CSF Specimens Research Articles

Prevalence, Genotypes, Clinical Manifestations and Outcomes Associated with Human Parechovirus (HPeV) Infections in Infants up to 2 Months old in Central Ohio

BackgroundHPeV have been associated with severe disease in young infants. Of the 17 genotypes described, HPeV 1 and 3 have been the most frequently reported. The epidemiology and clinical features associated with different genotypes have not been well defined. We analyzed the prevalence, genotypes, and clinical manifestations and outcomes of HPeV in infants ≤ 60 days evaluated at Nationwide Children’s Hospital, Columbus, Ohio.MethodsWe retrospectively reviewed EMRs of all infants ≤ 60 days undergoing sepsis evaluation with a positive HPeV PCR from any site between July 2013 and September 2016. All available HPeV CSF, blood, and superficial site specimens were typed by PCR or Sanger sequencing (types assigned per GenBank®).ResultsOf 1,265 patients tested, 131 (10%) were positive for HPeV in at least one site, of which 100 had available isolates for genotyping. Median age was 30 days (IQR 19–39), 55% were male. HPeV3 was identified in 87 (87%), HPeV4 in 6, HPeV1 in 5, and HPeV5 and 6 were identified in one infant each. For comparisons we grouped types 1, 4, 5 and 6 into HPeV0 (n = 13). The circulation of HPeV peaked in the months of July to October independent of the type. However, while HPeV0 were identified only in second half of the year, HPeV3 was detected year round. HPeV3 patients had higher temperatures (P < 0.05). There were no significant differences between HPeV3 vs. HPeV0 in age, gender, presenting symptoms, length of stay, PICU admission and CBC. ALT values were higher in HPeV0 patients (P < 0.01). CSF indices were also similar in both groups. Of the positive CSF isolates for HPeV, 43% had no pleocytosis; all CSF isolates typed were HPeV3. HPeV4 was found in blood and superficial sites. HPeV1, 5 and 6 were only found in superficial sites and more commonly with coinfections (enterovirus [EV], rhinovirus, group B streptococcus). There were 4 PICU admissions, 3 of them had HPeV3 and 1 had HPeV1 (patient also had Rhinovirus/EV bronchiolitis). All patients recovered at the time of discharge.ConclusionHPeV was commonly identified in infants ≤ 60 days undergoing sepsis evaluation. HPeV3 was the most common type in this age group. HPeV4 also caused viremia, while other infrequent types were identified with coinfections.Disclosures O. Ramilo, Abbvie: Board Member, Consulting fee; Regeneron: Board Member, Consulting fee; Janssen: Board Member and Investigator, Consulting fee and Research grant; NIH: Grant Investigator, Research grant; A. Leber, BioFIre Diagnostics: Research Contractor and Scientific Advisor, Research support, Speaker honorarium and Travel expenses

Intercellular Adhesion Molecular-5 as Marker in HIV Associated Neurocognitive Disorder.

Despite the use of antiretroviral drugs HIV associated neurocognitive disorders (HAND) are still common in HIV-seropositive patients. Identification of HIV patients with cognitive impairment in early-stage might benefit a great deal from disease progression monitoring and treatment adjustment. Intercellular adhesion molecule-5 (ICAM5), characteristically expressed on neuron, may suppress immune functions by inhibition of T cell activation in central nervous system. Previous studies have shown that ICAM5 could be detected in patients with brain injury. To investigate the relationship between cognitive impairment and ICAM5 in HIV patients, we compared soluble ICAM5 levels in paired CSF and plasma specimens from HIV-infected individuals with or without neurocognitive impairment. sICAM5 concentrations were measured by ICAM5 ELISA kit. A total of 41 Patients were classified into HIV infected with normal cognition (HIV-NC) and impaired cognition groups (HIV-CI) based on Memorial Sloan-Kettering Scale. CSF and plasma levels of sICAM5 in HIV-CI patients were significantly higher than HIV-NC group (p<0.0001, p=0.0054 respectively). sICAM5 concentrations in plasma strongly correlated with sICAM5 in CSF (r=0.7250, p<0.0001) and S100B in CSF (r=0.3812, p<0.0139). Among 6 follow-up patients we found that sICAM5 levels in CSF and plasma might change consistently with HAND progression. In summary, we have shown that the expressions of sICAM5 in CSF and plasma may correlate with neurocognitive impairment in HIV infected patients. The elevation of sICAM5 in plasma were correspond with that in CSF as a consequence of blood-brain barrier permeability changes. ICAM5 can serve as a potential and readily accessible biomarker to predict HIV associated neurocognitive disorder.

