Capacity building permitting comprehensive monitoring of a severe case of Lassa hemorrhagic fever in Sierra Leone with a positive outcome: Case Report
- Jessica N Grove†1,
- Luis M Branco†1, 2,
- Matt L Boisen3,
- Ivana J Muncy3,
- Lee A Henderson4,
- John S Schieffellin5,
- James E Robinson5,
- James J Bangura6, 7,
- Mbalu Fonnie8,
- Randal J Schoepp9,
- Lisa E Hensley10,
- Alhassan Seisay6,
- Joseph N Fair7 and
- Robert F Garry1Email author
© Grove et al; licensee BioMed Central Ltd. 2011
Received: 3 June 2011
Accepted: 20 June 2011
Published: 20 June 2011
Lassa fever is a neglected tropical disease with a significant impact on the health care system of endemic West African nations. To date, case reports of Lassa fever have focused on laboratory characterisation of serological, biochemical and molecular aspects of the disease imported by infected individuals from Western Africa to the United States, Canada, Europe, Japan and Israel. Our report presents the first comprehensive real time diagnosis and characterization of a severe, hemorrhagic Lassa fever case in a Sierra Leonean individual admitted to the Kenema Government Hospital Lassa Fever Ward. Fever, malaise, unresponsiveness to anti-malarial and antibiotic drugs, followed by worsening symptoms and onset of haemorrhaging prompted medical officials to suspect Lassa fever. A recombinant Lassa virus protein based diagnostic was employed in diagnosing Lassa fever upon admission. This patient experienced a severe case of Lassa hemorrhagic fever with dysregulation of overall homeostasis, significant liver and renal system involvement, the interplay of pro- and anti-inflammatory cytokines during the course of hospitalization and an eventual successful outcome. These studies provide new insights into the pathophysiology and management of this viral illness and outline the improved infrastructure, research and real-time diagnostic capabilities within LASV endemic areas.
Lassa virus (LASV), a member of the Arenaviridae family, is the etiologic agent of Lassa fever, an acute and often fatal illness endemic to West Africa. There are an estimated 300,000 - 500,000 cases of Lassa fever each year [1–3] with a mortality rate of 15%-20% for hospitalized patients, which can become as high as 50% during epidemics [4, 5]. Presently, there is no licensed vaccine or immunotherapy available for prevention or treatment of this disease. Although the antiviral drug ribavirin is somewhat beneficial, it must be administered at an early stage of infection to successfully alter disease outcome, thereby limiting its utility [6, 7]. Furthermore, there is no commercially available Lassa fever diagnostic assay, which hampers early detection and rapid implementation of existing treatment regimens (e.g. ribavirin administration). The severity of the disease, its ability to be transmitted by aerosol droplets and the lack of a vaccine or therapeutic drug led to its classification as a National Institutes of Allergy and Infectious Diseases (NIAID) Category A pathogen and biosafety level-4 (BSL-4) agent. Several imported Lassa fever cases have been described since 1973 primarily from foreign nationals displaying signs of the disease upon returning to native countries or having been evacuated after falling ill abroad [8–34]. To date, and despite the often severe nature of Lassa fever in Western African nations, resources have not been available to perform comprehensive daily analysis of blood samples from suspected and confirmed patients in-country. Continuous infrastructure improvements at the Kenema Government Hospital (KGH) Lassa Fever Laboratory (LFL) by Tulane University, Department of Defense (DoD) and the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) since 2005 have allowed for the implementation of sophisticated diagnostic and research capabilities at this location. Currently the KGH LFL diagnoses Lassa fever using ELISA and lateral flow immunoassay (LFI) platforms to detect viral antigen and virus-specific IgM and IgG levels in the serum of every suspected case presented to the LFW. Additionally, the laboratory can assess 14 serum analytes using a Piccolo® blood chemistry analyzer coupled with comprehensive metabolic panel disks. Flow cytometry powered by a 4-color Accuri® C6 cytometer is used to perform immunophenotyping and intracellular and bead-based secreted cytokine analysis. Together, these diagnostic assays and instruments enabled the analysis of metabolic and inflammatory functions in real time utilizing the sera of individuals discussed in this case report with concomitant appropriate medical intervention.
