Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material

Background

Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.

Method

We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.

Results

Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.

Conclusion

Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.     

Detection of Cytomegalovirus Antibodies Using a Biosensor Based on Imaging Ellipsometry

Background

Cytomegalovirus (CMV) is the most common infectious cause of mental disability in newborns in developed countries. There is an urgent need to establish an early detection and high-throughput screening method for CMV infection using portable detection devices.

Methods

An antibody analysis method is reported for the detection and identification of CMV antibodies in serum using a biosensor based on high spatial resolution imaging ellipsometry (BIE). CMV antigen (CMV-3A) was immobilized on silicon wafers and used to capture CMV antibodies in serum. An antibody against human immunoglobulin G (anti-IgG) was used to confirm the IgG antibody against CMV captured by the CMV-3A.

Results

Our results show that this assay is rapid and specific for the identification of IgG antibody against CMV. Further, patient serum was quantitatively assessed using the standard curve method, and the quantitative results were in agreement with the enzyme-linked immunosorbent assay. The CMV antibody detection sensitivity of BIE reached 0.01 IU/mL.

Conclusions

This novel biosensor may be a valuable diagnostic tool for analysis of IgG antibody against CMV during CMV infection screening.     

Influence of Nano-HA Coated Bone Collagen to Acrylic (Polymethylmethacrylate) Bone Cement on Mechanical Properties and Bioactivity

Objective

This research investigated the mechanical properties and bioactivity of polymethylmethacrylate (PMMA) bone cement after addition of the nano-hydroxyapatite(HA) coated bone collagen (mineralized collagen, MC).

Materials & Methods

The MC in different proportions were added to the PMMA bone cement to detect the compressive strength, compression modulus, coagulation properties and biosafety. The MC-PMMA was embedded into rabbits and co-cultured with MG 63 cells to exam bone tissue compatibility and gene expression of osteogenesis.

Results

15.0%(wt) impregnated MC-PMMA significantly lowered compressive modulus while little affected compressive strength and solidification. MC-PMMA bone cement was biologically safe and indicated excellent bone tissue compatibility. The bone-cement interface crosslinking was significantly higher in MC-PMMA than control after 6 months implantation in the femur of rabbits. The genes of osteogenesis exhibited significantly higher expression level in MC-PMMA.

Conclusions

MC-PMMA presented perfect mechanical properties, good biosafety and excellent biocompatibility with bone tissues, which has profoundly clinical values.     

Low Interferon Relative-Response to Cytomegalovirus Is Associated with Low Likelihood of Intrauterine Transmission of the Virus

Background

Congenital Cytomegalovirus (CMV) is a very common intrauterine infection which can cause severe mental and hearing impairments. Notably, only 40% of primarily infected women transmit CMV to the fetus. CMV-specific T-cell response has a role in CMV disease but individual immune heterogeneity precludes reliable correlation between measurable T-cells response and intrauterine transmission.

Study Aim

To establish a correlation between maternal T-cells response and fetal CMV transmission using an individual normalized immune response.

Methods

We analyzed IFN-γ secretion upon whole blood stimulation from primary CMV-infected pregnant women, with either CMV-peptides or PHA-mitogen.

Results

We established a new normalization method of individual IFN-γ response to CMV by defining the ratio between specific-CMV response and non-specific mitogen response (defined as IFN-γ relative response, RR), aiming to overcome high person-to-person immune variability. We found a unique subpopulation of women with low IFN-γ RR strongly correlated with absence of transmission. IFN-γ RR lower than 1.8% (threshold determined by ROC analysis) reduces the pre-test probability of transmission from 40% to 8%, revealing an unexpected link between low IFN-γ RR and non-transmission.

Conclusion

In pregnant women with primary CMV infection, low IFN-γ RR is associated with low risk of transmission.     

Hepatitis E virus as a Cause of Acute Hepatitis in The Netherlands

Background

Recent studies indicate that 27% of Dutch blood donors have evidence of past infection with HEV. However, the low number of diagnosed HEV infections indicates either an asymptomatic course or under diagnosis.

Objectives

We investigated whether HEV is a cause of acute hepatitis in Dutch patients and which diagnostic modality (serology or PCR) should be used for optimal detection.