Herpes Simplex Virus Genotyping in Neurological Abnormalities-Clinical Relevance for Disease Monitoring

Herpes Simplex Virus (HSV) is an important etiological agent for Neurological disorders like meningitis, seizures etc. Current work includes the molecular reveling of HSV 1 and 2 in CSF specimens. 13 cases of CSF were processed for the typing of HSV by Real Time PCR and out of which 03 cases came positive. All the positive cases were HSV genotype-1. Advanced biotechnological tools promises fast, accurate and timely diagnosis of such viral diseases for further prognosis.

Molecular epidemiological study of enteroviruses associated with encephalitis in children from Hangzhou, China.

Enterovirus (EV) has over 100 serotypes of species A–D, which can cause various symptoms in infants. Enterovirus encephalitis (EVE) is serve disease with high morbidity and mortality in children. To well define the epidemiology of EVE, we wanted to know more about EV and EV molecular typing by conducting this study in Hangzhou.Cerebrospinal fluid samples were collected from children with diagnosis of encephalitis. Meanwhile, one-step real-time RT-PCR was used for the detection of EV, and we also identified the serotypes of EV by using gene sequencing of VP1 or 5′UTR region.A total of 126 CSF specimens were tested and EV was detected in 26 specimens (20.6%). The molecular typing results showed different types of EV strains including Coxsackievirus B2, Coxsackievirus B3, Echovirus 5, Echovirus 16, Echovirus 18, Echovirus 30, and all EV isolates belonging to the human EV species B.According to the sequence of VP1 and 5′UTR region, E30 may be major cause of children's EVE in Hangzhou, China.

ASCIA‐P70: IS TESTING FOR AQP4 ANTIBODIES IN CSF AND SERUM MORE SENSITIVE THAN SERUM ALONE?

Neuromyelitis Optica (NMO) describes a spectrum of inflammatory disorders of the central nervous system characterised by optic neuritis and longitudinal extensive transverse myelitis. Antibodies against aquaporin 4 (AQP4) water channels have been identified as a disease specific marker of NMO, and are included in the diagnostic criteria. AQP4 antibodies can be detected via indirect immunofluorescence on cerebellum tissue, but more recently the transfected HEK293 cell based assay offers superior sensitivity and specificity. The majority of requests for testing are on serum samples, but some clinicians also request testing on CSF specimens. To determine if testing for AQP4 antibodies on CSF and serum is more sensitive than testing serum alone. Laboratory requests for AQP4 antibodies at PathWest (QEII) between 2012 and 2016 were audited to identify patients for whom both serum and CSF samples had been submitted for testing. Samples were tested on either primate cerebellum or the cell based assay. The AQP4 antibody results in serum and CSF samples were compared. A total of 25 patients were identified who underwent AQP4 antibody testing on matched CSF and serum samples. There were 4 patients who were positive for AQP4 antibodies on the serum only, and only 1 patient who was positive on both the serum and CSF. No CSF sample was positive in isolation. Testing CSF specimens for AQP4 antibodies does not appear to pick up additional cases compared to testing serum samples alone.

Comparative evaluation of in-house Real time IS 6110, nested MPT 64 PCR and Roche AMPLICOR 16s rRNA PCR for diagnosing tuberculous meningitis

