is evidence of strain specific antibody responses, both neutralizing (directed against the glycoproteins) and non-neutralizing (directed against the nucleoprotein) in LASV infections (Emmerich et al., 2008). Using serum samples collected from suspected cases of Lassa fever originating from different geographical locations across West Africa, Emmerich and colleagues found that depending on the serological method employed, up to one third of individuals previously infected with LASV would give a false-negative result if a heterologous LASV strain was used as antigen. These data suggest that regional specific antigen or a pool of LASV antigens are optimal for accurate diagnostic results; however, the impact of non-homologous LASV antigen may only affect seroprevalence studies aimed at identifying previously infected individuals since the strain specific responses appear to develop after convalescence.
The genetic diversity associated with LASV strains also hampers molecular diagnostic assays. Depending on the coding region analysed, genetic variation among LASV strains can be as high as 27% at the nucleotide level (Bowen et al., 2000). Not surprisingly, a universal LASV specific real-time RT-PCR assay is not available. Currently, molecular diagnostic assays rely on targeting conserved regions of Old World arenaviruses, typically utilizing a nested approach (Strong et al., 2006; Vieth et al., 2007). Further complicating matters, at this time no commercially available diagnostic test exists for Lassa fever, meaning individual facilities rely on in-house preparation and validation of specific assays.
The identification of imported cases of Lassa fever from countries connecting the two endemic regions, as well as the recently confirmed cases of Lassa fever in Ghana highlight the importance of increased surveillance for LASV across West Africa. Through analysis of incidence of disease and environmental factors, predictive modelling has identified risk areas for Lassa fever in countries outside the historical endemic regions including Côte d’Ivoire, Togo, Benin and the southern tip of Mali where we recently confirmed the presence of LASV-infected rodents (Fichet-Calvet and Rogers, 2009; Safronetz et al., 2010). We have recently started to map LASV prevalence in Mali; however, additional studies need to be conducted to address the prevalence of LASV in the other predicted risk areas to close some of the knowledge gaps in the near future.
Logistically, ecological studies aimed at identifying highly pathogenic viruses in remote areas of Africa present unique challenges, both physically and politically. Establishing collaborations between local and international research groups is critical to facilitate this work and will help to navigate around important issues including implementation of appropriate biosafety, and where applicable, biosecurity measures and social impact. Despite these challenges, ecological studies in Western Africa should be considered a priority since they will not only improve our understanding of the endemicity of LASV in Western Africa, but potentially several other rodent-borne pathogens.