Study design

Serum samples were retrospectively selected from non-severely immuno-compromised patients from a university hospital population, suspected of having an infectious hepatitis. Criteria were: elevated alanine aminotransferase (ALT> 34 U/l) and request for antibody testing for CMV, EBV or Hepatitis A (HAV).

Results

All samples were tested for HEV using ELISA and PCR. Ninety patients/sera were tested, of which 22% were HEV IgG positive. Only one serum was IgM positive. HEV PCR was positive in two patients: one patient was both HEV IgM and IgG positive, the other patient was only IgG positive. Both HEV RNA positive samples belonged to genotype 3. Evidence of recent infection with CMV, EBV and HAV was found in 13%, 10% and 3% respectively.

Conclusions

Although our study is limited by small numbers, we conclude that HEV is a cause of acute hepatitis in hospital associated patients in The Netherlands. Moreover, in our study population the prevalence of acute HAV (3%) was almost similar to acute HEV (2%). We propose to incorporate HEV testing in panels for acute infectious hepatitis. Negative results obtained for HEV IgM in a HEV PCR positive patient, indicates that antibody testing alone may not be sufficient and argues for PCR as a primary diagnostic tool in hospital associated patients. The high percentage of HEV IgG seropositivity confirms earlier epidemiological studies.     

Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

Background

Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence microscopy and nucleic acid analogues have been proposed so far.

Methods and Results

Here we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least 1000-fold above the potential detection limit.

Conclusion

Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay, which allows for an accurate estimation of mutant abundance when the detection limit requirement is met. Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly in the biological sample of choice.     

Association of Cytomegalovirus End-Organ Disease with Stroke in People Living with HIV/AIDS: A Nationwide Population-Based Cohort Study

Objectives

Cytomegalovirus (CMV) infection might increase the risk of cardiovascular event. However, data on the link between incident stroke and co-infections of CMV and human immunodeficiency virus (HIV) are limited and inconsistent. This nationwide population-based cohort study analyzed the association of CMV end-organ disease and stroke among people living with HIV/AIDS (PLWHA).

Methods

From January 1, 1998, this study identified adult HIV individuals with and without CMV end-organ disease in the Taiwan National Health Insurance Research Database. All patients were observed for incident stroke and were followed until December 31, 2012. Time-dependent analysis was used to evaluate associations of CMV end-organ disease with stroke.

Results

Of the 22,581 PLWHA identified (439 with CMV end-organ disease and 22,142 without CMV end-organ disease), 228 (1.01%) had all-cause stroke during a mean follow-up period of 4.85 years, including 169 (0.75%) with ischemic stroke and 59 (0.26%) with hemorrhagic stroke. After adjusting for age, sex, comorbidities, opportunistic infections after HIV diagnosis, and antiretroviral treatment, CMV end-organ disease was found to be an independent risk factor for incident all-cause stroke (adjusted hazard ratio [AHR], 3.07; 95% confidence interval [CI], 1.70 to 5.55). When stroke type was considered, CMV end-organ disease was significantly positively associated with the risk of ischemic stroke (AHR, 3.14; 95% CI, 1.49 to 6.62) but not hemorrhagic stroke (AHR, 2.52; 95% CI, 0.64 to 9.91).

Conclusions

This study suggested that CMV end-organ disease was an independent predictor of ischemic stroke among PLWHA.     

A Prospective Monitoring Study of Cytomegalovirus Infection in Non-Immunosuppressed Critical Heart Surgery Patients

Background

Reactivation of cytomegalovirus (CMV) has been reported occasionally in immnunocompetent patients in the intensive care unit (ICU). The epidemiology and association of CMV infection with adverse outcome is not well defined in this population. Patients undergoing major heart surgery (MHS) are at a particularly high risk of infection. CMV infection has not been systematically monitored in MSH-ICU patients.

Methods

We assessed CMV plasma viremia weekly using a quantitative polymerase chain reaction assay in a prospective cohort of immunocompetent adults admitted to the MHS-ICU for at least 72 hours between October 2012 and May 2013. Risk factors for CMV infection and its potential association with continued hospitalization or death by day 30 (composited endpoint) were assessed using univariate and multivariate logistic regression analyses.

