Convalescent Samples Research Articles

Chikungunya Virus Growth and Fluorescent Labeling: Detection of Chikungunya Virus by Immunofluorescence Assay.

Immunofluorescence assay (IFA) is a highly versatile and sensitive assay for detection and titration of chikungunya virus (CHIKV). The IFA technique requires virus-infected cells (viral antigen) and antibodies specific to the viral antigens for detection. Suitable antibodies for detection include monoclonal antibodies specific to CHIKV structural and nonstructural proteins, polyclonal antibodies, and convalescent serum samples. Here, the details of virus antigen preparation, detection by IFA method, and applications are described. The described IFA method is potentially useful in a wide range of studies including virus growth kinetics and virus infection mechanism studies. Additionally, the described IFA method can be modified for applications in arbovirus diagnosis, including CHIKV.

SEVERITY OF BRONCHIOLITIS ASSOCIATED WITH ATYPICAL PATHOGENS IN HOSPITALIZED INFANTS IN GEORGIA

Introduction: Bronchiolitis is the most common reason for hospitalization worldwide. Respiratory syncytial virus (RSV), human Metapneumovirus, human Rhinoviruses, human Bocavirus have been shown to predominate. A few studies however have attempted to determine whether other pathogens, particularly Mycoplasma Pneumoniae (Mpn) and Chlamydophila pneumoniae (Cpn), are associated with bronchiolitis in children under 2 years of age. The aim of this study was to determine the prevalence and severity of Mpn and Cpn infection in children under the age of two years presenting to the Iashvili Central Children Hospital in Tbilisi.
 Material and Methods: Acute and convalescent serum samples were tested by ELISA for IgM and IgG antibodies to RSV, Cpn and Mpn. 37 children under two years of age were studied. In 19 patients out of 37 (51.35%) etiological diagnosis were established and in 18 patients (48.65%) no pathogens were found. 11 patients (29.72%) had either Cpn or Mpn and 8 patients (21.62%) had RSV.
 Results: Children infected with Cpn and Mpn had less severe bronchiolitis than those infected with RSV. There were no statistically significant differences between groups with respect to length of hospital stay.
 Conclusion: Our study underlines the importance of atypical bacterial pathogens in acute bronchiolitis in children under two years of age and highlights the complex epidemiology and clinical features of these pathogens in this age group.

Serological Evidence of Chikungunya Virus among Acute Febrile Patients in Southern Mozambique.

BackgroundIn the last two decades, chikungunya virus (CHIKV) has rapidly expanded to several geographical areas, causing frequent outbreaks in sub-Saharan Africa, South East Asia, South America, and Europe. Therefore, the disease remains heavily neglected in Mozambique, and no recent study has been conducted.MethodsBetween January and September 2013, acute febrile patients with no other evident cause of fever and attending a health center in a suburban area of Maputo city, Mozambique, were consecutively invited to participate. Paired acute and convalescent serum samples were requested from each participant. Convalescent samples were initially screened for anti-CHIKV IgG using a commercial indirect immunofluorescence test, and if positive, the corresponding acute sample was screened using the same test.ResultsFour hundred patients were enrolled. The median age of study participants was 26 years (IQR: 21–33 years) and 57.5% (224/391) were female. Paired blood samples were obtained from 209 patients, of which 26.4% (55/208) were presented anti-CHIKV IgG antibodies in the convalescent sample. Seroconversion or a four-fold titer rise was confirmed in 9 (4.3%) patients.ConclusionThe results of this study strongly suggest that CHIKV is circulating in southern Mozambique. We recommend that CHIKV should be considered in the differential diagnosis of acute febrile illness in Mozambique and that systematic surveillance for CHIKV should be implemented.

