Molecular Diagnostics for Lassa Fever at Irrua Specialist Teaching Hospital,Nigeria: Lessons Learnt from Two Years of Laboratory Operation

Background

Lassa fever is a viral hemorrhagic fever endemic in West Africa. However, none of the hospitals in the endemic areas of Nigeria has the capacity to perform Lassa virus diagnostics. Case identification and management solely relies on non-specific clinical criteria. The Irrua Specialist Teaching Hospital (ISTH) in the central senatorial district of Edo State struggled with this challenge for many years.

Methodology/Principal Findings

A laboratory for molecular diagnosis of Lassa fever, complying with basic standards of diagnostic PCR facilities, was established at ISTH in 2008. During 2009 through 2010, samples of 1,650 suspected cases were processed, of which 198 (12%) tested positive by Lassa virus RT-PCR. No remarkable demographic differences were observed between PCR-positive and negative patients. The case fatality rate for Lassa fever was 31%. Nearly two thirds of confirmed cases attended the emergency departments of ISTH. The time window for therapeutic intervention was extremely short, as 50% of the fatal cases died within 2 days of hospitalization—often before ribavirin treatment could be commenced. Fatal Lassa fever cases were older (p = 0.005), had lower body temperature (p<0.0001), and had higher creatinine (p<0.0001) and blood urea levels (p<0.0001) than survivors. Lassa fever incidence in the hospital followed a seasonal pattern with a peak between November and March. Lassa virus sequences obtained from the patients originating from Edo State formed—within lineage II—a separate clade that could be further subdivided into three clusters.

Conclusions/Significance

Lassa fever case management was improved at a tertiary health institution in Nigeria through establishment of a laboratory for routine diagnostics of Lassa virus. Data collected in two years of operation demonstrate that Lassa fever is a serious public health problem in Edo State and reveal new insights into the disease in hospitalized patients.     

Early onset of neurological features differentiates two outbreaks of Lassa fever in Ebonyi state,Nigeria during 2017–2018

Lassa fever (LF) is an acute viral haemorrhagic illness with various non-specific clinical manifestations. Neurological symptoms are rare at the early stage of the disease, but may be seen in late stages, in severely ill patients.The aim of this study was to describe the epidemiological evolution, socio-demographic profiles, clinical characteristics, and outcomes of patients seen during two Lassa fever outbreaks in Ebonyi State, between December 2017 and December 2018.Routinely collected clinical data from all patients admitted to the Virology Centre of the hospital during the period were analysed retrospectively. Out of a total of 83 cases, 70(84.3%) were RT-PCR confirmed while 13 (15.7%) were probable cases. Sixty-nine (83.1%) patients were seen in outbreak 1 of whom 53.6% were urban residents, while 19%, 15%, and 10% were farmers, students and health workers respectively. There were 14 (16.8%) patients, seen in second outbreak with 92.9% rural residents. There were differences in clinical symptoms, signs and laboratory findings between the two outbreaks. The case fatality rates were 29.9% in outbreak 1 and 85.7% for outbreak 2. Neurological features and abnormal laboratory test results were associated with higher mortality rate, seen in outbreak 2. This study revealed significant differences between the two outbreaks. Of particular concern was the higher case fatality during the outbreak 2 which may be from a more virulent strain of the Lassa virus. This has important public health implications and further molecular studies are needed to better define its characteristics.     

拉沙病毒分子流行病学特征和跨境传播风险

拉沙热主要流行于西非,经鼠传播,人群普遍易感,病死率高,暴发疫情频发,跨境传播时有发生。拉沙病毒易于传播,病毒分离、培养需在生物安全四级实验室(BSL-4)。2018年,世界卫生组织将其列为年度重点关注传染病,需加快研制防治关键技术手段。随着中非关系的日益紧密,贸易往来频繁,中国面临拉沙热的威胁显著增加。本文检索20世纪50年代首例拉沙热病例报道以来公开发表的主要文献,归纳拉沙热临床表现、病原学特征、流行病学特征、实验室检测以及跨境传播风险等,并对GenBank发布的全部287条拉沙病毒S基因编码区全长序列进行复核分析,以加强人们对拉沙热的了解,提高防控意识。     