Results

CMV viremia at any level was recorded in 16.5% of patients at a median of 17 days (range, 3-54 days) after admission to the MHS-ICU. Diabetes (adjusted OR, 5.6; 95% CI, 1.8-17.4; p=0.003) and transfusion requirement (>10 units) (adjusted OR, 13.7; 95% CI, 3.9-47.8; p<0.001) were independent risk factors associated with CMV reactivation. Reactivation of CMV at any level was independently associated with the composite endpoint (adjusted OR, 12.1; 95% CI, 2.3-64; p=0.003).

Conclusion

Reactivation of CMV is relatively frequent in immunocompetent patients undergoing MHS and is associated with prolonged hospitalization or death.     

Genital Cytomegalovirus Replication Predicts Syphilis Acquisition among HIV-1 Infected Men Who Have Sex with Men

Objective

Sexually transmitted infections (STI) are common among HIV-infected men who have sex with men (MSM). While behavioral factors are important in STI acquisition, other biological factors such as immune modulation due to chronic viral infection may further predispose to STI acquisition.

Design

Post Hoc analysis including data collected over 12 months of follow-up from 131 HIV-infected MSM receiving antiretroviral therapy and screened for incident bacterial STI every 3 months.

Methods

Genital secretions collected at baseline were used to measure herpesvirus replication and inflammatory cytokines. Baseline predictors of STI were determined using survival analysis of time to incident STI.

Results

All participants were seropositive for cytomegalovirus (CMV), and 52% had detectable genital CMV at baseline. Thirty-five individuals acquired STI during follow-up, sometimes with multiple pathogen (17 syphilis, 21 gonorrhea, 14 chlamydia). Syphilis acquisition was associated with genital CMV replication at baseline (19.1% CMV-shedders versus 4.8% non-shedders, p=0.03) and younger age (p=0.02). Lower seminal MCP-1 was associated with higher seminal CMV levels and with syphilis acquisition (p<0.01). For syphilis acquisition, in multivariable Cox-Proportional Hazard model adjusted hazard rates were 3.56 (95%CI:1.00–12.73) for baseline CMV replication and 2.50 (0.92–6.77) for younger age.

Conclusions

This post hoc analysis suggest that CMV-associated decrease in seminal MCP-1 levels might predispose HIV-infected MSM to syphilis acquisition, but not other STI. Future studies should determine underlying mechanisms and if a causal association exists.     

Oral Valganciclovir as a Preemptive Treatment for Cytomegalovirus (CMV) Infection in CMV-Seropositive Liver Transplant Recipients

Objectives

Cytomegalovirus (CMV) infections in liver transplant recipients are common and result in significant morbidity and mortality. Intravenous ganciclovir or oral valganciclovir are the standard treatment for CMV infection. The present study investigates the efficacy of oral valganciclovir in CMV infection as a preemptive treatment after liver transplantation.

Methods

Between 2012 and 2013, 161 patients underwent liver transplantation at Samsung Medical Center. All patients received tacrolimus, steroids, and mycophenolate mofetil. Patients with CMV infection were administered oral valganciclovir (VGCV) 900mg/day daily or intravenous ganciclovir (GCV) 5mg/kg twice daily as preemptive treatment. Stable liver transplant recipients received VGCV.

Results

Eighty-three patients (51.6%) received antiviral therapy as a preemptive treatment because of CMV infection. The model for end-stage liver disease (MELD) score and the proportions of Child-Pugh class C, hepatorenal syndrome, and deceased donor liver transplantation in the CMV infection group were higher than in the no CMV infection group. Sixty-one patients received GCV and 22 patients received VGCV. The MELD scores in the GCV group were higher than in the VGCV group, but there were no statistical differences in the pretransplant variables between the two groups. AST, ALT, and total bilirubin levels in the GCV group were higher than in the VGCV group when CMV infection occurred. The incidences of recurrent CMV infection in the GCV and VGCV groups were 14.8% and 4.5%, respectively (P=0.277).

Conclusion

Oral valganciclovir is feasible as a preemptive treatment for CMV infection in liver transplant recipients with stable graft function.     