Abstract O.23: Lysyl Oxidase Expression In The Patients With Kawasaki Disease

Background: Histologic study of inflamed coronary arteries from the Kawasaki disease (KD) patients shows infiltration by immune effector cells, IL-17-producing myofibroblasts, and breaks in the internal elastic lamina. Transcripts from genes associated with cell trafficking (e.g. paxillin) are elevated in whole blood from acute KD patients. Lysyl oxidase (LOX) is a multifunctional protein affecting affecting “monocyte and smooth muscle cell migration, elastogenesis, and endothelial-to-mesenchymal transition. LOX promotes cell motility through paxillin phosphorylation, which influences T-cell trafficking via the calcium signaling pathway. We investigated the role of LOX in the pathogenesis of KD. Methods: Agilent microarray data using whole blood were analyzed for differential expression of enzymes in the LOX family (LOX, LOX-like (L)1-4) in acute and convalescent KD samples (n=19). LOXL1 results were validated using RT-PCR in an independent cohort (n=20). LOXL1 transcripts levels were measured in KD whole blood, peripheral blood mononuclear cells (PBMC), T-cell clones, B-cell lines, and myeloid dendritic cells from acute KD patients. Immunohistochemical staining for LOXL1 was performed on coronary arteries from a KD patient who died on illness day 7. Result: LOXL1 expression by microarray was 3.2-fold higher in acute than convalescent samples (p<0.001) and was validated in an independent cohort by RT-PCR (5.4-fold higher, p<0.001). LOXL1 transcripts levels were 2-35 times higher in PBMC compared to whole blood (n=8). KD T cell clones had higher LOXL1 transcript levels than B-cells and dendritic cells. Immunohistochemical staining showed LOXL1-expressing inflammatory cells and smooth muscle cells in the coronary artery walls. Conclusion: LOXL1 may increase inflammation in acute KD by promoting the motility of infiltrating T cells and smooth muscle cells through focal adhesion formation. However, LOXL1 may also participate in arterial wall repair by crosslinking collagen and elastin. Statins inhibit focal adhesion formation and thus cell trafficking but not the crosslinking mediated by LOXL1. Studies of LOXL1 protein levels and paxillin phosphorylation in PBMC may further our understanding of their role in KD pathogenesis.

Detection of serum anti-NV-F antibodies in the convalescent phase of severe hepatitis in patients positive for tissue NV-F antigen and novel virus-like particles.

Previously, a non-human DNA fragment named NV-F was isolated from a patient with non-A-E fulminant hepatitis. This sequence encoded an incomplete open reading frame (the NV-F antigen). In this study, we developed a western blot assay to detect serum anti-NV-F antibodies. Serum samples from 347 patients with severe hepatitis (ALT > fivefold ULN) were analyzed to understand the prevalence and distribution of the NV-F associated virus (HnFV) infection. Of these patients, acute HnFV infection was diagnosed (by positive serum NV-F DNA) in 34 patients (9.8%). However, none of these 34 serum samples were positive for serum anti-NV-F antibodies. In the remaining patients negative for serum NV-F DNA, 62 (17.9%) were positive for serum anti-NV-F antibodies. Liver biopsy samples from 35 severe hepatitis patients were submitted for immunohistochemistry and electron microscopy examination. Of them, seven were positive for hepatic NV-F antigen expression. Electron microscopy identified a novel virus-like particle in all of the seven NV-F antigen-positive liver tissues but not in the remaining 28 NV-F antigen-negative liver tissues. Longitudinal serum sample analysis revealed transient positivity of serum NV-F DNA in three of the seven patients during the clinical courses. Seroconversion of anti-NV-F antibody from negative to positive was found in four of the seven patients and all positive anti-NV-F antibodies were detected in the convalescent phases. In conclusion, in patients with severe hepatitis, a novel hepatotropic virus, temporarily named HnFV, was found in liver tissues expressing the NV-F antigen. Serum anti-NV-F antibodies were detected in the convalescent serum samples.

Guideline on the management of acute chest syndrome in sickle cell disease.

1. Department of Haematology, Guy’s and St Thomas’ NHS Foundation Trust 2. Lane Fox Respiratory Unit, St Thomas’ Hospital, Guy’s and St Thomas’ NHS Foundation Trust 3. Department of Haematology, North Middlesex Hospital 4. Department of Paediatric Haematology, Bristol Royal Hospital for Children 5. Department of Haematological Medicine, King’s College Hospital 6. Department of Haematology, Whittington Hospital

Antibody responses to influenza viruses in paediatric patients and their contacts at the onset of the 2009 pandemic in Mexico.