Background: Early diagnosis of tuberculous meningitis (TBM) is still a diagnostic challenge due to paucibacillary nature of the disease and conventional methods being quite insensitive and time consuming. We evaluated performance of three real time PCR assays targeting MPT64, IS6110 and 16s rRNA gene sequences for early diagnosis of TBM Methods & Materials: A total of 100 consecutive probable TBM patients and 50 non TBM patients were enrolled from an ongoing prospective study on tuberculous meningitis (July 2012 to Dec 2014). CSF specimens were subjected to microscopy and automated BACTEC MGIT culture. In-house Real time nested MPT64 and IS6110 PCR were standardized using H37Rv spiked CSF and evaluated for diagnostic utility along with commercially available Roche Amplicor Real time PCR assay. Results: Out of 100 clinical specimens processed, the sensitivity of smear microscopy and culture was 4% and 42% respectively. The sensitivity, specificity of Real time IS6110 PCR, Nested MPT 64 assay and Roche Amplicor Real time PCR assay was 71%, 98%; 69%, 98% and 25%, 100% respectively against probable TBM as reference standard. The Real time IS6110 PCR, Nested MPT 64 assay and Roche Amplicor Real time PCR assay could detect M. tuberculosis in only 84%, 77% and 30% of culture positive patient's respectively Conclusion: Real time IS6110 PCR proved to be a simple and rapid method to diagnose TBM with sensitivity, specificity of 71%, 98% only. Nested MPT 64 assay though had comparable sensitivity and specificity was much more difficult due to nested design and higher risks of contamination. None of the PCR was 100% sensitive for detecting all culture positive samples suggesting that for TBM diagnosis there is no single rule out test and all the tests are contingent upon their ability to pick the target in tested volume of CSF.

Development of Real-Time PCR Methods for the Detection of Bacterial Meningitis Pathogens without DNA Extraction.

Neisseria meningitidis (Nm), Haemophilus influenzae (Hi), and Streptococcus pneumoniae (Sp) are the lead causes of bacterial meningitis. Detection of these pathogens from clinical specimens using traditional real-time PCR (rt-PCR) requires DNA extraction to remove the PCR inhibitors prior to testing, which is time consuming and labor intensive. In this study, five species-specific (Nm-sodC and -ctrA, Hi-hpd#1 and -hpd#3 and Sp-lytA) and six serogroup-specific rt-PCR tests (A, B, C, W, X, Y) targeting Nm capsular genes were evaluated in the two direct rt-PCR methods using PerfeCTa and 5x Omni that do not require DNA extraction. The sensitivity and specify of the two direct rt-PCR methods were compared to TaqMan traditional rt-PCR, the current standard rt-PCR method for the detection of meningitis pathogens. The LLD for all 11 rt-PCR tests ranged from 6,227 to 272,229 CFU/ml for TaqMan, 1,824–135,982 for 5x Omni, and 168–6,836 CFU/ml for PerfeCTa. The diagnostic sensitivity using TaqMan ranged from 89.2%-99.6%, except for NmB-csb, which was 69.7%. For 5x Omni, the sensitivity varied from 67.1% to 99.8%, with three tests below 90%. The sensitivity of these tests using PerfeCTa varied from 89.4% to 99.8%. The specificity ranges of the 11 tests were 98.0–99.9%, 97.5–99.9%, and 92.9–99.9% for TaqMan, 5x Omni, and PerfeCTa, respectively. PerfeCTa direct rt-PCR demonstrated similar or better sensitivity compared to 5x Omni direct rt-PCR or TaqMan traditional rt-PCR. Since the direct rt-PCR method does not require DNA extraction, it reduces the time and cost for processing CSF specimens, increases testing throughput, decreases the risk of cross-contamination, and conserves precious CSF. The direct rt-PCR method will be beneficial to laboratories with high testing volume.

The Interleukin-1 Balance During Encephalitis Is Associated With Clinical Severity, Blood-Brain Barrier Permeability, Neuroimaging Changes, and Disease Outcome.

Encephalitis is parenchymal brain inflammation, commonly due to herpes simplex virus (HSV). Key host inflammatory mediators and their relationship to blood-brain barrier (BBB) permeability, neuroimaging changes, and disease outcome are poorly understood. We measured levels of 38 mediators in serum (n = 78) and cerebrospinal fluid (n = 37) specimens from patients with encephalitis, including 17 with disease due to HSV infection. Outcome measures were Glasgow coma and outcome scores; CSF to serum albumin ratio, reflecting BBB permeability; and, in patients with HSV infection, magnetic resonance imaging-based temporal lobe volume. Serum interleukin 1 receptor antagonist (IL-1RA) levels were elevated in patients with a good outcome (P= .004). Among patients infected with HSV, the ratio of CSF IL-1β to IL-1RA was associated with a worse outcome (P= .009); a ratio of ≥0.55 pg/mL had high specificity and sensitivity for a poor outcome (100% and 83%;P= .015). Temporal lobe volume had a negative correlation with serum IL-1RA level (P= .012) and a positive correlation with serum IL-1α level (P= .0003) and CSF IL-1β level (P= .007). A normal coma score was associated with an elevated interleukin 10 (IL-10) level in serum specimens from HSV-infected patients (P= .007) and CSF specimens from all patients (P= .016); the IL-10 level correlated inversely with BBB permeability (P= .005). A proinflammatory cytokine response is associated with greater clinical severity, BBB permeability, and neuroimaging damage during encephalitis. IL-1 antagonists should be investigated as adjunctive treatment in encephalitis.