The main patient case discussed in this report was closely monitored for nine days during his hospitalization, during which time his condition stabilized; he began walking with supervision and was nearing the end of ribavirin treatment. These studies herald a new era in real time diagnosis and management of Lassa hemorrhagic fever in resource poor, endemic areas of Western Africa. They represent a novel platform toward more efficient and broader control of the effects of this disease in the population at large.
Human Subjects: Suspected LF patients, close contacts, and healthy volunteers were eligible to participate in these studies as outlined in Tulane University's Institutional Review Board (IRB) protocol for this project, National Institutes of Health/National Institutes of Allergy and Infectious Diseases guidelines governing the use of human subject for research, and Department of Health and Human Services/National Institutes of Health/National Institute of Allergy and Infectious Diseases Challenge and Partnership Grant Numbers AI067188 and AI082119. This project was approved by the Tulane University IRB. Adult patients in this manuscript have given written informed consent for the publication of their case details. Written informed consent was obtained from the adult guardian of patient G-1180 for publication of this case report.
Normal and positive control sera: Serum from one Sierra Leonean and two Caucasian American volunteers were used in these studies as normal controls. A serum sample collected from a 20-year-old pregnant woman who succumbed to Lassa fever in the KGH Maternity Ward on August 29, 2010 was used as a positive control. A single serum sample was collected from this subject before expiration, and assigned the coded designation G-1177.
Detection of Lassa virus antigen and Lassa virus-specific antibodies: Serum levels of Lassa nucleoprotein (NP)-specific antigen were first determined using LFI modules currently under pre-clinical development by Corgenix Medical Corp., Broomfield, CO, U.S.A., and the Lassa fever consortium (see acknowledgements). The LFI modules utilize LASV NP specific murine monoclonal antibodies in a capture line and gold-conjugated detection reagent. Downstream of the capture line is an anti-murine polyclonal control line. Serum samples are added to a sample well followed by buffer solution to initiate lateral flow through the reagent pads and across the capture and control lines. The formation of Lassa NP antigen immune-complexes by the reagents produces a red signal due to gold conjugate deposition, allowing visual interpretation or measurement by reflectance. The red signal can be seen within 3-5 minutes with full signal development between 15-25 minutes. The positive Lassa fever diagnosis is then confirmed with a sensitive antigen-capture ELISA employing either a murine monoclonal or caprine polyclonal capture antibody, followed by a peroxidase-labeled caprine reagent and tetramethylbenzidine (TMB) substrate. A standard curve was generated with recombinant LASV NP in order to quantify serum levels of virus-associated NP.
IgM and IgG levels to recombinant LASV proteins (NP alone and NP - glycoprotein 1 (GP1) - glycoprotein complex (GPC) combination) ELISA: Individual LASV proteins and combinations optimized for detection of virus-specific IgM and IgG levels in serum were coated in stripwell plates, blocked, dried, and packaged with desiccating packs (Corgenix Medical Corp.). For analysis, sera were diluted 1:100 in a proprietary sample dilution buffer and incubated in wells for 30 minutes at room temperature, washed, and incubated with optimized HRP-labeled anti-human IgG or IgM conjugates for an additional 30 minutes. After washing, detection was performed with TMB substrate for 10 minutes at room temperature, stopped with sulphuric acid, and read at A450 in a BioTek ELISA plate reader.
IgM and IgG endpoint titer determination: Sera were analyzed in 3-fold serial dilutions, starting at 1:50, in optimized LASV NP, GP1, GP2, Z protein combination ELISA, as outlined above. Reciprocal titers were calculated using mean +3 standard deviation (S.D.) cutoffs established with similarly diluted normal serum controls.