On April 2009, the Mexican Ministry of Health received notification of cases of severe pneumonia mostly affecting young healthy people; this was the beginning of the first influenza pandemic of the 21st century. The nature of the immune response to the influenza A(H1N1)2009 pandemic strain in Mexico at the beginning of the pandemic outbreak has not been completely defined. We describe the serological response to the 2009 pandemic influenza virus in paediatric patients with influenza-like illness, their household contacts (HHCs), and exposed health-care workers (HCWs) at the beginning of the pandemic outbreak in Mexico City. thirty pre-epidemic and 129 epidemic samples were collected and serum antibodies were measured against A(H1N1)2009 pandemic virus and two non-pandemic swine influenza viruses by an haemagglutination inhibition assay . 91% (29/32) of the convalescence samples from confirmed patients had an antibody titre ≥ 10 (GMT 25), 63% (41/65) of the HHCs (GMT 12), 41% of HCWs (GMT 6) and 13% (4/30) of pre-epidemic samples (GMT 6) for the pandemic influenza virus. Of the 32 confirmed cases, 60% had an antibody titre ≥ 40 for the pandemic strain, 53% for the A/swine/Iowa(H1N1) virus (GMT 62) and 43% for the A/swine/Texas(H3N2) virus (GMT 66). The antibody response to 2009 pandemic influenza virus was widespread in convalescence samples from patients with confirmed pandemic influenza infection but the GMT was below the protective titre. There was no evidence that antibodies to the swine influenza viruses had cross-protective effect against the 2009 pandemic influenza virus.

Outbreak of scrub typhus in Puducherry & Tamil Nadu during cooler months

Background & objectives:The southern part of India has witnessed an increase in scrub typhus (ST) during the past ten years. ST outbreaks occurred during winter months but at intervals of one to three years. With only a few reports of ST in Puducherry, this study was undertaken to look for the persistence of ST cases in Puducherry and Tamil Nadu in the winter months.Methods:During relatively cooler months of September, 2012 to March, 2013, a total of 45 patients with fever and clinical suspicion of ST and who provided both acute and convalescent blood samples were included. Total WBC, platelet counts, serum creatinine, liver enzymes levels and a rapid immunochromatographic test (RICT) for ST were first done. Paired serum samples were analysed by two specific tests - ST IgM and IgG ELISA- and a non-specific, but widely used Weil-Felix (WF) test.Results:Of the 45 patients, 21 adults and seven children were confirmed as ST based on clinical and laboratory findings, and positivity in specific serological test(s). Setting ST IgM and IgG ELISA as reference, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for RICT were 91.67, 85.71 per cent; 90.48, 100 per cent; 91.67, 100 per cent and 90.48, 80.95 per cent, respectively. Similarly, for WF the values were 83.33, 75 per cent; 95.24, 100 per cent; 95.24, 100 per cent and 83.33, 70.83 per cent, respectively.Interpretation & conclusions:ST continues to persist in the cooler months in Puducherry and neighbouring Tamil Nadu with fever and myalgia as prominent features. None of the tests evaluated in this study was found to be ideal, but ST IgM/IgG ELISA was useful for batch testing and the non-specific WF test can be used in resource poor settings.

Amino acid substitutions in hemagglutinin of the 2009 pandemic influenza A(H1N1) viruses that might affect the viral antigenicity.