CSF Nrf2 and HSPA8 in Parkinson's disease patients with and without LRRK2 gene mutations.

Leucine-rich repeat kinase 2 (LRRK2) gene mutations are the most common genetic cause of Parkinson's disease (PD). CSF specimens from LRRK2+PD patients and healthy LRRK2 mutation carriers are, therefore, useful for biomarker studies. This study examined the hypothesis that differences are present between subjects with sporadic PD (sPD), PD carriers of LRRK2 mutations (LRRK2+PD), healthy control subjects lacking LRRK2 mutations (CTL), and LRRK2 mutation-carrying healthy controls (LRRK2+CTL) for CSF concentrations of six potential PD biomarkers. Two of these proteins, nuclear factor (erythroid-derived 2)-like 2 ("Nrf2") and heat shock 70kDa protein 8 ("HSPA8"), were detected in preliminary ELISAs, then measured in a larger cohort (60 sPD, 10 LRRK2+PD, 23 CTL, 31 LRRK2+CTL). No statistically significant differences were found between the groups (Nrf2 p=0.13, HSPA8 p=0.21). Nrf2 concentrations in LRRK2+PD subjects were strongly positively associated with Unified Parkinson's Disease Rating Scale (UPDRS) total and motor scores [Spearman rho=0.77 (p=0.012) and 0.83 (p=0.005)] and negatively associated with Montreal Cognitive Assessment (MoCA) scores (rho=-0.57; p=0.11). Partial correlation coefficient calculations indicated that disease duration contributed to the associations of Nrf2 levels with UPDRS scores and with MoCA scores in this group. While CSF Nrf2 and HSPA8 do not appear to offer diagnostic biomarkers for PD, the associations between Nrf2 levels and UPDRS scores in LRRK2+PD patients merit further investigation.

Association of antibodies to the NR1 subunit of N-methyl-D-aspartate receptors with neuropsychiatric systemic lupus erythematosus

Objective: To determine epitope reactivity of autoantibodies to N-methyl-D-aspartate (NMDA) receptor NR1 subunit and their association with neuropsychiatric systemic lupus erythematosus (NPSLE).Methods: Paired serum and CSF specimens were obtained from 41 patients with NPSLE (22 with diffuse psychiatric/neuropsychological syndromes [diffuse NPSLE] and 19 with neurologic syndromes or polyneuropathy [focal NPSLE]), 21 patients with various rheumatic diseases other than SLE (non-SLERD). Sera were also obtained from 27 SLE patients without neuropsychiatric manifestations (non-CNS SLE). Antibodies to murine NR1 (mNR1) or to 4 different preparations of synthetic 25-amino-acid (AA) peptides of human NR1 were measured by enzyme-linked immune sorbent assay (ELISA).Results: Serum anti-mNR1 levels were significantly higher in NPSLE than in non-SLERD. Sera from NPSLE patients bound efficiently to the AA residues 19–44 from the N-terminus of NR1 (NR1-A) or 56–81 (NR1-C). Accordingly, serum anti-NR1-A and anti-NR1-C were also elevated in NPSLE compared with non-SLERD. Of note, anti-NR1-A as well as anti-NR1-C levels in CSF, but not in sera, were significantly elevated in diffuse NPSLE compared with focal NPSLE or with non-SLERD.Conclusion: These results suggest that autoantibodies to NMDA receptor NR1, especially to the AA residues 19–44 and 56–81 from the N-terminus play a pivotal role in the pathogenesis of diffuse NPSLE.

A Five Years Study of Tuberculous Meningitis in Iran.