Piccolo®: The kinetics of fourteen serum analytes were analyzed daily using a Piccolo® blood chemistry analyzer (Abaxis, Inc., Union City, CA) with Comprehensive Metabolic Reagent Discs. Normal values were determined for a male in the age range of the patient using established clinical guidelines. Blood was collected in serum vacutainer tubes from patients and control donors and allowed to coagulate for 20 minutes at room temperature, followed by centrifugation in a tabletop centrifuge. The serum fraction was collected for analysis and aliquots were stored in cryovials at -20°C for future use.
Flow Cytometry: The kinetics of eleven serum cytokines were analyzed daily using an Accuri® C6 benchtop cytometer (Accuri Cytometers Inc., Ann Harbor, MI) with an eBioscience FlowCytomix Human Th1/Th2 11-plex Kit (Bender MedSystems GmbH, Vienna, Austria). Serum aliquots collected and frozen throughout the monitoring timeline were analyzed concurrently at the end of the study.
Treatment and hospital course
Intravenous (IV) ribavirin was administered upon LFI Ag positive diagnosis: a loading dose of 30mg per kilogram followed by 15mg/kg every 6 hours for 4 days and 7.5mg/kg every 8 hours for an additional 6 days. Broad spectrum antibiotics (ceftriaxone, ciprofloxacin and metronidazole) and anti-malarials (artemether and quinine) were also started. A single dose of dexamethasone was given. Five percent and 50% glucose boluses were given as needed. Upon slight recovery, the patient requested energy drinks, which were provided (Lucozade, a sports-like drink similar to Gatorade in composition). Despite therapy, G-1180 continued to experience bleeding abnormalities for the next two weeks including hematochezia and hemoptysis. Ciprofloxacin was restarted on the eighth day post onset of illness to treat a catheter-related infection of his penis, which was resolved shortly thereafter. The patient required a blood transfusion on day 8 of illness due to severe anaemia (Hgb = 7.0 g/dL, Figure 3B). The donor was a Caucasian American female of the same blood type (A+). A second blood transfusion was administered on day 21 of illness (Hgb = 6.5 g/dL, Figure 3B) from a type O+ Caucasian American male donor. On this day the patient was alert, responsive, able to walk on his own and had stopped bleeding altogether.
Blood samples were collected daily, except for day 12 of illness. Day 14 post onset of illness was the last day that diagnostic, metabolic and inflammatory tests were conducted on patient G-1180. The LFI tests detected LASV NP in G-1180 through day 8 of illness (Figure 5) and NP Ag capture ELISA detected LASV NP through day 11 (Figure 6A; P < 0.05, N = 2). LASV NP antigen dropped rapidly over 3 days following the onset of ribavirin treatment (Figure 6A).
Despite the severity of the disease, most of G-1180's chemistry levels were near or within the normal range for a male his age by the end of our monitoring period. The originally low levels of Na+, Cl- and TCO2 returned to normal levels; though, the Alb and TP remained consistently low. The liver function panel decreased significantly, although it did not return to within normal range. BUN and BUN:Cre levels, on the other hand, returned to normal levels. The analytes that were initially within the normal range remained stable throughout the monitoring period except for K+, which declined.
Cytokine profiles were performed on serum samples collected daily. G-1180's IL-12p70, IL-6 and IL-10 levels were elevated upon admission (day 6) but returned to normal by day 7. On day 10, a sudden increase in IL-6, IL-8, IL-10 and tumor necrosis factor (TNF) -alpha levels was noted. These levels all decreased to background values the following day. IL-2 was increased on day 11 only. IL-6 was again elevated on day 13.
Two blood samples have been collected from G-1180 since his discharge from the KGH LFW. A blood sample for follow-up testing was obtained on day 74 post onset of illness. At that time, IgM titers against GPC and NP had risen relative to day 14. Endpoint titers, determined by NP, GP1, GP2 and Z combination ELISA diagnostics, revealed a three-fold increase in IgM titer and an eighteen-fold increase in IgG titer from day 6 to 14 post onset of ilness, and a three-fold increase in both IgM and IgG titer from day 14 to 74 (additional file 2 table S1). Additionally, his complete metabolic panel had returned to the normal range except for a slightly elevated Na+ and TP, and low Cl- and Ca2+. The liver function panel had also normalized. A second follow-up sample obtained on day 108 post onset of illness revealed slightly elevated Na+ and K+, and low Cl- (data not shown). The Cre level was below normal and both ALP and AST were slightly elevated (data not shown). TP also remained elevated since the previous analysis.