BackgroundDuring 2009 to 2012, Thailand had encountered 4 distinctive waves of the 2009 pandemic influenza A(H1N1) (H1N1pdm) outbreaks. Considering the RNA nature of the influenza viral genome, a mutation in hemagglutinin (HA) gene which led to change in antigenicity of the strains circulating during those epidemic periods is anticipated. It is also uncertain whether the A/California/07/2009 (H1N1) (CA/07) vaccine strain still confers protective immunity against those evolved viruses, the causative agents of the later epidemic waves.MethodsHA gene segments of 10 H1N1pdm isolates obtained during 2009 to 2012 were sequenced and phylogenetically analysed using ClustalW and MEGA5 programs. A total of 124 convalescent serum samples collected from patients naturally infected during 3 epidemic waves were employed as tools to investigate for antigenic change in HA of these 10 circulating H1N1pdm viruses by hemagglutination inhibition (HI) assay.ResultsA phylogenetic analysis showed that the 10 virus isolates were grouped into 4 clusters corresponding to the time of 4 consecutive outbreaks. An accumulation of amino acid substitutions in HA was observed in viruses derived from the late epidemic waves. Significantly lower antibody titers were observed when CA/07 was tested against convalescent sera collected from the 3 waves (p < 0.05) compared to most of Thai isolates; and significantly lower antibody titers were also obtained when virus isolates, retrieved from the third epidemic wave were tested against convalescent sera collected during the first and second wave. These results were suggestive of change in antigenicity of the evolved viruses. Our results also showed some mutation position residing outside the previously reported antigenic site that may involve in an alteration of the viral antigenicity.ConclusionsOur study demonstrated that convalescent sera collected from individuals naturally infected with H1N1pdm virus were successfully used to reveal a statistically significant change in antibody titers against the currently evolved H1N1pdm viruses as determined by HI assay. Nevertheless, the antibody titers of individual serum against various viruses were less than 4-folded difference as compared to that against the CA/07 vaccine strain. Therefore, CA/07 is still a potent vaccine strain for those evolved H1N1pdm viruses.

Evidence of West Nile virus infection in Nepal.

BackgroundAcute febrile illness is common among those seeking medical care and is frequently treated empirically with the underlying illness remaining undiagnosed in resource-poor countries. A febrile illness study was conducted 2009-2010 to identify known and unknown pathogens circulating in Nepal.MethodStudy methods included diagnostic testing and preliminary ELISA screening of acute and convalescent samples for diseases both known and unknown to be circulating in Nepal, including West Nile virus (WNV). The molecular assays including Polymerase Chain Reaction (PCR), Sanger sequencing and ultra deep sequencing on MiSeq Illumina Platform were conducted to further confirm the presence of WNV.ResultsThe study enrolled 2,046 patients presenting undifferentiated febrile illness with unknown etiology. Sera from 14 out of 2,046 patients were tested positive for west nile virus (WNV) by nested Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Only two out of 14 cases were confirmed for the presence of WNV by sequencing and identified as WNV lineage 1 phylogentically. The two patients were adult males with fever and no neurological symptoms from Kathmandu and Bharatpur, Nepal.ConclusionTwo out of 2,046 serum samples contained fragments of WNV genome resembling WNV lineage 1, which is evidence of the continued spread of WNV which should be considered a possible illness cause in Nepal.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0606-0) contains supplementary material, which is available to authorized users.

Abstract 1384: Promoter methylation of tumor suppressor genes in convalescent saliva samples from patients with head and neck squamous cell carcinoma