Tuberculous meningitis (TBM) is a severe form of extra pulmonary tuberculosis with high mortality and morbidity rate in all age group patients specific in adults and children. The incidence and prevalence are not exactly known in Iran. In this study, we tried to evaluate the role of rapid diagnosis and to find out the highest risk group patients. Totally, 1783-suspected patients with tuberculous meningitis whose CSF specimens were admitted at Noor Pathobiology Laboratory, Tehran, Iran were enrolled in this study from January 2009 until December 2013. All specimens were checked for MTB by direct examination, culture and PCR tests, and for the adenosine deaminase (ADA). Confirmed positive cases were aged from 13 to 82 yr old with mean age 46.63 yr (SD±18.84). The number of diagnosed positive MTB was different by the 3 applied protocol, 64 by PCR, 28 by culture and 33 by direct examination. Considering the result of PCR protocol the TBM was approved in 64 patients with rate of 3.59%. Two patients had other infection as well, one 56 years old with VZV and the other patient who was HIV positive was 27 years old. Increased ADA titer higher than cutoff was relevant with other results of positive samples except in two cases. Analysis of the results proved adults are more at risk for tuberculous meningitis than children in Iran are. It is also confirmed PCR method provide the most efficient, rapid and reliable results for these patients who are at the critical situations.

The immune response in the CNS in Theiler's virus induced demyelinating disease switches from an early adaptive response to a chronic innate-like response.

Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is an important model of the progressive disability caused by irreversible CNS tissue injury, and provides an example of how a CNS pathogen can cause inflammation, demyelination, and neuronal damage. We were interested in which molecules, especially inflammatory mediators, might be upregulated in the CNS throughout TMEV-IDD. We quantitated by a real-time RT-PCR multi-gene system the expression of a pathway-focused panel of genes at 30 and 165days post infection, characterizing both the early inflammatory and the late neurodegenerative stages of TMEV-IDD. Also, we measured 32 cytokines/chemokines by multiplex Luminex analysis in CSF specimens from early and late TMEV-IDD as well as sham-treated mice. Results indicate that, in the later stage of TMEV-IDD, activation of the innate immune response is most prominent: TLRs, type I IFN response genes, and innate immunity-associated cytokines were highly expressed in late TMEV-IDD compared to sham (p ≤ 0.0001) and early TMEV-IDD (p < 0.05). Conversely, several molecular mediators of adaptive immune response were highly expressed in early TMEV-IDD (all p ≤ 0.001). Protein detection in the CSF was broadly concordant with mRNA abundance of the corresponding gene measured by real-time RT-PCR in the spinal cord, since several cytokines/chemokines were increased in the CSF of TMEV-IDD mice. Results show a clear shift from adaptive to innate immunity from early to late TMEV-IDD, indicating that adaptive and innate immune pathways are likely involved in the development and progression of the disease to different extents. CSF provides an optimal source of biomarkers of CNS neuroinflammation.

THE ANTI-TB DRUG SENSITIVITY OF Mycobacterium tuberculosis FROM CEREBROSPINAL FLUID AND BONE TISSUE BIOPSY SPECIMENS OF PATIENTS SUSPECTED TUBERCULOUS MENINGITIS AND SPINAL TB IN Dr SOETOMO HOSPITAL INDONESIA

Tuberculous meningitis (TBM) is an infection of meningens which potentially life threatening with significant morbidity and mortality. Spinal TB has the same problem with TBM, infection in bone and joint, the delayed diagnosis worsens the prognosis. The rapid and accurate diagnosis plus promt adequate treatment is essential for the good outcome. The aim of this research is to study the first line drug sensitivity of Mycobacterium tuberculosis isolated from specimens of cerebrospinal fluid from suspected tuberculous meningitis patients and bone tissue biopsy from suspected spinal TB patients. The method of this research is TB Laboratory examination in Department of Clinical Microbiology – Dr. Soetomo General Hospital, Indonesia, using the gold standard liquid culture method MGIT 960 System (Becton Dickinson) and solid culture method with Lowenstein-Jensen medium. The specimens CSF from 50 TBM patients at January 2013 until May 2014. Positive isolate detection of Mycobacterium tuberculosis complex were 11 isolates (22%), which sensitivity 100% (11/11 isolates) to Rifampin (R), Pyrazinamide (Z), Ethambutol (E), and Streptomycin (S); one isolate resistant to Isoniazid, sensitivity to Isoniazid 90,90% (10/11); and received 21 specimens of bone tissue biopsy which positive 5 isolates (23%), all isolates sensitive 100% (5/5 isolates) to Rifampin and Pyrazinamide, and 1 isolates resistant to Isoniazid, Ethambutol, and Streptomycin, in which sensitivity 80% (4/5 isolates) to Isoniazid, Ethambutol, and Streptomycin. The conclusion of this research is positivity detection 22% of CSF specimens, and 23% of bone tissue biopsy were low. All isolates sensitive 100% to Rifampin and Pyrazinamide, and 80-90% sensitive to Isoniazid.

