Diagnosis of pathogens causing bacterial meningitis using Nanopore sequencing in a resource-limited setting.

AIM: The aim of the present study is to compare the performance of 16S rRNA Nanopore sequencing and conventional culture in detecting infectious pathogens in patients with suspected meningitis in a resource-limited setting without extensive bioinformatics expertise. METHODS: DNA was isolated from the cerebrospinal fluid (CSF) of 30 patients with suspected bacterial meningitis. The isolated DNA was subjected to 16S sequencing using MinION™. The data were analysed in real time via the EPI2ME cloud platform. The Nanopore sequencing was done in parallel to routine microbiological diagnostics. RESULTS: Nanopore sequencing detected bacterial pathogens to species level in 13 of 30 (43%) samples. CSF culture showed 40% (12/30) positivity. In 21 of 30 patients (70%) with suspected bacterial meningitis, both methods yielded concordant results. About nine of 30 samples showed discordant results, of these five were false positive and four were false negative. In five of the culture negative results, nanopore sequencing was able to detect pathogen genome, due to the higher sensitivity of the molecular diagnostics. In two other samples, the CSF culture revealed Cryptococcus neoformans and Streptococcus pneumoniae, which were not detected by Nanopore sequencing. Overall, using both the cultures and 16S Nanopore sequencing, positivity rate increased from 40% (12/30) to 57% (17/30). CONCLUSION: Next-generation sequencing could detect pathogens within six hours and could become an important tool for both pathogen screening and surveillance in low- and middle-income countries (LMICs) that do not have direct access to extensive bioinformatics expertise.

A multi-phenotype genome-wide association study of clades causing tuberculosis in a Ghanaian- and South African cohort.

Despite decades of research and advancements in diagnostics and treatment, tuberculosis remains a major public health concern. New computational methods are needed to interrogate the intersection of host- and bacterial genomes. Paired host genotype datum and infecting bacterial isolate information were analysed for associations using a multinomial logistic regression framework implemented in SNPTest. A cohort of 853 admixed South African participants and a Ghanaian cohort of 1359 participants were included. Two directly genotyped variants, namely rs529920 and rs41472447, were identified in the Ghanaian cohort as being statistically significantly associated with risk for infection with strains of different members of the MTBC. Thus, a multinomial logistic regression using paired host-pathogen data may prove valuable for investigating the complex relationships driving infectious disease.

Pathogens That Cause Acute Febrile Illness Among Children and Adolescents in Burkina Faso, Madagascar, and Sudan.

BACKGROUND: The etiology and optimal clinical management of acute febrile illness (AFI) is poorly understood. METHODS: Blood samples taken from study participants with acute fever (≥37.5°C) or a history of fever and recruited into the previous Typhoid Fever Surveillance in Africa (TSAP) study were evaluated using a polymerase chain reaction (PCR)-based TaqMan-Array Card designed to detect a panel of bacterial, viral, and parasitic pathogens. Clinical metadata were also assessed. RESULTS: A total of 615 blood samples available for analysis originated from Burkina Faso (n = 53), Madagascar (n = 364), and Sudan (n = 198) and were taken from participants ranging in age from 0-19 years. Through the TaqMan-Array Card, at least 1 pathogen was detected in 62% (33 of 53), 24% (86 of 364), and 60% (118 of 198) of specimens from Burkina Faso, Madagascar, and Sudan, respectively. The leading identified pathogen overall was Plasmodium spp., accounting for 47% (25 of 53), 2.2% (8 of 364), and 45% (90 of 198) of AFI at the respective sites. In Madagascar, dengue virus was the most prevalent pathogen (10.2%). Overall, 69% (357 of 516) of patients with clinical diagnoses of malaria, respiratory infection, or gastrointestinal infection were prescribed a World Health Organization guideline-recommended empiric antibiotic, whereas only 45% (106 of 237) of patients with pathogens detected were treated with an antibiotic exerting likely activity. CONCLUSIONS: A PCR approach for identifying multiple bacterial, viral, and parasitic pathogens in whole blood unveiled a diversity of previously undetected pathogens in AFI cases and carries implications for the appropriate management of this common syndrome.

Hepatitis E virus genome detection in commercial pork livers and pork meat products in Germany.

The hepatitis E virus (HEV) is one of the most common causes of hepatitis worldwide. HEV is also widespread in many developed countries, where the number of infections is steadily increasing. In those countries, the virus is transmitted mainly through consumption of undercooked or raw food or through contact with animals. Especially, pigs serve as a main reservoir of HEV. Here, we investigated the prevalence of HEV RNA in pork livers and pork meat products to assess the actual risk of HEV infection through food consumption in Germany. A total of 131 pork products were collected from grocery stores and butcher shops between October 2019 and February 2020 and screened for HEV RNA using nested PCR and subsequent sequencing. Overall, 10% of the samples were positive for HEV, including pork livers (5%), spreadable liver sausages (13%) and liver pâté samples (15%). Sequence analyses indicated that the large majority of HEV strains belonged to subtype HEV-3c, representing the most frequent subtype in Germany. One sample belonged to subtype HEV-3f. Further sequence analysis revealed large sequence variation between the samples; however, most of the mutations identified were synonymous. Although infectivity of the virus was not tested, the results suggest a considerable risk of HEV infection through food consumption. Therefore, preventive measures should be taken according to a One Health approach.