Abstract Purpose: Silencing of tumor suppressor genes (TSGs) plays an important role in head and neck carcinogenesis. Methylation of CpG islands in the promoter regions of genes acts as a significant mechanism of epigenetic gene silencing. In this study, we assessed the feasibility of epigenetic alterations in TSGs in post-treatment DNA shed from the oral mucosa as a prognostic marker for persistent/recurrent head and neck cancer. A panel of four genes (KIF1A, EDNRB, p16, DCC) was evaluated in this study. Experimental Design: Hypermethylation of the promoter regions was investigated by bisulfite modification and quantitative methylation-specific PCR (Q-MSP) which provides more objective and rapid estimation of gene methylation status. Fresh tumor samples and pre- and post-treatment salivary rinses were collected from 83 patients with HNSCC. Post-treatment saliva samples were collected once in every 3 months. In addition, 46 normal saliva and 16 normal mucosal samples from healthy individuals were obtained. Then we evaluated the correlation of the results with the clinical parameters. Results: KIF1A, EDNRB, p16 and DCC genes were methylated in 94%, 95%, 37% and 84% of primary HNSCC tissues, respectively and were only methylated in 2%, 6.8%, 5% and 10% of the salivary rinses from normal subjects. In addition, KIF1A, EDNRB, p16 and DCC were methylated in 35%, 69%, 8.4% and 38.6% of pre-treatment salivary rinses from HNSCC patients, respectively. We evaluated whether methylation levels of these genes in the post treatment saliva may predict the recurrence/prognosis or the chance of cure after treatment. Our data indicate that these genes may be potential biomarkers for HNSCC detection and follow up. Sixteen of 83 patients had recurrent tumors and KIF1A+EDNRB+p16+DCC methylation was observed in 37.5% of the patients in this group. The three gene panel (KIF1A, EDNRB and DCC) was also observed in 37.5% of the recurrent group and 46% of the overall patients, respectively. Conclusion: Methylation of the KIF1A, EDNRB and DCC gene promoters is a frequent event in HNSCC and these genes are not methylated in normal salivary rinses, demonstrating potential that they may act as biomarkers in detection strategies. Patients with presence of methylation in surveillance salivary rinses are at significant risk for recurrence. Quantitative measurement of salivary methylated DNA may have promise for surveillance and early detection of recurrence. Citation Format: Semra Demokan, Jatinder Kaur, Alice Y. Chuang, Wojciech K. Mydlarz, Kavita M. Pattani, Wayne M. Koch, David Sidransky, Nejat Dalay, Joseph A. Califano. Promoter methylation of tumor suppressor genes in convalescent saliva samples from patients with head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1384. doi:10.1158/1538-7445.AM2014-1384

High rate of A(H1N1)pdm09 infections among rural Thai villagers, 2009-2010.

BackgroundPandemic influenza A(H1N1)pdm09 emerged in Thailand in 2009. A prospective longitudinal adult cohort and household transmission study of influenza-like illness (ILI) was ongoing in rural Thailand at the time of emergence. Symptomatic and subclinical A(H1N1)pdm09 infection rates in the cohort and among household members were evaluated.MethodsA cohort of 800 Thai adults underwent active community-based surveillance for ILI from 2008–2010. Acute respiratory samples from ILI episodes were tested for A(H1N1)pdm09 by qRT-PCR; acute and 60-day convalescent blood samples were tested by A(H1N1)pdm09 hemagglutination inhibition assay (HI). Enrollment, 12-month and 24-month follow-up blood samples were tested for A(H1N1)pdm09 seroconversion by HI. Household members of influenza A-infected cohort subjects with ILI were enrolled in household transmission investigations in which day 0 and 60 blood samples and acute respiratory samples were tested by either qRT-PCR or HI for A(H1N1)pdm09. Seroconversion between annual blood samples without A(H1N1)pdm09-positive ILI was considered as subclinical infection.ResultsThe 2-yr cumulative incidence of A(H1N1)pdm09 infection in the cohort in 2009/2010 was 10.8% (84/781) with an annual incidence of 1.2% in 2009 and 9.7% in 2010; 83.3% of infections were subclinical (50% in 2009 and 85.9% in 2010). The 2-yr cumulative incidence was lowest (5%) in adults born ≤1957. The A(H1N1)pdm09 secondary attack rate among household contacts was 47.2% (17/36); 47.1% of these infections were subclinical. The highest A(H1N1)pdm09 secondary attack rate among household contacts (70.6%, 12/17) occurred among children born between 1990 and 2003.ConclusionSubclinical A(H1N1)pdm09 infections in Thai adults occurred frequently and accounted for a greater proportion of all A(H1N1)pdm09 infections than previously estimated. The role of subclinical infections in A(H1N1)pdm09 transmission has important implications in formulating strategies to predict and prevent the spread of A(H1N1)pdm09 and other influenza virus strains.