Evaluation of a multiplex polymerase chain reaction for early diagnosis of ventriculostomy-related infections.

Diagnosis of ventriculostomy-related infections (VRIs) is challenging due to the lack of rapid, sensitive assays for pathogen detection. The authors report the development of a multiplex polymerase chain reaction (PCR) assay for differential diagnosis of common VRI pathogens. MassTag PCR was used to develop a multiplex assay for detection of 11 VRI pathogens. The assay was established and optimized using cloned template standards and spiked samples and was then evaluated on CSF specimens from ventricular drains. Subjects were grouped into definite VRI, possible VRI, or no VRI based on conventional microbiology, CSF evaluation, and clinical parameters. CSF specimens were obtained from 45 subjects (median age 49 years, interquartile range 32-63 years; 51% were male). The assay detected 10-100 genome copies. It detected a pathogen in 100% (6 of 6) of definite VRI cases in which a pathogen targeted by the assay was present; these represented 67% of all definite VRIs (6 of 9). Among subjects with a possible VRI, the assay detected a pathogen in 29% (5 of 17). In subjects without overt infection the presence of a pathogen was detected in 32% of subjects (6 of 19), albeit with lower signal compared with the VRI group. MassTag PCR enabled parallel testing of CSF specimens for 11 pathogens of VRI. The high sensitivity of PCR combined with possible device colonization, specimen contamination, and concurrent antibiotic treatments limit the clinical value of the assay, similar to other current diagnostic approaches. With further optimization, multiplex PCR may provide timely identification of multiple possible VRI pathogens and guide management, complementing classic culture approaches.

Echovirus 30 meningitis epidemic followed by an outbreak-specific RT-qPCR

BackgroundAn outbreak of enteroviral aseptic meningitis emerged in Southwestern Finland in August 2009. The same enterovirus reappeared with increasing incidence of meningitis in other parts of Finland in 2010. ObjectivesTo identify the incidence and molecular epidemiology of enteroviral meningitis outbreak. Study designThe causative agent was identified as echovirus 30 (E-30) by sequencing partial viral protein 1 capsid genome, and a virus type-specific RT-qPCR was set up for sensitive detection of the virus in cerebrospinal fluid specimens. Enterovirus positive CSF specimens were subjected to the E-30-specific assay to investigate this unusual occurrence of aseptic meningitis and facilitate case confirmation during the outbreaks between August 2009 and September 2010. ResultsE-30 was detected in 106 (72%) enterovirus positive cerebrospinal fluid specimens. All the meningitis cases in 2009 and most of them in 2010 were among adolescents and several were members of sport teams. ConclusionsBetween August 2009 and September 2010, E-30 caused an extensive outbreak with two peaks in Finland. Type-specific RT-PCR allowed rapid diagnostic follow-up of the epidemic.

Epidemiological Investigation of an Outbreak of Acute Encephalitis Syndrome (AES) in Malda District of West Bengal, India