Chemistries of Healthy Volunteers and a Fatal Case of Lassa Fever
In order to establish the capabilities and reliability of the Piccolo®, complete chemistries were performed on blood drawn from healthy volunteers as well as a single sample from a patient who succumbed to Lassa Fever. This last sample (G-1177) was drawn less than 12 hours prior to expiry.
The chemistries of the three healthy volunteers were in the normal range as specified by the manufacturer (Abaxis, Inc.) except for a few values. One subject had a low serum Na+, two subjects had low Cl-, one had a low TCO2 one had an elevated TP (Figure 3, additional file 1 figure S1).
Subject G-1177 had extremely abnormal labs prior to expiring. Many of her analytes were above the normal range specified by Abaxis, Inc., including Na+, K+, Cl-, ALP, ALT, TBil, BUN and Cre (Figure 3, additional file 1 figure S1). Conversely, the TCO2, Alb, BUN:Cre and TP levels were low. Only the corrected Ca2+ fell within the normal range. The AST level was zero. When compared to healthy volunteers, she presented with high levels of circulating IL-1beta, IL-12p70, IL-8, IL-6, and IL-10.
On the night of September 2nd patient G-1180 was admitted to the KGH LFW and as routine procedure in the KGH Lassa fever program, the outreach team was dispatched to the village of origin to further investigate the case. Three prior deaths of relatives of G-1180 living in the same dwelling were reported to the outreach team at that time. These deaths were attributed to an undetermined febrile illness and all occurred within the previous month. The deceased family members included a 69-year-old woman, a 42-year-old man and a third individual whose symptoms before death were not known. The histories of the 2 known individuals are as follows: On July 9th the 69-year-old female reportedly experienced persistent high fever, vomiting, diarrhoea and severe headaches. A few days after the initial onset of symptoms the woman travelled to Kenema to seek medical treatment. The 69-year-old woman was admitted at the Arab Hospital, Hangha Road, Kenema for treatment but succumbed on July 16, 2010 without definitive diagnosis. The body was subsequently transported to Hangha and buried at the town cemetery (Figure 1). Two weeks after the death of the 69-year-old woman, her 42-year-old son living in the same dwelling as G-1180 fell ill with similar presentations, including profuse bleeding from all orifices, and succumbed on August 7, 2010 without visiting a health facility. The 42-year-old man was buried at Sembehun Town cemetery. The history of the third, expired relative is unknown.
The outreach team performed a close investigation of the dwelling, which revealed rodent faeces and holes in the walls of all rooms, poor food and water storage and congested home settings. At this time, eight close contacts of G-1180 were identified and blood samples were collected and transported to KGH LFL for LASV Ag and IgM analyses. None of the subjects had a recent travelling history outside the township.
Two of the contacts (G-1180-A, female, age 20, and G-1180-B, female, age 38) tested positive for LASV Ag and IgM, and were subsequently transported to the KGH LFW for ribavirin treatment. Despite detecting LASV Ag and IgM in both patient sera, neither presented with classical symptoms of the disease. G-1180-A and -B, provided only three serum samples each during their 10 day stay in the ward on three consecutive days starting with date of admission on September 4, 2010. Patient G-1180-A was admitted with an elevated TP that declined during ribavirin therapy. Her Alb and Ca2+ remained low. The daily cytokine profile was unremarkable when compared to the healthy volunteers except for a one day increase in IL-8 observed on day two. Patient G-1180-B developed several abnormalities during the 3 day monitoring period. Her TP and TBil increased drastically; though, her cytokine profile was unremarkable.