Differential pathogenesis of closely related 2018 Nigerian outbreak clade III Lassa virus isolates

Nigeria continues to experience ever increasing annual outbreaks of Lassa fever (LF). The World Health Organization has recently declared Lassa virus (LASV) as a priority pathogen for accelerated research leading to a renewed international effort to develop relevant animal models of disease and effective countermeasures to reduce LF morbidity and mortality in endemic West African countries. A limiting factor in evaluating medical countermeasures against LF is a lack of well characterized animal models outside of those based on infection with LASV strain Josiah originating form Sierra Leone, circa 1976. Here we genetically characterize five recent LASV isolates collected from the 2018 outbreak in Nigeria. Three isolates were further evaluated in vivo and despite being closely related and from the same spatial / geographic region of Nigeria, only one of the three isolates proved lethal in strain 13 guinea pigs and non-human primates (NHP). Additionally, this isolate exhibited atypical pathogenesis characteristics in the NHP model, most notably respiratory failure, not commonly described in hemorrhagic cases of LF. These results suggest that there is considerable phenotypic heterogeneity in LASV infections in Nigeria, which leads to a multitude of pathogenesis characteristics that could account for differences between subclinical and lethal LF infections. Most importantly, the development of disease models using currently circulating LASV strains in West Africa are critical for the evaluation of potential vaccines and medical countermeasures.     

Characterization of human CD4(+) T-cell clones recognizing conserved and variable epitopes of the Lassa virus nucleoprotein

T cells must play the major role in controlling acute human Lassa virus infection, because patients recover from acute Lassa fever in the absence of a measurable neutralizing antibody response. T cells alone seem to protect animals from a lethal Lassa virus challenge, because after experimental vaccination no neutralizing antibodies are detectable. In order to study human T-cell reactivity to single Lassa virus proteins, the nucleoprotein (NP) of Lassa virus, strain Josiah, was cloned, expressed in Escherichia coli, and affinity purified. Peripheral blood mononuclear cells (PBMC) obtained from 8 of 13 healthy, Lassa virus antibody-positive individuals living in the Republic of Guinea, western Africa, were found to proliferate in response to the recombinant protein (proliferation index >/=10). PBMC obtained from one individual with a particularly high proliferative response were used to generate 50 NP-specific T-cell clones (TCC). For six of these the epitopes were mapped with overlapping synthetic peptides derived from the sequence of the NP. These CD4(+) TCC displayed high specific proliferation and produced mainly gamma interferon upon stimulation with NP. Because variation of up to 15% in the amino acid sequences of the structural proteins of naturally occurring Lassa virus variants has been observed, the reactivity of the TCC with peptides derived from the homologous epitopes of the Nigeria strain of Lassa virus and of the eastern Africa arenavirus Mopeia was tested. With the Nigeria strain of Lassa virus the levels of homology were 100% for two of these epitopes and 85% for three of them, whereas homology with the respective Mopeia epitopes ranged from 92 to 69%. Reactivity of the TCC with peptides derived from the variable epitopes of the Nigeria strain and of Mopeia was reduced or completely abolished. This report shows for the first time that seropositive individuals from areas of endemicity have very strong memory CD4(+) T-cell responses against the NP of Lassa virus, which are partly strain specific and partly cross-reactive with other Lassa virus strains. Our findings may have important implications for the strategy of designing recombinant vaccines against this mainly T-cell-controlled human arenavirus infection.     

Geographic Distribution and Genetic Characterization of Lassa Virus in Sub-Saharan Mali

Background

Lassa fever is an acute viral illness characterized by multi-organ failure and hemorrhagic manifestations. Lassa fever is most frequently diagnosed in Nigeria, Sierra Leone, Liberia, and Guinea, although sporadic cases have been recorded in other West African countries, including Mali. The etiological agent of Lassa fever is Lassa virus (LASV), an Arenavirus which is maintained in nature and frequently transmitted to humans by Mastomys natalensis. The purpose of this study was to better define the geographic distribution of LASV-infected rodents in sub-Saharan Mali.