Background: An unusual outbreak of acute encephalitis syndrome (AES) with high case fatality was reported from Kaliachak- I, II and III Blocks of Malda District of West Bengal in the month of June 2014 affecting 72 children with 34 deaths. The purpose of this study was to investigate the outbreak in the light of epidemiological as well as etiological determinants. Methods: The investigating team collected clinical and epidemiological data from the cases admitted at Malda Medical College and at the Kaliachak BPHC. Different clinical samples, (serum, CSF etc) collected from cases as well as control population were screened for different pathological, biochemical and microbiological parameters. Additionally, the CSF specimens were also processed for the isolation of viruses by inoculating in the chorio-allantoic membrane (CAM) of embryonated chick eggs and intracerebral inoculation of suckling mice. Statistical methods included calculation of proportions (percentages), different test of significance (t-test, chi square etc). Results: All children were from age group of 9 months to 10 years (median=3, mean=3.73, SD=1.98) and belonged to low socioeconomic background of litchi growing belt of Malda. Most of the cases were male (65% approx). Case Fatality Rate was 47.2%. The main presenting features were sudden onset of convulsions (100%) in the early hours of dawn followed by rapid progression to unconsciousness (100%) and decerebrate rigidity (47%). Fever was present in around one third of cases. Hypoglycaemia and leucocytosis were two predominant features. Clinical samples subjected to molecular and serological testing, were all found negative for known viruses causing acute encephalitis. 3 out of 4 CSF samples produced demonstrable pocks in Chorio allantoic membrane of the embryonated eggs although the pock count varied from 4- 22 per CAM. Significantly low blood glucose level was found in the controls from litchi belt areas as compared to the controls of non-litchi belt areas of Malda. Conclusion: The evidence gathered so far pointed towards a viral etiology although the causative virus remained unidentified. Hypoglycaemia probably induced by litchi fruit might have aggravated the encephalitis rather than actually causing it.

Retrospective study of the epidemiology and clinical manifestations of Cryptococcus gattii infections in Colombia from 1997-2011.

BackgroundCryptococcosis due to Cryptococcus gattii is endemic in various parts of the world, affecting mostly immunocompetent patients. A national surveillance study of cryptococcosis, including demographical, clinical and microbiological data, has been ongoing since 1997 in Colombia, to provide insights into the epidemiology of this mycosis.Methodology/Principal FindingsFrom 1,209 surveys analyzed between 1997–2011, 45 cases caused by C. gattii were reported (prevalence 3.7%; annual incidence 0.07 cases/million inhabitants/year). Norte de Santander had the highest incidence (0.81 cases/million/year), representing 33.3% of all cases. The male: female ratio was 3.3∶1. Mean age at diagnosis was 41±16 years. No specific risk factors were identified in 91.1% of patients. HIV infection was reported in 6.7% of patients, autoimmune disease and steroids use in 2.2%. Clinical features included headache (80.5%), nausea/vomiting (56.1%) and neurological derangements (48.8%). Chest radiographs were taken in 21 (46.7%) cases, with abnormal findings in 7 (33.3%). Cranial CT scans were obtained in 15 (33.3%) cases, with abnormalities detected in 10 (66.7%). Treatment was well documented in 30 cases, with most receiving amphotericin B. Direct sample examination was positive in 97.7% cases. Antigen detection was positive for all CSF specimens and for 75% of serum samples. C. gattii was recovered from CSF (93.3%) and respiratory specimens (6.6%). Serotype was determined in 42 isolates; 36 isolates were serotype B (85.7%), while 6 were C (14.3%). The breakdowns of molecular types were VGII (55.6%), VGIII (31.1%) and VGI (13.3%). Among 44 strains, 16 MLST sequence types (ST) were identified, 11 of them newly reported.Conclusions/SignificanceThe results of this passive surveillance study demonstrate that cryptococcosis caused by C. gattii has a low prevalence in Colombia, with the exception of Norte de Santander. The predominance of molecular type VGII is of concern considering its association with high virulence and the potential to evolve into outbreaks.

Evaluation of the performance of 2 DNA-based methods for the detection of extra-pulmonary tuberculosis in comparison with the conventional culture technique