The outreach team conducted sensitization meetings with the community, including a close door conference with village heads and health committee members. General education on Lassa fever and preventative measures were conveyed to the community through a film on the subject. The team also set rodent traps during their overnight stay, but Mastomys species, the known rodent reservoir of Lassa fever virus, were not trapped. The team was informed that shortly after the onset of G-1180's illness, rat poison was applied in the dwelling.
The NP is the most abundant polypeptide in Lassa virions, with approximately 60 molecules per particle. Thus, detection of NP antigen may be the most sensitive diagnostic for LASV, particularly in samples presenting with low antigenemia. Thus LFI and NP Ag detection ELISA were developed into rapid and confirmatory diagnostic platforms, respectively. Additionally, sensitive assays for the detection of LASV-specific IgM and IgG (Corgenix Medical Corp.), based on recombinant LASV protein ELISA, were used to qualitatively measure immunoglobulin levels (Figure 6B, C). Data from these sensitive assays suggest that G-1180 was naive to LASV exposure prior to this incident as he presented with very low NP-specific IgM levels on day 6, which progressively increased throughout the monitoring period. Conversely, GP1 and GP2-specific IgM titers were not statistically above background levels throughout the same period. On day 10, low IgG titers were detectable in G-1180 as determined by endpoint titers, which increased three-fold through day 14. Immunoglobulins were again measured on day 74 post onset of illness in G-1180, and surprisingly IgM titers against glycoprotein and NP had risen relative to day 14, rather than subsiding (additional file 2 table S1). An expected increase in IgG titers to LASV proteins was also observed. Immunoglobulin levels in G-1180 measured on day 108 post onset of illness revealed still elevated IgM titers as well as IgG titers against NP (data not shown). The IgM titer to NP dropped to approximately one third the qualitative level measured on day 74, whereas IgG rose slightly compared to the same time point. IgM and IgG levels against GP1 and GP2 were not significant on days 74 and 108. This phenomenon of persistently high IgM titers against LASV antigens for weeks, months or longer, in a significant proportion of convalescent patients is currently under investigation (Garry et al., unpublished data).
Concurrent with this observed increase in pro-inflammatory cytokines, a significant spike was measured in the IL-10 levels, an anti-inflammatory cytokine and potent inhibitor of Th1 cytokines and cellular responses. This spike coincided with the onset of a significant increase in the levels of LASV-specific IgM and IgG. Notably, IL-10 levels were elevated throughout the seven days of monitoring. Following the spike in pro- and anti-inflammatory cytokines, body temperature, respiration and pulse rates on day 10, the patient stabilized. His temperature did not return to febrile levels again during the course of these studies, and average respiration and pulse rates stabilized within normal levels. Thus, it appears that G-1180 experienced a dynamic interplay of pro- and anti-inflammatory cytokines and their physiological effects were ongoing throughout the extended analysis period of seven days. More importantly these results strengthen the hypothesis, as previously proposed by others [35, 37, others], that an imbalance between pro- and anti-inflammatory cytokines plays an important role in the development of Lassa hemorrhagic shock, with poor outcome. Notably, the marked absence of TNF-α, a potent inducer of endothelial damage via apoptosis  and thrombocytopenia  throughout the monitored course of illness in G-1180, with the exception of a spike on day10, suggests a somewhat regulated and effective immune response at play. This applies to the marked absence of IL-1β during the same period. It has been suggested that an early pro-inflammatory cytokine response followed by downregulation to baseline levels, which has been characterized in Ebola patients, may also be important in a regulated and balanced immune response and outcome in Lassa fever infections . IL-4, a strong mediator of Th2 CD4+ T cells that stimulate B cells and enhance the humoral response, and IL-5, which functions as a B cell stimulator leading to increase in immunoglobulin secretion were not detected throughout the course of illness .