Molecular detection of blaCTX-M gene to predict phenotypic cephalosporin resistance and clinical outcome of Escherichia coli bloodstream infections in Vietnam.

BACKGROUND: Blood stream infections (BSI) caused by Extended Spectrum Beta-Lactamases (ESBLs) producing Enterobacteriaceae is a clinical challenge leading to high mortality, especially in developing countries. In this study, we sought to describe the epidemiology of ESBL-producing Escherichia coli strains isolated from Vietnamese individuals with BSI, to investigate the concordance of genotypic-phenotypic resistance, and clinical outcome of ESBL E. coli BSI. METHODS: A total of 459 hospitalized patients with BSI were screened between October 2014 and May 2016. 115 E. coli strains from 115 BSI patients were isolated and tested for antibiotic resistance using the VITEK®2 system. The ESBL phenotype was determined by double disk diffusion method following the guideline of Clinical and Laboratory Standards Institute. Screening for beta-lactamase (ESBL and carbapenemase) genes was performed using a multiplex-PCR assay. RESULTS: 58% (67/115) of the E. coli strains were ESBL-producers and all were susceptible to both imipenem and meropenem. Resistance to third-generation cephalosporin was common, 70% (81/115) were cefotaxime-resistant and 45% (52/115) were ceftazidime-resistant. blaCTX-M was the most common ESBL gene detected (70%; 80/115) The sensitivity and specificity of blaCTX-M-detection to predict the ESBL phenotype was 87% (76-93% 95% CI) and 54% (39-48% 95% CI), respectively. 28%% (22/80) of blaCTX-M were classified as non-ESBL producers by phenotypic testing for ESBL production. The detection of blaCTX-M in ESBL-negative E. coli BSI was associated with fatal clinical outcome (27%; 6/22 versus 8%; 2/26, p = 0.07). CONCLUSION: A high prevalence of ESBL-producing E. coli isolates harbouring blaCTX-M was observed in BSI patients in Vietnam. The genotypic detection of blaCTX-M may have added benefit in optimizing and guiding empirical antibiotic therapy of E. coli BSI to improve clinical outcome.

Impact of diagnostic methods on efficacy estimation - a proof-of-principle based on historical examples.

INTRODUCTION: Accurate diagnostic methods are essential for evaluating treatment efficacy in clinical trials, including vaccine trials. Although a plethora of studies assessing novel or modified treatment options is available, clinical trials evaluating the sensitivity and specificity of diagnostic methods in compliance with the demands of drug registration trials are scarce. We assessed the accuracy of diagnostic methods in two vaccine trials conducted in 1995 and 2009 to demonstrate the impact of sensitivity and specificity on efficacy estimations. METHODS: We applied the sensitivity- and specificity-adjusted vaccine efficacy estimator of Lachenbruch for modelling the impact of test characteristics on the outcome of the two vaccine trials by varying diagnostic specificity. RESULTS: Because of non-ideal diagnostic sensitivity and specificity, the estimation of vaccine efficacy is compromised. We demonstrate the impact of diagnostic accuracy on efficacy estimations with increasing confidence limits. CONCLUSIONS: Because sensitivity and specificity less than one have a direct impact on efficacy estimations in clinical trials, evaluation of diagnostic methods should lead to a level of evidence comparable with the efficacy assessment of novel treatment options. Furthermore, statistical methods adjusted for sensitivity and specificity of diagnostic methods should be applied for efficacy estimations, or this lack of confidence has to be taken into account when interpreting the results of trials.

INTRODUCTION: Des méthodes de diagnostic précises sont essentielles pour évaluer l'efficacité du traitement dans les essais cliniques, y compris les essais de vaccins. Bien qu'une pléthore d'études évaluant des options de traitement nouvelles ou modifiées soit disponible, les essais cliniques évaluant la sensibilité et la spécificité des méthodes de diagnostic conformément aux exigences des essais pour l'enregistrement des médicaments sont rares. Nous avons évalué la précision des méthodes de diagnostic dans deux essais vaccinaux menés en 1995 et 2009 afin de démontrer l'impact de la sensibilité et de la spécificité sur les estimations d'efficacité. MÉTHODE: Nous avons appliqué l'estimateur d'efficacité vaccinale ajusté en fonction de la sensibilité et de la spécificité de Lachenbruch pour modéliser l'impact des caractéristiques des tests sur les résultats de deux essais vaccinaux en faisant varier la spécificité diagnostique. RÉSULTAT: En raison de la sensibilité et de la spécificité diagnostiques non idéales, l'estimation de l'efficacité du vaccin est compromise. Nous démontrons l'impact de la précision du diagnostic sur les estimations d'efficacité avec l'augmentation des limites de confiance. CONCLUSION: Etant donné que la sensibilité et la spécificité inférieures à un ont un impact direct sur les estimations d'efficacité dans les essais cliniques, l'évaluation des méthodes de diagnostic devrait conduire à un niveau d'évidence comparable à l'évaluation de l'efficacité de nouvelles options de traitement. En outre, des méthodes statistiques ajustées en fonction de la sensibilité et de la spécificité des méthodes de diagnostic doivent être appliquées pour les estimations de l'efficacité, ou alors ce manque de confiance devrait être pris en compte lors de l'interprétation des résultats des essais.