IntroductionDiagnosing extra-pulmonary tuberculosis continues to be a challenge for both infectious disease specialists and microbiologists. ObjectivesThis prospective study was done to evaluate the performance of two DNA-based methods for the detection of extra-pulmonary tuberculosis in comparison with the conventional culture technique. MethodsAll extra-pulmonary specimens received by the Kuwait National Tuberculosis Reference from October 2011 until August 2013 were included in the study. Smears were stained by Ziehl Neelson (Merck, Germany) followed by inoculation of the specimens into GeneXpert MTB/RIF assay (Cepheid, USA), ProbTec ET PCR (Becton Dickinson) and MGIT 960 (Becton Dickinson). Urine was inoculated into Lowenstein Jensen media (MAST). ResultsA total of 1674 extra-pulmonary specimens (pleural fluid 553, ascetic fluid 194, cerebrospinal fluid [CSF] 85, Urine 67, other sterile body fluids 153, fine needle aspirates [FNA] 301, pus 181, tissue 102, swabs 27 and stool 11) were evaluated. Out of 155 extra-pulmonary specimens that grew Mycobacterium tuberculosis (MTB) by culture, 143 were positive by GeneXpert compared with 128 by ProbTec with a sensitivity of 92% and 83%, respectively. Out of 1517 specimens that did not grow by culture, 52 were detected by GeneXpert while 46 were detected by ProbTec with specificity of 96.5% and 96.9%, respectively. All the 4 smear-negative CSF samples which grew MTB were positive by GeneXpert with a sensitivity of 100% compared with only 2 detected by ProbTec with a sensitivity of 50%. Additionally, all CSF specimens that did not grow by culture were negative by both the molecular methods showing 100% specificity. Of the 3 smear-positive urine specimens that grew by culture, all were positive, and of the 64 samples that did not grow by culture, all were negative by both the molecular methods with a sensitivity and specificity of 100%. For other sterile body fluids the sensitivity and specificity of both the methods were 68% and 99%, respectively. Finally, for FNA, pus and tissue, the sensitivity of GeneXpert was 97% compared with 86% for ProbTec. ConclusionDNA-based technology looks promising for the rapid diagnosis of extra-pulmonary tuberculosis with an overall better performance of GeneXpert over ProbTec.

Comparative profile of circulating antigenic peptides in CSF, serum & urine from patients with neurocysticercosis diagnosed by immunoblotting.

Traditionally serum and/or CSF specimens have been used for detection of either specific antibodies or antigens as a supportive diagnosis of NCC. However, in recent days, much interest has been shown employing noninvasive specimens such as urine. In our study, we identified and compared a profile of circulating antigenic peptides of parasite origin in three different body fluids (CSF, serum and urine) obtained from confirmed NCC cases and control subjects. The circulating antigenic peptides were resolved by SDS-PAGE and subjected to immunoblotting. For confirmation of their origin as parasite somatic or excretory secretory (ES) material, immunoreactivity was tested employing affinity purified polyclonal Taenia solium metacestode anti-somatic or ES antibodies, respectively. Only lower molecular weight antigenic peptides were found circulating in urine in contrast to serum and CSF specimens. Few somatic peptides were identified to be 100% specific for NCC (19·5 kDa in all three specimens; 131, 70 kDa in CSF and serum only; 128 kDa in CSF only). Similarly, the specific ES peptides detected were 32 kDa (in all three specimens), 16·5 kDa (in serum and CSF only), and 15 kDa (urine only). A test format detecting either one or more of these specific peptides would enhance the sensitivity in diagnosis of NCC.

26: Murine melioidosis with CNS syndrome induced by CD11bCD62L cells harboring Burkholderia pseudomallei

Backgrounds As an infectious agent, Burkholderia pseudomallei is capable of persisting in CD11b phagocytes and escapes from innate immunity during subacute or chronic melioidosis. We hypothesized that those infected cells were used as a vehicle that facilitate the bacterial invading to CNS (central nerve system). Materials and methods The BALB/c and C57BL/6 respectively as subacute and chronic infectious models were used to evaluate the murine melioidosis with CNS syndromes via an intravenous injection of B. pseudomallei GFP or through an adoptively transferred by CD11b cells harboring B. pseudomallei GFP. Observation on occurrence of CNS melioidosis was performed by histological examination, cytokine analysis and bacterial loads in brain. Results We found that, during an initial 14 d-infection, by injecting B. pseudomallei GFP, the sL-selectin, sP-selectin, sICAM-1, MCP-1 and IFN-gamma were increased in blood and CSF specimens for the mice with melioidosis. After a 14 d-infection, the CD11b + CD45 + subpopulation of brain infiltrating leukocytes (BILs) were significantly increased. Approximately 6.5% of BILs were harbored with B. pseudomallei GFP. After adoptive transfer of infected CD11b cells that collected from C57BL/6 WT (wild-type) with melioidosis, the bacterial colonization in the brain and neutrophil infiltration in meninges were obviously occurred while they did not occur in recipients after adoptively transferred if the donor cells were isolated from C57BL sel −/− (selectin knock-out mice). Conclusions/Significance We suggest that B. pseudomallei -infected CD11b + CD62L + cells play an important role in inducing melioidosis with meningitis that could be due to the migration of infected leukocyte involving in a selectin-dependent manner.