Resistance of polio to its eradication in Pakistan
Background
This study is based on EPI (Expanded Program on Immunization) immunization surveys and surveillance of polio, its challenges in immunization and the way forward to overcome these challenges.
Methods
Several Government documents, survey reports and unpublished program documents were studied and online search was made to find information on EPI Pakistan. SPSS 16 and Microsoft Excel 2007 were used for the statistical analysis.
Results
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Immunization against polio is higher in urban areas as compared to rural areas. Marked variation in vaccination has been observed in different provinces of Pakistan in the last decade. Secondly 10-20% of the children who have received their first dose of trivalent polio vaccine were deprived of their 2nd and 3rd dose because of poor performance of EPI and Lack of information about immunization.
Conclusion
In spite of numerous successes, such as the addition of new vaccines and raising immunization to over 100% in some areas, EPI is still struggling to reach its polio eradication goals. Inadequate service delivery, lack of information about immunization and limited number of vaccinators were found to be the key reason for poor performance of immunization and for large number of cases reported each year due to the deficiency of second and third booster dose.
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Introduction
Many epidemics are caused by poliovirus in the last three centuries. About 100,000 new polio cases are reported each year worldwide. Europe and North America were the targets of Epidemic poliomyelitis in 1890s. Now a day most of these cases occur in Asia and Africa. German physician Jakob Heine recognized Poliomyelitis as a distinct condition for the first time in 1840. Poliovirus was identified by Austrian physicians Karl Landsteiner and E. Popper in 1908. The three serotypes 1, 2, and 3 infect cells via a specific receptor, PVR (polio virus receptors): CD-155. These receptors are only present in human cell that is why humans are the only reservoirs of this virus. The serological relationship is present between serotype 1 and serotype 2. This is conferred by significant protection against type 2 by the antibodies which were produced against serotype 1. Immunologically Type 2 is broad and it is the first serotype to disappear during vaccination campaigns. Albert Sabin and Jonas Salk developed effective vaccines against Polio virus in the early 1960s with different approaches. The Sabin oral vaccine which contains live attenuated polio virus is superior to the Salk inactivated vaccine in two ways, firstly it is easily administered; secondly it provides a long-lasting immunization. Pakistan use trivalent OPV that contain all the three serotypes in attenuated form.
Novel snake papillomavirus does not cluster with other non-mammalian papillomaviruses. Part 3
Furthermore, Needle alignments which reflect global alignments of the entire genes in contrast to the alignment used for the tree were made. Upon these pairwise alignments on the nucleotide level of the L1 ORF the highest percentage of sequence identities was found with a human PV (HPV5; 57.9%) and with TmPV1 on the E1 ORF (55.6%). Consequently MsPV1 may be regarded as rather a close to the root prototype of a new PV clade that might establish a new genus regarding to the guidelines of PV classification.
While many PVs have been described in humans and other mammalian species, the number of known PVs infecting non-mammalian species is limited. The described MsPV1 genome represents the first PV isolated from a snake. It contains the characteristic ORFs E6, E7, E1, E2, L1 and L2, a large non-coding region between L1 and E6 as well as a small non-coding region between E2 and L2. The size of the viral genome (7048 bp) is comparable with the two turtle PV genomes (CcPV1; 7020 bp and CmPV1; 6953 bp), which are all relatively small. However, while the latter two viruses have short versions of E1, E2, L2 and E6 ORFs as well as the NCR, the small size of MsPV1 is primarily due to comparably short E2 and L2 ORFs and also comparably short NCRs.
The genomic sequences of the PVs from three bird and two turtle species have previously been published. According to our and other’s phylogenetic analysis, they cluster together. However, our newly discovered PV genome isolated from the snake did not at all cluster with the five other sauropsid PVs (CcPV1, CmPV1, FcPV1, FlPV1 and PePV1). Upon pairwise alignment of individual ORFs, MsPV1 shares much higher percentage of sequence identities with mammalian PVs than with any of the known sauropsid PVs. This finding raises interesting questions in the context of PV evolution. While co-evolution with the host has been suggested and demonstrated to play a role in PV evolution it has also been shown that PV evolution is probably a complex matter and other mechanism such as crossing of species barriers and adaptive radiation have to be considered as well.