Access to CD4 Testing for Rural HIV Patients: Findings from a Cohort Study in Zimbabwe

Background

CD4 cell count measurement remains an important diagnostic tool for HIV care in developing countries. Insufficient laboratory capacity in rural Sub-Saharan Africa is frequently mentioned but data on the impact at an individual patient level are lacking. Urban-rural discrepancies in CD4 testing have not been quantified to date. Such evidence is crucial for public health planning and to justify new yet more expensive diagnostic procedures that could circumvent access constraints in rural areas.

Objective

To compare CD4 testing among rural and urban HIV patients during the first year of treatment.

Methods

Records from 2,145 HIV positive adult patients from a Médecins sans Frontières (Doctors without Borders) HIV project in Beitbridge, Zimbabwe, during 2011 and 2012 were used for a retrospective cohort analysis. Covariate-adjusted risk ratios were calculated to estimate the effects of area of residence on CD4 testing at treatment initiation, six and 12 months among rural and urban patients.

Findings

While the proportion of HIV patients returning for medical consultations at six and 12 months decreased at a similar rate in both patient groups, CD4 testing during consultations dropped to 21% and 8% for urban, and 2% and 1% for rural patients at six and 12 months, respectively. Risk ratios for missing CD4 testing were 0.8 (95% CI 0.7-0.9), 9.2 (95% CI 5.5-15.3), and 7.6 (95% 3.7-17.1) comparing rural versus urban patients at treatment initiation, six and 12 months, respectively.

Conclusions

CD4 testing was low overall, and particularly poor in rural patients. Difficulties with specimen transportation were probably a major factor underlying this difference and requires new diagnostic approaches. Our findings point to severe health system constraints in providing CD4 testing overall that need to be addressed if effective monitoring of HIV patients is to be achieved, whether by alternative CD4 diagnostics or newly-recommended routine viral load testing.     

Dystroglycan Depletion Impairs Actin-Dependent Functions of Differentiated Kasumi-1 Cells

Background

Dystroglycan has recently been characterised in blood tissue cells, as part of the dystrophin glycoprotein complex involved in the differentiation process of neutrophils.

Purpose

In the present study we have investigated the role of dystroglycan in the human promyelocytic leukemic cell line Kasumi-1 differentiated to macrophage-like cells.

Methods

We characterised the pattern expression and subcellular distribution of dystroglycans in non-differentiated and differentiated Kasumi-1 cells.

Results

Our results demonstrated by WB and flow cytometer assays that during the differentiation process to macrophages, dystroglycans were down-regulated; these results were confirmed with qRT-PCR assays. Additionally, depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated Kasumi-1 cells, including morphology, migration and phagocytic activities although secretion of IL-1β and expression of markers of differentiation are not altered.

Conclusion

Our findings strongly implicate dystroglycan as a key membrane adhesion protein involved in actin-based structures during the differentiation process in Kasumi-1 cells.     

Association of CMV-Specific T Cell-Mediated Immunity with CMV DNAemia and Development of CMV Disease in HIV-1–Infected Individuals

Background

Among HIV-1–infected individuals, cytomegalovirus (CMV) reactivation and disease occur in the setting of advanced immunosuppression. The value of a standardized assessment of CMV-specific T-cell mediated immunity by the CMV QuantiFERON assay (CMV-QFT) has not yet been thoroughly investigated in HIV-1–infected subjects.

Methods

Prospective, longitudinal study in 153 HIV-1–infected subjects with a CD4⁺ T cell count < 350/μL who simultaneously underwent CMV-QFT, CMV serology testing and CMV-DNA quantification. Factors associated with CMV-QFT were evaluated. Clinical screening for CMV manifestations was then performed every 3 months.

Results

Among the 141 CMV IgG-seropositive individuals the CMV-QFT assay yielded reactive results in 84% (118/141), negative results in 15% (21/141) and indeterminate (negative mitogen IFN-gamma response) results in 1% (2/141) of subjects. The mean actual CD4⁺ T cell count was significantly higher in CMV-QFT reactive subjects, when compared to CMV-QFT non-reactive individuals (183 ± 102 vs. 126 ± 104 cells/μL, P = 0.015). A significantly lower proportion of CMV-QFT reactive vs. non-reactive patients displayed CMV DNAemia > 100 copies/mL (23% (27/118) vs. 48% (11/23), P = 0.02). Furthermore, a statistically significant inverse association between mitogen IFN-gamma response and CMV-DNAemia > 1000 copies/mL was observed (P < 0.001). During the observational period, 5 CMV end-organ manifestations were observed. In three of the CMV cases the CMV-QFT yielded indeterminate results.