Methodologies/Principal Findings

Small mammals were live-trapped at various locations across Mali for the purpose of identifying potential zoonotic pathogens. Serological and molecular assays were employed and determined LASV infected rodents were exclusively found in the southern Mali near the border of Côte d''Ivoire. Overall, 19.4% of Mastomys natalensis sampled in this region had evidence of LASV infection, with prevalence rates for individual villages ranging from 0 to 52%. Full-length genomic sequences were determined using high throughput sequencing methodologies for LASV isolates generated from tissue samples of rodents collected in four villages and confirmed the phylogenetic clustering of Malian LASV with strain AV.

Conclusions/Significance

The risk of human infections with LASV is greatest in villages in southern Mali. Lassa fever should be considered in the differential diagnosis for febrile individuals and appropriate diagnostic techniques need to be established to determine the incidence of infection and disease in these regions.     

Genetic diversity among Lassa virus strains

The arenavirus Lassa virus causes Lassa fever, a viral hemorrhagic fever that is endemic in the countries of Nigeria, Sierra Leone, Liberia, and Guinea and perhaps elsewhere in West Africa. To determine the degree of genetic diversity among Lassa virus strains, partial nucleoprotein (NP) gene sequences were obtained from 54 strains and analyzed. Phylogenetic analyses showed that Lassa viruses comprise four lineages, three of which are found in Nigeria and the fourth in Guinea, Liberia, and Sierra Leone. Overall strain variation in the partial NP gene sequence was found to be as high as 27% at the nucleotide level and 15% at the amino acid level. Genetic distance among Lassa strains was found to correlate with geographic distance rather than time, and no evidence of a "molecular clock" was found. A method for amplifying and cloning full-length arenavirus S RNAs was developed and used to obtain the complete NP and glycoprotein gene (GP1 and GP2) sequences for two representative Nigerian strains of Lassa virus. Comparison of full-length gene sequences for four Lassa virus strains representing the four lineages showed that the NP gene (up to 23.8% nucleotide difference and 12.0% amino acid difference) is more variable than the glycoprotein genes. Although the evolutionary order of descent within Lassa virus strains was not completely resolved, the phylogenetic analyses of full-length NP, GP1, and GP2 gene sequences suggested that Nigerian strains of Lassa virus were ancestral to strains from Guinea, Liberia, and Sierra Leone. Compared to the New World arenaviruses, Lassa and the other Old World arenaviruses have either undergone a shorter period of diverisification or are evolving at a slower rate. This study represents the first large-scale examination of Lassa virus genetic variation.     

A Recombinant Vesicular Stomatitis Virus-Based Lassa Fever Vaccine Protects Guinea Pigs and Macaques against Challenge with Geographically and Genetically Distinct Lassa Viruses

Background

Lassa virus (LASV) is endemic in several West African countries and is the etiological agent of Lassa fever. Despite the high annual incidence and significant morbidity and mortality rates, currently there are no approved vaccines to prevent infection or disease in humans. Genetically, LASV demonstrates a high degree of diversity that correlates with geographic distribution. The genetic heterogeneity observed between geographically distinct viruses raises concerns over the potential efficacy of a “universal” LASV vaccine. To date, several experimental LASV vaccines have been developed; however, few have been evaluated against challenge with various genetically unique Lassa virus isolates in relevant animal models.

Methodologies/principle findings

Here we demonstrate that a single, prophylactic immunization with a recombinant vesicular stomatitis virus (VSV) expressing the glycoproteins of LASV strain Josiah from Sierra Leone protects strain 13 guinea pigs from infection / disease following challenge with LASV isolates originating from Liberia, Mali and Nigeria. Similarly, the VSV-based LASV vaccine yields complete protection against a lethal challenge with the Liberian LASV isolate in the gold-standard macaque model of Lassa fever.

Conclusions/significance

Our results demonstrate the VSV-based LASV vaccine is capable of preventing morbidity and mortality associated with non-homologous LASV challenge in two animal models of Lassa fever. Additionally, this work highlights the need for the further development of disease models for geographical distinct LASV strains, particularly those from Nigeria, in order to comprehensively evaluate potential vaccines and therapies against this prominent agent of viral hemorrhagic fever.     

Exotic viral diseases

Marburg virus disease, Lassa fever, monkeypox, and Ebola virus diseases of humans have all been recognized since 1967. These are examples of some of the exotic virus diseases which through importation may present a potential public health problem in the United States. Some of these viruses are also highly hazardous to laboratory and medical personnel. This paper is a review of the general characteristics, the epidemiology, and laboratory diagnosis of the exotic viruses which have been described during the last 25 years.     