Turtles as putative representatives of the Anapsida and snakes as representatives of the Diapsida are phylogenetically distinct with a common ancestor dating back more than 200 million years. However, the three PVs isolated from birds (FcPV1, FlPV1 and PePV1), which also belong to the Diapsida, are phylogenetically much closer to the turtle PVs (CcPV1, CmPV1) than to MsPV1. As the snake PV appears closer to mammalian PVs, one explanation could be that it belongs to a lineage of PVs, which existed already in Amniota species, that lived before the split of Synapsida (mammals and mammal-like reptiles) and Sauropsida. The five other sauropsid PVs could under these circumstances go back to a second distinct ancestral lineage. However, an alternative explanation could be that some ancestor of this virus had been able to cross species barriers between the Sauropsida and the Synapsida, even long after their separation.
Novel snake papillomavirus does not cluster with other non-mammalian papillomaviruses. Part 2
Total DNA from a 25 mg tissue sample was isolated using a QIAamp DNA extraction kit (Qiagen) according to the manufacturer’s recommendations. One microliter of the extracted DNA was used for RCA, using a TempliPhi Amplification kit (General Electrics Biosciences). Slight modifications were applied to the protocol supplied by the manufacturer: 1 μl of 10 mM dNTPs was added and the reaction time was prolonged to 16 h at 30°C. Amplified DNA was cloned into the EcoRI or XhoI site of pBluescript II KS+ (Stratagene) using standard procedures.
The nucleotide sequence of cloned DNA and of precipitated RCA product was determined (Microsynth) on both strands by cycle sequencing using an ABI 377 sequencer (Applied Biosystems). The primary sequences were assembled using Contigexpress software (Vector NTI Informax).
Analyses of the cloned sequence confirmed that papillomavirus DNA had actually been detected. The amplified genome consists of 7048 bp and has a GC content of 41%. ORFs putatively encoding E6, E7, E1, E2, E4, L2 and L1 but no E5 were identified. Deduced amino acid sequences of the putative proteins revealed a degenerate ATP-dependent helicase motif GQPNTGKS in E1, two putative metal-binding motifs in E6, one such motif in E7, and one pRb binding domain. The deduced amino acid sequences of both structural proteins L1 and L2 are predicted to harbour a basic tail at their C termini.
A non coding region (NCR1) between the stop-codon of the L1 ORF and the start-codon of the E6 ORF was 473 nt in length. A second non coding region (NCR2) of 178 nt was between the stop-codon of the E2 and the start-codon of the L2 ORF.
In addition, papillomavirus-specific DNA motifs were identified in the readily determined genome sequence. Four putative consensus sequences for E2 binding (ACCN5-7GGT) were detected; two of these were located in the NCR1 (positions 6919-6931 and 32-43) and two were located within the predicted L1 ORF (positions 5936-5948 and 5990-6000). Within the NCR1, a putative origin of DNA replication was identified, consisting of two E2-binding regions flanking a region with 54% A/T content. Two polyadenylation consensus sequences (AATAAA) were predicted, one within the NCR1 (position 6729-6734) and the other within the NCR2 (position 3816-3821).
In order to possibly allocate the novel PV in the evolutionary context, phylogeny based on the aligned E1-E2-L2-L1 sequences was determined. Sequences of fifty PVs, representing all presently classified genera and MsPV1 were included in these analyses. While all other sauropsid PVs clustered together, MsPV1 was located far from any of them. Interestingly, MsPV1 was found in relative proximity to the PV (TmPV1) of a marine mammal, the manatee (sea cow).