Serological profile among patients with malaria in secondary community hospitals in India - a multicenter cross sectional study

Materials and methods Serological tests from patients with malaria in an observational fever study were analyzed. During April 2011November 2012, 1564 patients aged ≥5 years with fever for 2-14 days were consecutively included at seven secondary community hospitals in Tezpur (Assam), Raxaul (Bihar), Mungeli (Chhattisgarh), Ratnagiri (Maharashtra), Anantapur (Andhra Pradesh), Oddanchatram and Ambur (Tamil Nadu). Scrub typhus IgM ELISA (In Bios, USA), Leptospira IgM ELISA (Alere, Australia), Chikungunya IgM ELISA (NIV, India), Dengue rapid NS1 antigen and IgM Combo test (SD bioline, USA) were performed at the hospital sites. At the monitoring site (Benjamin M Pulimood Laboratories, Christian Medical College, Vellore, India), malaria genus mitochondrial PCR was performed on all samples and 18S species-specific PCR or sequencing performed on genus positive samples with methods as described previously (Haanshuus et al., Malaria Journal 2013). At the monitoring site Dengue IgM capture ELISA (MAC-ELISA), Scrub typhus immunofluorescence (IFA) on Scrub ELISA positive samples and Leptospira Microscopic Agglutination Test (MAT) on Leptospira ELISA positive samples were also performed. Convalescence samples for test of rise in titer were not collected due to logistical challenges. Malaria PCR was considered gold standard for the diagnosis of malaria.

Are patients with erythema migrans who have leukopenia and/or thrombocytopenia coinfected with Anaplasma phagocytophilum or tick-borne encephalitis virus?

Lyme borreliosis (LB), tick-borne encephalitis (TBE) and human granulocytic anaplasmosis (HGA) are endemic in central part of Slovenia. We tested the hypothesis that patients with erythema migrans (EM) from this region, who have leukopenia and/or thrombocytopenia (typical findings in HGA and in the initial phase of TBE but not in patients with LB) are coinfected with Anaplasma phagocytophilum and/or with TBE virus, i.e. that cytopenia is a result of concomitant HGA or the initial phase of TBE. Comparison of clinical and laboratory findings for 67 patients with EM who disclosed leukopenia/thrombocytopenia with the corresponding results in sex- and age-matched patients with EM and normal blood cell counts revealed no differences. In addition, patients with typical EM and leukopenia and/or thrombocytopenia tested negative for the presence of IgM and IgG antibodies to TBE virus by ELISA as well as for the presence of specific IgG antibodies to A. phagocytophilum antigens by IFA in acute and convalescent serum samples. Thus, none of 67 patients (95% CI: 0 to 5.3%) with typical EM (the presence of this skin lesion attests for early Lyme borreliosis and is the evidence for a recent tick bite) was found to be coinfected with A. phagocytophilum or had a recent primary infection with TBE virus. The findings in the present study indicate that in Slovenia, and probably in other European countries endemic for LB, TBE and HGA, patients with early LB are rarely coinfected with the other tick-transmitted agents.

CLINICAL MANIFESTATIONS SEEMED MORE SEVERE AMONG PATIENTS WITH ANTIBODY 4-FOLD OR A GREATER INCREASE IN TITER BOTH AGAINST pH1N1 AND AGAINST SEASONAL INFLUENZA THAN THOSE WHOSE ANTIBODY 4-FOLD OR GREATER INCREASE IN TITER WAS ONLY AGAINST ONE TYPE OF SEASONAL INFLUENZA

June 24, 2013To the editorIn 2009, after an emergence of pandemic influenza A(H1N1) (pH1N1), one of our serological investigations showed 23 students whose paired serum antibodies simultaneously presented 4-fold or greater increase in titer both against pH1N1 and against seasonal influenza (SI). This caught our attention and in order to further understand clinical features of these cases, a case control study was conducted. From October 2009 to September 2012, we collected the acute and convalescent phase serum samples from patients whose throat swabs were positive for pH1N1 or SI by real-time reverse transcription polymerase chain reaction (RT-PCR)

Pneumococcal Aetiology and Serotype Distribution in Paediatric Community-Acquired Pneumonia