Viral infections in travellers from tropical Africa.

Examination of sera from 86 travellers to Britain from tropical Africa disclosed evidence of past infection with 10 identifiable viruses, of which the most important were O''nyong-nyong, dengue, chikungunya, and Ntaya. The findings indicate that infection with O''nyong-nyong may be acquired sporadically in Nigeria, Ghana, and Sierra Leone, where it has not previously been identified. Chikungunya infection had not been recorded in West Africa other than Nigeria and Senegal. Patients from Sierra Leone and contiguous Liberia had antibodies to this infection. An outbread of dengue fever in the Seychelles in early 1977 was confirmed. Ntaya virus, though known in Uganda, Cameroon, and Zaire, appears also to be transmitted in Kenya, Nigeria, and Zambia. Clinical studies indicated that chikungunya infection may present with alimentary features, possibly with jaundice. The clinical features of Ntaya infection may include kizarre neurological manifestations in addition to fever. The absence of Lassa antibodies among these travellers suggested that this infection is not a common hazard among such persons.     

Monitoring of clinical and laboratory data in two cases of imported Lassa fever

During 2000, four cases of fatal Lassa fever were imported from Africa to Europe. In two patients, consecutive serum samples were available for monitoring of virus load and cytokine levels in addition to standard laboratory data. Both patients had non-specific early clinical symptoms including high fever. Patient 1 developed multi-organ failure and died of hemorrhagic shock on day 15 of illness, while patient 2 died of respiratory failure due to aspiration without hemorrhage on day 16. Ribavirin was administered to both patients beginning only on day 11. High serum aspartate aminotransferase and lactate dehydrogenase (LDH) levels were remarkable in both patients. Patient 1 had an initial virus load of 10(6) S RNA copies/ml as measured by real-time RT-PCR. Viremia increased steadily and reached a plateau of approximately 10(8)-10(9) copies/ml 4 days before death, while IFN-gamma and TNF-alpha rose to extremely high levels only shortly before death. In contrast, in patient 2 the virus load decreased from 10(7) to 10(6) copies/ml during the late stage of illness which was paralleled by a decrease in the IFN-gamma and TNF-alpha levels. The IL-10 level increased when specific IgM and IgG appeared. These data suggest that a high virus load and high levels of pro-inflammatory cytokines in the late stage of Lassa fever play an important role in the pathogenesis of hemorrhage, multi-organ failure, and shock in Lassa fever.     

拉沙热研究进展

拉沙热(Lassa fever,LF)由拉沙病毒(Lassa virus,LASV)引起,是一种通过鼠类传播给人的急性出血性动物源性传染病,病死率高。主要在西非地区流行,但在全球多个国家发现输入性病例。本文主要针对拉沙病毒的病原学特点、疾病流行现状、临床特征、实验室检测、治疗及预防进展进行综述。同时,建议在全球经济一体化的大形势下,加强对拉沙热国际防控的关注度,建立国际联防联控防治策略和措施,并将其纳入全球防控体系。     

Health seeking behavior after the 2013–16 Ebola epidemic: Lassa fever as a metric of persistent changes in Kenema District,Sierra Leone