Novel snake papillomavirus does not cluster with other non-mammalian papillomaviruses
Papillomaviruses (PVs) are associated with the development of neoplasias and have been found in several different species, most of them in humans and other mammals. We identified, cloned and sequenced PV DNA from pigmented papilloma-like lesions of a diamond python (Morelia spilota spilota). This represents the first complete PV genome discovered in a Squamata host (MsPV1). It consists of 7048 nt and contains the characteristic open reading (ORF) frames E6, E7, E1, E2, L1 and L2. The L1 ORF sequence showed the highest percentage of sequence identities to human PV5 (57.9%) and Caribbean manatee (Trichechus manatus) PV1 (55.4%), thus, establishing a new clade. According to phylogenetic analysis, the MsPV1 genome clusters with PVs of mammalian rather than sauropsid hosts.
The members of the family Papillomaviridae are non-enveloped, icosahedral viral particles with a diameter of 50 to 55 nm and a small (~8 kbp), circular, double stranded DNA genome, which is transcribed into one single direction. The family may be divided into at least 29 genera with a vast number of species, types, subtypes, and variants of papillomaviruses (PVs), based on nucleotide identities of the L1 open reading frames (ORFs). Due to the high genetic diversity and the host range it is anticipated, that the family Papillomaviridae has a long evolutionary history, but details remain yet vague.
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DNA of PVs can be detected in a wide range of vertebrate species, thus far including humans, various other mammals, birds, and turtles. PVs have been found to play a role in several human diseases of the skin and mucous membranes. The DNA of PVs can be amplified from samples of clinical asymptomatic individuals, but more importantly their influence on the development of certain benign and malignant disorders was demonstrated repeatedly.
The sequences of three PVs from birds, namely the Chaffinch (Fringgilla coelebs), the Yellownecked Francolin (Francolinus leucoscepus) and the Timneh African gray parrot (Psittacus erithacus timneh), have been determined. Various data suggest the existence of sauropsid-specific PVs in association with papillomas in lizards, snakes, crocodiles and turtles. Sequences of the entire genome from two PVs of turtles were determined recently from the Loggerhead turtle (Caretta caretta) and the Green seaturtle (Chelonia mydas). Upon phylogenetic analysis, these five sauropsid PVs cluster together and appear clearly distinct from the PVs infecting mammalian species.
Here, we report on the cloning, sequence determination and phylogenetic analysis of a PV-specific DNA from the Australian diamond python (Morelia spilota spilota).
Samples from a diamond python with small black papillated and pedunculated exophytic skin proliferations were taken and stored at -20°C until processing.
Contact Tracing. Part 3
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, 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. 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.
Conclusions
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 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.
Contact Tracing. Part 2
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. 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. 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).
The most remarkable aspect of the cytokine profile obtained at the time of admission was the significant level of IL-12p70 measured in serum. This pleitropic proinflammatory cytokine is produced by phagocytic cells, B cells, and other antigen-presenting cells that modulate adaptive immune responses, promotes Th1 cell development and is a potent inducer of IFN-γ and other cytokines in peripheral blood T and NK cells. IFN-γ further enhances production of additional IL-12 and other pro-inflammatory cytokines by phagocytic cells. IL-12 induced IFN-γ acts in a positive feedback loop providing an amplification mechanism in the inflammatory response. Remarkably on day 7, IL-12p70 was not detected, but its decrease coincided with a rise in IFN-γ levels, which remained elevated through day 10, but were undetectable on day 11. Another increase in IL-12p70 levels was measured on days 11 and 12, the latter coinciding with a second spike in IFN-γ. Though IL-12p70 may have some impact on pathogenesis of Lassa fever, its role in arenaviral pathogenesis has not been described. Despite this profile early in the monitoring period other pro-inflammatory cytokines, such as IL-1β and TNF-α were undetectable, and IL-6 and IL-8 fluctuated in the lower end of the assay range. Elevated levels of IL-8 have been previously reported by Mahanty et al., 2001 to correlate with a positive outcome in acute Lassa fever infections, in addition to IFN-induced IP-10, which was not measured in these studies. A very notable albeit short increase in pro-inflammatory cytokines was measured on day 10, when IL-6 and IL-8 spiked to very high levels, in addition to a small spike in IL-1β and TNF-α. The increase of endogenous pyrogens IL-1β, TNF-α, IL-6, IL-8 on day 10 coincided with re-development of a mild fever, and significantly increased respiratory and pulse rates. These levels dropped to baseline or were undetectable 24 hours later. A single spike in IL-2, a cytokine important in the differentiation and survival of cytotoxic T cells, and a facilitator of immunoglobulins by B cells, was measured on day 11.