Community-acquired pneumonia (CAP) is a major cause of morbidity in children. This study estimated the proportion of children with pneumococcal CAP among children hospitalised with CAP in Belgium and describes the causative serotype distribution after implementation of the 7-valent pneumococcal conjugate vaccine. Children 0–14 years hospitalised with X-ray-confirmed CAP were prospectively enrolled in a multicentre observational study. Acute and convalescent blood samples were collected. Pneumococcal aetiology was assessed by conventional methods (blood or pleural fluid cultures with Quellung reaction capsular typing or polymerase chain reaction [PCR] in pleural fluid), and recently developed methods (real-time PCR in blood and World Health Organization-validated serotype-specific serology). A total of 561 children were enrolled. Pneumococcal aetiology was assessed by conventional methods in 539, serology in 171, and real-time PCR in blood in 154. Pneumococcal aetiology was identified in 12.2% (66/539) of the children by conventional methods alone but in 73.9% by the combination of conventional and recently developed methods. The pneumococcal detection rate adjusted for the whole study population was 61.7%. Serotypes 1 (42.3%), 5 (16.0%), and 7F(7A) (12.8%) were predominant. In conclusion, Streptococcus pneumoniae remains the predominant bacteria in children hospitalised for CAP in Belgium after implementation of 7-valent pneumococcal conjugate vaccine, with non-vaccine-serotypes accounting for the majority of cases. The use of recently developed methods improves diagnosis of pneumococcal aetiology.

A fluorescent multiplexed bead-based immunoassay (FMIA) for quantitation of IgG against Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis protein antigens

Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are pathogens commonly associated with infectious diseases in childhood. This study aimed to develop a fluorescent multiplexed bead-based immunoassay (FMIA) using recombinant proteins for the quantitation of serum IgG antibodies against these bacteria. Eight pneumococcal proteins (Ply, CbpA, PspA1, PspA2, PcpA, PhtD, SP1732-3 and SP2216-1), 3 proteins of H. influenzae (NTHi Protein D, NTHi0371-1, NTHi0830), and 5 proteins of M. catarrhalis (MC Omp CD, MC_RH4_2506, MC_RH4_1701, MC_RH4_3729-1, MC_RH4_4730) were used to develop the FMIA. Optimal coupling concentrations for each protein, comparison of singleplex and multiplex assays, specificity, reproducibility, and correlation to ELISA for six pneumococcal antigens were determined for validation. FMIA was then used to analyze acute and convalescent paired serum samples of 50 children with non-severe pneumonia. The coupling concentrations varied for different antigens, ranging from 1.6 to 32μg of protein/million beads. Correlation between singleplexed and multiplexed assays was excellent, with R≥0.987. The FMIA was specific, reaching >92% homologous inhibition for all specificities; heterologous inhibition ≥20% was found only in six cases. The assay was repeatable, with averages of intra-assay variation ≤10.5%, day-to-day variation ≤9.7% and variation between technicians ≤9.1%. Comparison with ELISA for pneumococcal antigens demonstrated good correlation with R ranging from 0.854 (PspA2) to 0.976 (PcpA). The samples from children showed a wide range of antibody concentrations and increases in convalescent samples. In conclusion, the FMIA was sensitive, specific, and repeatable, using small amounts of recombinant proteins and sera to detect antibodies against S. pneumoniae, H. influenzae and M. catarrhalis. The methodology would be suitable for studies investigating etiological diagnosis and in experimental vaccine studies.

Abstract 38: Herpes Viruses in Childhood Arterial Ischemic Stroke: Interim Results of the VIPS Study

Background: Varicella zoster virus (VZV) is a known cause of cerebral arteriopathy and arterial ischemic stroke (AIS) in children, and scant evidence suggests other herpes viruses may also play a role. An objective of the Vascular effects of Infection in Pediatric Stroke (VIPS) study was to determine whether recent infection with herpes viruses_as measured by serologies_is associated with childhood AIS. Methods: VIPS is an international prospective 35-center case-control study enrolling 350 children (28 days-18 years) with AIS and 120 unmatched trauma controls. Case confirmation and vascular imaging review are performed centrally. Acute blood samples are collected ≤3 weeks after stroke/trauma; convalescent samples 7-28 days after the acute draw in cases only. Samples were tested for IgG and IgM antibodies to herpes simplex virus (HSV) 1 and 2, cytomegalovirus (CMV), Epstein Barr virus (EBV), and VZV. An algorithm developed a priori by a pediatric infectious disease expert (C.A.G.) classified acute infection as a dichotomous variable. Results: Of 310 cases enrolled thus far, 141 cases with paired samples and 44 controls underwent serologic analysis. Median age (IQR) was 7.0 years (2.9, 14.3) for cases and 8.3 (3.3, 13.3) for controls (P=0.90). Evidence of an acute herpes infection was found in 41% of cases versus 9% of controls (P&lt;0.0001; Table). The most commonly identified virus was HSV-1. After adjusting for age in a multivariable logistic regression analysis, herpes exposure remained a significant predictor of AIS (OR 9.0, 95% CI 3.1, 38.7; P=0.0004). Of the 58 cases positive for herpes, 36% had a definite arteriopathy, and 33% a possible arteriopathy, compared to 34% and 24%, respectively, of 83 negative cases. Conclusions: In this interim analysis, acute exposure to a herpes virus appears associated with childhood AIS, present in 41% of cases that were tested.