BackgroundThe West African Ebola epidemic of 2013–2016 killed nearly 4,000 Sierra Leoneans and devastated health infrastructure across West Africa. Changes in health seeking behavior (HSB) during the outbreak resulted in dramatic underreporting and substantial declines in hospital presentations to public health facilities, resulting in an estimated tens of thousands of additional maternal, infant, and adult deaths per year. Sierra Leone’s Kenema District, a major Ebola hotspot, is also endemic for Lassa fever (LF), another often-fatal hemorrhagic disease. Here we assess the impact of the West African Ebola epidemic on health seeking behaviors with respect to presentations to the Kenema Government Hospital (KGH) Lassa Ward, which serves as the primary health care referral center for suspected Lassa fever cases in the Eastern Province of Sierra Leone.Methodology/Principal findingsPresentation frequencies for suspected Lassa fever presenting to KGH or one of its referral centers from 2011–2019 were analyzed to consider the potential impact of the West African Ebola epidemic on presentation patterns. There was a significant decline in suspected LF cases presenting to KGH following the epidemic, and a lower percentage of subjects were admitted to the KGH Lassa Ward following the epidemic. To assess general HSB, a questionnaire was developed and administered to 200 residents from 8 villages in Kenema District. Among 194 completed interviews, 151 (78%) of respondents stated they felt hospitals were safer post-epidemic with no significant differences noted among subjects according to religious background, age, gender, or education. However, 37 (19%) subjects reported decreased attendance at hospitals since the epidemic, which suggests that trust in the healthcare system has not fully rebounded. Cost was identified as a major deterrent to seeking healthcare.Conclusions/SignificanceAnalysis of patient demographic data suggests that fewer individuals sought care for Lassa fever and other febrile illnesses in Kenema District after the West African Ebola epidemic. Re-establishing trust in health care services will require efforts beyond rebuilding infrastructure and require concerted efforts to rebuild the trust of local residents who may be wary of seeking healthcare post epidemic.     

The impact of human conflict on the genetics of Mastomys natalensis and Lassa virus in West Africa

Environmental changes have been shown to play an important role in the emergence of new human diseases of zoonotic origin. The contribution of social factors to their spread, especially conflicts followed by mass movement of populations, has not been extensively investigated. Here we reveal the effects of civil war on the phylogeography of a zoonotic emerging infectious disease by concomitantly studying the population structure, evolution and demography of Lassa virus and its natural reservoir, the rodent Mastomys natalensis, in Guinea, West Africa. Analysis of nucleoprotein gene sequences enabled us to reconstruct the evolutionary history of Lassa virus, which appeared 750 to 900 years ago in Nigeria and only recently spread across western Africa (170 years ago). Bayesian demographic inferences revealed that both the host and the virus populations have gone recently through severe genetic bottlenecks. The timing of these events matches civil war-related mass movements of refugees and accompanying environmental degradation. Forest and habitat destruction and human predation of the natural reservoir are likely explanations for the sharp decline observed in the rodent populations, the consequent virus population decline, and the coincident increased incidence of Lassa fever in these regions. Interestingly, we were also able to detect a similar pattern in Nigeria coinciding with the Biafra war. Our findings show that anthropogenic factors may profoundly impact the population genetics of a virus and its reservoir within the context of an emerging infectious disease.     

Controlling the exotic diseases: 1. Isolation facilities.

The exotic diseases are highly virulent transmissible conditions that include Lassa fever, some viral hemorrhagic fevers, smallpox and plague. Any of these diseases could be brought into or diagnosed in Canada as the result of natural or laboratory acquired infection. The patients must be isolated until the presumptive diagnosis is proved. High-security isolation is necessary and needs to be backed up by high-security laboratory services. In Canada facilities for high-security isolation are generally not available; therefore, hospitals must preplan and be ready to effect the best possible isolation under the existing conditions. The plan should address construction, ventilation, filtration, temperature and humidity, together with protective measures for staff and careful handling of laboratory specimens. Materials the patient has contacted and areas or vehicles he or she has been in will have to be decontaminated, and appropriate, safe disposal of corpses must be considered.     

Lassa fever,Marburg and Ebola virus diseases and other exotic diseases: is there a risk to Canada?

There are seven exotic diseases of concern; three of these, the most unpredictable and least understood, are Lassa fever, Marburg virus disease and Ebola virus disease. In this article the epidemiologic aspects of these diseases are discussed, with particular emphasis on exportation from their indigenous areas in Africa and on the occurrence of secondary cases. Any of these conditions could be brought into Canada either by aeromedical evacuation or inadvertently. Between 1972 and 1978 there were seven occasions when Canada could have been involved with handling cases of Lassa fever. The Government of Canada has purchased several containment bed and transit isolators. These units, with filtered air under negative pressure, accommodate infectious patients being transported and cared for without contaminating medical attendants or the environment.     

The association between farmers’ participation in herd health programmes and their behaviour concerning treatment of mild clinical mastitis

Background

Methicillin-resistant Staphylococcus aureus (MRSA) in animals is a rare finding in Sweden. In horses, MRSA was first detected in a screening survey in 2007. In 2008, six clinical cases occurred in an equine hospital, indicating an outbreak.