Contact Tracing
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.
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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.
Chemistries of Healthy Volunteers and a Fatal Case of Lassa Fever
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.
Clinical Chemistry
On presentation to KGH LFW, his sodium (Na+), chloride (Cl-), total carbon dioxide (TCO2), albumin (Alb) and total protein (TP) were all below the normal values for a male his age. The liver function panel revealed an aspartate aminotransferase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) drastically above the normal range. Additionally, the blood urea nitrogen (BUN) and BUN: creatinine (Cre) levels were elevated. His potassium (K+), total bilirubin (TBil), corrected calcium (Ca2+) and Cre levels, on the other hand, all were within the upper normal range. His hemoglobin (Hb) levels were low throughout hospitalization.
At the time of admission to KGH LFW, a cytokine profile indicated that interleukin (IL) -12p70, IL-6 and IL-10 levels were elevated compared to the healthy controls.
Diagnosis
A blood specimen collected on the day of admission was positive for Lassa NP antigen (Ag) by LFI, and positive by quantitative NP antigen-capture ELISA, with a level of 13 μg/mL NP. Additionally, IgM levels to recombinant Lassa proteins (NP alone and NP, GP1, GPC combination) were determined by ELISA, with low but detectable levels of immunoglobulin to NP and GPC. IgG levels were not above background detection upon initial diagnosis.
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. The donor was a Caucasian American female of the same blood type (A+). A second blood transfusion was administered on day 21 of illness 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 and NP Ag capture ELISA detected LASV NP through day 11. LASV NP antigen dropped rapidly over 3 days following the onset of ribavirin treatment.
Capacity building permitting comprehensive monitoring of a severe case of Lassa hemorrhagic fever in Sierra Leone with a positive outcome. Part 3
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.
Presentation
Case 1: On September 1, 2010 the KGH LFW was contacted by the medical officer of Gondama Hospital in Bo district, Sierra Leone, concerning a suspected case of Lassa hemorrhagic fever. The patient was an eight-year-old male from Sembehun town, Malegohun chiefdom, Kenema district, Sierra Leone who had been first seen at the local health facility on August 30, 2010 (Figure 1). He presented to the health facility 2 days after the onset of symptoms with a history of persistent fever, headache, and profuse oral, nasal, and rectal bleeding. He was then referred to the Gondama Hospital where he could receive free medical care. After two days of treatment with anti-malarial and antibiotic drugs of unknown type and no improvement, the patient was referred to the KGH LFW as a suspected Lassa case. Upon arrival by ambulance at the KGH LFW on September 2, 2010, the patient reported having multiple symptoms including anorexia, malaise, headache, nausea, abdominal pain, loose black stools, hematemesis, dysuria, cough, sore throat and retrosternal pain. He presented with a body temperature of 38°C, a pulse rate of 140 beats/minute, and a respiration rate of 30. On examination, he was in obvious pain and lethargic. Bleeding from the mouth and nose was noted along with conjunctival injection, facial edema and hepatomegaly. During the first 24 hours after his admission to KGH LFW, he had multiple episodes of grossly bloody stools as well as hematemesis, hemoptysis and hemeturia. This patient was assigned the coded designation G-1180 upon initial diagnosis by NP LFI at KGH LFW, which will be used henceforth.