Conclusions

While CMV-QFT reactivity indicates CMV-specific immunity, indeterminate results due to negative mitogen IFN-gamma response might reflect HIV-1-induced immunodeficiency. Thus, dependency upon CD4⁺ T cell count should be considered when interpreting CMV-QFT results.     

The Antibacterial Activity of Acetic Acid against Biofilm-Producing Pathogens of Relevance to Burns Patients

Introduction

Localised infections, and burn wound sepsis are key concerns in the treatment of burns patients, and prevention of colonisation largely relies on biocides. Acetic acid has been shown to have good antibacterial activity against various planktonic organisms, however data is limited on efficacy, and few studies have been performed on biofilms.

Objectives

We sought to investigate the antibacterial activity of acetic acid against important burn wound colonising organisms growing planktonically and as biofilms.

Methods

Laboratory experiments were performed to test the ability of acetic acid to inhibit growth of pathogens, inhibit the formation of biofilms, and eradicate pre-formed biofilms.

Results

Twenty-nine isolates of common wound-infecting pathogens were tested. Acetic acid was antibacterial against planktonic growth, with an minimum inhibitory concentration of 0.16–0.31% for all isolates, and was also able to prevent formation of biofilms (at 0.31%). Eradication of mature biofilms was observed for all isolates after three hours of exposure.

Conclusions

This study provides evidence that acetic acid can inhibit growth of key burn wound pathogens when used at very dilute concentrations. Owing to current concerns of the reducing efficacy of systemic antibiotics, this novel biocide application offers great promise as a cheap and effective measure to treat infections in burns patients.     

Modelling the Impact and Cost-Effectiveness of Biomarker Tests as Compared with Pathogen-Specific Diagnostics in the Management of Undifferentiated Fever in Remote Tropical Settings

Background

Malaria accounts for a small fraction of febrile cases in increasingly large areas of the malaria endemic world. Point-of-care tests to improve the management of non-malarial fevers appropriate for primary care are few, consisting of either diagnostic tests for specific pathogens or testing for biomarkers of host response that indicate whether antibiotics might be required. The impact and cost-effectiveness of these approaches are relatively unexplored and methods to do so are not well-developed.

Methods

We model the ability of dengue and scrub typhus rapid tests to inform antibiotic treatment, as compared with testing for elevated C-Reactive Protein (CRP), a biomarker of host-inflammation. Using data on causes of fever in rural Laos, we estimate the proportion of outpatients that would be correctly classified as requiring an antibiotic and the likely cost-effectiveness of the approaches.

Results

Use of either pathogen-specific test slightly increased the proportion of patients correctly classified as requiring antibiotics. CRP testing was consistently superior to the pathogen-specific tests, despite heterogeneity in causes of fever. All testing strategies are likely to result in higher average costs, but only the scrub typhus and CRP tests are likely to be cost-effective when considering direct health benefits, with median cost per disability adjusted life year averted of approximately $48 USD and $94 USD, respectively.

Conclusions

Testing for viral infections is unlikely to be cost-effective when considering only direct health benefits to patients. Testing for prevalent bacterial pathogens can be cost-effective, having the benefit of informing not only whether treatment is required, but also as to the most appropriate antibiotic; this advantage, however, varies widely in response to heterogeneity in causes of fever. Testing for biomarkers of host inflammation is likely to be consistently cost-effective despite high heterogeneity, and can also offer substantial reductions in over-use of antimicrobials in viral infections.     

Cue Recognition and Integration – Eye Tracking Evidence of Processing Differences in Sentence Comprehension in Aphasia

Purpose

We aimed at further elucidating whether aphasic patients’ difficulties in understanding non-canonical sentence structures, such as Passive or Object-Verb-Subject sentences, can be attributed to impaired morphosyntactic cue recognition, and to problems in integrating competing interpretations.

Methods

A sentence-picture matching task with canonical and non-canonical spoken sentences was performed using concurrent eye tracking. Accuracy, reaction time, and eye tracking data (fixations) of 50 healthy subjects and 12 aphasic patients were analysed.