Method

All MRSA isolates detected, 11 spa-type t011 and one t064 (n = 12), in infected horses (n = 10) and screening of horses (n = 2) in Sweden from December 2007 to March 2010 were retrospectively analysed with pulsed-field gel electrophoresis (PFGE) using Cfr9I and ApaI restriction enzymes, to study relationship between the isolates. Medical records of infected horses and outbreak investigation notes were scrutinised to monitor the clinical outcome and other aspects of the outbreak.

Results

Eight of the 10 infected horses were linked to one equine hospital and two to another hospital in the same region. The six horses infected with MRSA in 2008 underwent surgery during the period 22 May-7 July in one of the hospitals. Four more infections linked to the two hospitals were notified between 2009 and March 2010. Nine of the 11 spa-type t011 isolates had identical Cfr9I and ApaI PFGE pattern. All six infected horses from 2008 presented with this MRSA. Two t011 isolates differed in one and two bands, respectively, in PFGE. Nine horses suffered from surgical site infections (SSI). No antimicrobials were used following the MRSA diagnosis and the infections cleared. The time from surgery to MRSA diagnosis differed greatly between the horses (range 15-52 days).

Conclusions

Association in time and space of six horses infected with an identical MRSA strain of spa-type t011 confirmed an outbreak. Two isolates found in 2009 and 2010 in the outbreak hospital were closely related to the outbreak strain, indicating one circulating strain. Both spa-type t011 and t064 have been reported in horses in Europe prior to these findings. The observation that the infections cleared although antimicrobials were not used is encouraging for future prudent use of antimicrobials. The time from surgery to bacteriological diagnosis was not acceptable in most cases, as contagious spread was a risk. Sampling when symptoms of infection are noticed and accurate analysis are thus important.     

基于暴露分析与关键控制点方法对医院开展严重急性呼吸综合征冠状病毒2型检测的可行性研究

评估2019新型冠状病毒病暴发期间在医院开展严重急性呼吸综合征冠状病毒 2 型(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)核酸检测的可行性,为最终在医院开展核酸检测提供参考。熟悉暴露分析和关键点控制(exposure analysis and critical control points,EACCP)工作框架的专业人员在基于医院现实条件下,对SARS-CoV-2检测过程中可能的感染暴露风险和途径进行梳理,建立整个检测流程,验证在配备有发热门诊的医院开展的可行性,并明确降低暴露风险的关键控制点。高风险是在发热门诊标本的采集和灭活处理,中风险是未灭活标本的储运,低风险是灭活标本的储运和检测。优化检验流程能降低检测过程中感染暴露风险,对于高风险的操作,可在生物安全二级实验室(发热门诊或移动采集点等)和相应的安全防护等级下进行操作; EACCP分析方法可用于新发感染性疾病暴发期间的管理。     

A prospective study of maternal and fetal outcome in acute Lassa fever infection during pregnancy.

Several viral infections have been reported to result in more severe disease in pregnant than non-pregnant women, but the relative risks have not been well characterised. This has now been done for Lassa fever in a prospective study of 68 pregnant and 79 non-pregnant women who were admitted to hospital in Sierra Leone with confirmed Lassa fever. Lassa fever was the main cause of maternal mortality in the hospital, accounting for 25% of maternal deaths. Twelve of 40 patients in the third trimester died, compared with two of 28 in the first two trimesters and 10 of 79 non-pregnant women. The odds ratio for death in the third trimester compared with the first two trimesters was 5.57 (95% confidence intervals 1.02 to 30.26). The condition of the mother improved rapidly after evacuation of the uterus, whether by spontaneous abortion, evacuation of retained products of conception, or normal delivery; 10 of 26 women without uterine evacuation died, but only four of 39 women with evacuation died (p = 0.0016). The odds ratio for death with pregnancy intact was 5.47 (95% confidence interval 1.35 to 22.16). Fetal and neonatal loss was 87%. The risk of death from Lassa fever in the third trimester is significantly higher than that in the first two trimesters and higher than that for non-pregnant women, but evacuation of the uterus can significantly improve the mother''s chance of survival.