The two contacts of G-1180 that were admitted to the KGH LFW presented with lower, albeit significant levels of LASV NP antigen compared to G-1180 (Figure 6A). Surprisingly, and despite clear and re-confirmed presence of NP antigen in the serum of both G-1180-A and -B contacts by ELISA, levels did not decrease over a two day period following treatment with ribavirin. Neither contact presented with or developed classical symptoms of Lassa fever during their 10-day hospitalization period. Contact G-1180-A presented with very low but detectable IgM titer to NP, and background level titers to GP1 and GP2. G-1180-B presented with significant IgM titer to GP2, but undetectable titers to GP1 and NP. In both contacts IgG titers were detectable from the outset, but remained unchanged throughout the three days of testing according to endpoint titers. Thus, potential previous exposure to LASV and development of protective humoral and/or cellular immunity may have contributed to the observed Ag positive but asymptomatic status of these two patients
Despite the poor outcome for patient G-1177, a positive control used throughout these studies, she presented with moderate IgM to GP2, low IgM to GP1 and NP, high IgG to NP, and undetectable IgG to glycoproteins. Additionally, G-1177 presented with highly elevated levels of ALP and ALT, but with undetectable AST. Analysis of cytokines in the single G-1177 sample revealed similarities with those seen with G-1180. Notably, elevated levels of IFN-γ, IL-12p70, IL-6, IL-8, and IL-10 were detected. The most remarkable difference was a highly elevated level of IL-1β in G-1177, which was only detected in low levels in G-1180 on day 10. Similar to G-1180's profile, cytokines IL-2, IL-4, IL-5, TNF-α, and TGF-β were not detected in G-1177.
Together, this case outlines the severe and prolonged multi-organ dysregulation, pro- and anti-inflammatory cytokine up- and down-regulation, and difficulty in management of acute Lassa fever infections. This case also delineates the necessity for prompt treatment with IV ribavirn, fluid management and maintenance of electrolyte balance to counter hypovolemia, hemorrhagic shock and malnutrition during the early stage of infection. Given the health status of G-1180 on day 6 post onset of symptoms, this study points to the possibility of positive outcomes in Lassa fever patients with severely compromised metabolic and immunological presentations.
Lassa fever has a high fatality rate if not diagnosed and treated promptly. Due to the low economic status of Lassa endemic areas, it often remains misdiagnosed and untreated. However, due to advances in diagnostics and laboratory research equipment at the KGH LFL, strides are being made towards better understanding the immunological impact of LASV. This case report exemplifies the utility of these methods in defining the host immune response to this pathogen. Our results combined with those of others [35, 37–40] that extensively characterized metabolic and immunological parameters of Lassa fever, point to a strong divergence in metabolites, cytokines and immunoglobulin levels in positive versus fatal outcomes. Further studies on both the cellular immune response and cytokine profiles early in LASV infections will be critical in deepening our understanding of Lassa fever and in closing the gap that currently exists between the initial time of infection and diagnosis and treatment.
This case report represents a snapshot of severe Lassa fever and exemplifies the capabilities of KGH LFL in diagnosing and monitoring the immunologic response during and post infection. This report dispels misconceptions that these studies are impossible in field hospital settings and demonstrates the potential to deploy diagnostic capabilities that will drastically impact treatment of Lassa fever by allowing the rapid identification, isolation and treatment of Lassa fever cases. Lastly the data in this manuscript provides insights into the clinical course of Lassa fever. Not only will these data inform clinical case management but they will also be critical to validating animal models currently being utilized for testing new candidate therapeutics and vaccines.
This work was supported by Department of Health and Human Services/National Institutes of Health/National Institute of Allergy and Infectious Diseases Challenge and Partnership Grant Numbers AI067188 and AI082119, and RC-0013-07 from the Louisiana Board of Regents. We thank the members of the Hemorrhagic Fever Diagnostic Consortium, and Lassa Fever - Mano River Union for ongoing support. Additionally we thank the Ministry of Health in Sierra Leone and the members of the KGH Lassa Fever team including Michael A. Gbakie, Alex G. Moiboi, Alice B. Kovoma, Patrick B. Sannoh, Veronica J. Koroma, Veronica Tucker, Edwin Konuwa, Vandy Sinnah, Fatima Kamara, Sidikie Saffa and Lansana Kanneh for their dedication to this project. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.
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