Results

Patients showed increased error rates and reaction times, as well as delayed fixation preferences for target pictures in non-canonical sentences. Patients’ fixation patterns differed from healthy controls and revealed deficits in recognizing and immediately integrating morphosyntactic cues.

Conclusion

Our study corroborates the notion that difficulties in understanding syntactically complex sentences are attributable to a processing deficit encompassing delayed and therefore impaired recognition and integration of cues, as well as increased competition between interpretations.     

A Population-Based Acute Meningitis and Encephalitis Syndromes Surveillance in Guangxi,China, May 2007- June 2012

Objectives

Acute meningitis and encephalitis (AME) are common diseases with the main pathogens being viruses and bacteria. As specific treatments are different, it is important to develop clinical prediction rules to distinguish aseptic from bacterial or fungal infection. In this study we evaluated the incidence rates, seasonal variety and the main etiologic agents of AME, and identified factors that could be used to predict the etiologic agents.

Methods

A population-based AME syndrome surveillance system was set up in Guigang City, Guangxi, involving 12 hospitals serving the study communities. All patients meeting the case definition were investigated. Blood and/or cerebrospinal fluid were tested for bacterial pathogens using culture or RT-PCR and serological tests for viruses using enzyme-linked immunosorbent assays. Laboratory testing variables were grouped using factor analysis. Multinomial logistic regression was used to predict the etiology of AME.

Results

From May 2007 to June 2012, the annual incidence rate of AME syndrome, and disease specifically caused by Japanese encephalitis (JE), other viruses, bacteria and fungi were 12.55, 0.58, 4.57, 0.45 and 0.14 per 100,000 population, respectively. The top three identified viral etiologic agents were enterovirus, mumps virus, and JE virus, and for bacteria/fungi were Streptococcus sp., Cryptococcus neoformans and Staphylococcus sp. The incidence of JE and other viruses affected younger populations and peaked from April to August. Alteration of consciousness and leukocytosis were more likely to be caused by JE, bacteria and fungi whereas CSF inflammation was associated with bacterial/fungal infection.

Conclusions

With limited predictive validity of symptoms and signs and routine laboratory tests, specific tests for JE virus, mumps virus and enteroviruses are required to evaluate the immunization impact and plan for further intervention. CSF bacterial culture cannot be omitted in guiding clinical decisions regarding patient treatment.     

Prevalence of Bloodstream Pathogens Is Higher in Neonatal Encephalopathy Cases vs. Controls Using a Novel Panel of Real-Time PCR Assays

Background

In neonatal encephalopathy (NE), infectious co-morbidity is difficult to diagnose accurately, but may increase the vulnerability of the developing brain to hypoxia-ischemia. We developed a novel panel of species-specific real-time PCR assays to identify bloodstream pathogens amongst newborns with and without NE in Uganda.

Methodology

Multiplex real-time PCR assays for important neonatal bloodstream pathogens (gram positive and gram negative bacteria, cytomegalovirus (CMV), herpes simplex virus(HSV) and P. falciparum) were performed on whole blood taken from 202 encephalopathic and 101 control infants. Automated blood culture (BACTEC) was performed for all cases and unwell controls.

Principal Findings

Prevalence of pathogenic bacterial species amongst infants with NE was 3.6%, 6.9% and 8.9%, with culture, PCR and both tests in combination, respectively. More encephalopathic infants than controls had pathogenic bacterial species detected (8.9%vs2.0%, p = 0.028) using culture and PCR in combination. PCR detected bacteremia in 11 culture negative encephalopathic infants (3 Group B Streptococcus, 1 Group A Streptococcus, 1 Staphylococcus aureus and 6 Enterobacteriacae). Coagulase negative staphylococcus, frequently detected by PCR amongst case and control infants, was considered a contaminant. Prevalence of CMV, HSV and malaria amongst cases was low (1.5%, 0.5% and 0.5%, respectively).

Conclusion/Significance

This real-time PCR panel detected more bacteremia than culture alone and provides a novel tool for detection of neonatal bloodstream pathogens that may be applied across a range of clinical situations and settings. Significantly more encephalopathic infants than controls had pathogenic bacterial species detected suggesting that infection may be an important risk factor for NE in this setting.