Anti-GQ1b ganglioside positive Miller Fisher syndrome – evidence of paranodal pathology on nerve biopsy

Miller Fisher syndrome is a regional variant of Guillain-Barre syndrome with a characteristic clinical triad of ophthalmoplegia, areflexia and ataxia and occasionally distal limb sensory loss. 90% of patients have associated antibodies to the GQ1b ganglioside. The pathophysiology of antibody-mediated peripheral nerve impairment remains uncertain. This report includes the first description of a peripheral sensory nerve biopsy in Miller Fisher syndrome. A single case report is described of a 46 year old woman who presented with 2 weeks of distal glove and stocking sensory loss to both deep and superficial sensory modalities, areflexia and weight loss. This was followed by rapid onset of ataxia, ophthalmoplegia, and bulbar impairment. Peripheral neurophysiology showed reduced sensory nerve amplitudes with preserved conduction velocities in keeping with an axonal pattern of impairment. Clinical concerns of a systemic inflammatory disorder led to a diagnostic peripheral nerve biopsy from the sensory branch of the radial nerve. However she subsequently made a complete recovery over 5 weeks. Combinatorial glycoarrays confirmed restricted serum binding for GQ1b in acute serum which later resolved in a convalescent sample. The nerve biopsy showed lengthening of nodes of Ranvier, myelin splitting and macrophage internodal axonal invasion without any features of demyelination. The pathological features were strikingly similar to those found in acute motor axonal neuropathy and indicate the region of the node of Ranvier to be a primary focus of GQ1b induced damage in Miller Fisher syndrome, at least in this particular overlap syndrome with prominent sensory nerve involvement.

Changes in full blood count parameters in leptospirosis: a prospective study.

BackgroundLeptospirosis presents diagnostic challenges to clinicians, in settings where other acute febrile illness are prevalent. The patterns of serial changes in haematological parameters in leptospirosis has not been evaluated previously.MethodsClinical and laboratory data were collected prospectively from patients with leptospirosis in two hospitals in Sri Lanka. Leptospirosis was diagnosed based on WHO clinical criteria with confirmation using Microscopic Agglutination Test titre > 400 or 4 fold rise between acute and convalescent samples. Full blood count parameters were analysed up to the 14th day of illness.ResultsData from 201 patients with leptospirosis were available. Leukocyte counts and absolute neutrophil counts showed a decline over the first 5 days of illness, then rose until the end of the second week. On day 3 of fever, the majority (75%) had normal leukocyte counts, and by day 5, leukocytosis was seen only in 38.1%; leucopenia was an uncommon finding. Lymphopenia was seen in over half on day 5, declining to just under a quarter of patients by day 10. Platelets declined over the first 6 days and then gradually rose. Thrombocytopenia was seen in nearly three-fourths of patients by day 5. Haemoglobin and haematocrit levels declined over the course of illness. Total white cell and neutrophil counts were higher, and haemoglobin and haematorcrit were significantly lower, in patients with severe disease.ConclusionsNeither leukocytosis nor lymphopenia were prominent features, while thrombocytopenia was seen during the 3rd to 5th day of illness, with dropping haemoglobin levels. Neutrophilia and low haemoglobin levels appear to predict severe disease. These findings may be of use to clinicians in differentiating leptospirosis from other acute infections like dengue, and could help in predicting severe leptospirosis.