Upregulation of Enzymes involved in ISGylation and Ubiquitination in patients with hepatocellular carcinoma.

Background: ISGylation is the conjugation of ISG15 with target proteins. ISGylation occurs through an enzymatic cascade, which is similar to that of ubiquitination. Through ISGylation, ISG15 can bind to proteins involved in cell proliferation and differentiation, thus promoting genesis and progression of malignancies. The present study aims to investigate expression of genes involved in ISGylation and ubiquitination in patients with hepatocellular carcinoma and to correlate gene expression with clinical laboratory parameters of these patients. Methods: mRNA expression of genes encoding enzymes involved in the ISGylation process (EFP, HERC5, UBA1, UBC and USP18) was evaluated by quantitative real-time PCR in 38 pairs of tumour and adjacent non-tumour tissues from patients with hepatocellular carcinoma and correlated with distinct clinical laboratory parameters. Results: Relative mRNA expression of EFP, HERC5, UBA1 and USP18 was significantly higher in tumour tissues compared to adjacent non-tumour tissues (P=0.006; 0.012; 0.02 and 0.039, respectively). The correlation pattern of mRNA expression between genes in the tumours differed from the pattern in adjacent non-tumour tissues. Relative expression of EFP, HERC5 and UBA1 in adjacent non-tumour tissues was positively associated with direct bilirubin levels (Spearman's rho=0.31, 0.33 and 0.45; P=0.06, 0.05 and 0.01, respectively) and relative expression of USP18 in adjacent non-tumour tissues correlated negatively with ALT levels (Spearman's rho= -0.33, P=0.03). Conclusions: EFP, HERC5, UBA1, and USP18 genes are upregulated in tumour tissues of patients with HCC and, thus, may be associated with the pathogenesis of hepatocellular carcinoma.

The Typhoid Fever Surveillance in Africa Program: Geospatial Sampling Frames for Household-based Studies: Lessons Learned From a Multicountry Surveillance Network in Senegal, South Africa, and Sudan.

BACKGROUND: Robust household sampling, commonly applied for population-based investigations, requires sampling frames or household lists to minimize selection bias. We have applied Google Earth Pro satellite imagery to constitute structure-based sampling frames at sites in Pikine, Senegal; Pietermaritzburg, South Africa; and Wad-Medani, Sudan. Here we present our experiences in using this approach and findings from assessing its applicability by determining positional accuracy. METHODS: Printouts of satellite imagery combined with Global Positioning System receivers were used to locate and to verify the locations of sample structures (simple random selection; weighted-stratified sampling). Positional accuracy was assessed by study site and administrative subareas by calculating normalized distances (meters) between coordinates taken from the sampling frame and on the ground using receivers. A higher accuracy in conjunction with smaller distances was assumed. Kruskal-Wallis and Dunn multiple pairwise comparisons were performed to evaluate positional accuracy by setting and by individual surveyor in Pietermaritzburg. RESULTS: The median normalized distances and interquartile ranges were 0.05 and 0.03-0.08 in Pikine, 0.09 and 0.05-0.19 in Pietermaritzburg, and 0.05 and 0.00-0.10 in Wad-Medani, respectively. Root mean square errors were 0.08 in Pikine, 0.42 in Pietermaritzburg, and 0.17 in Wad-Medani. Kruskal-Wallis and Dunn comparisons indicated significant differences by low- and high-density setting and interviewers who performed the presented approach with high accuracy compared to interviewers with poor accuracy. CONCLUSIONS: The geospatial approach presented minimizes systematic errors and increases robustness and representativeness of a sample. However, the findings imply that this approach may not be applicable at all sites and settings; its success also depends on skills of surveyors working with aerial data. Methodological modifications are required, especially for resource-challenged sites that may be affected by constraints in data availability and area size.

The Severe Typhoid Fever in Africa Program: Study Design and Methodology to Assess Disease Severity, Host Immunity, and Carriage Associated With Invasive Salmonellosis.

BACKGROUND: Invasive salmonellosis is a common community-acquired bacteremia in persons residing in sub-Saharan Africa. However, there is a paucity of data on severe typhoid fever and its associated acute and chronic host immune response and carriage. The Severe Typhoid Fever in Africa (SETA) program, a multicountry surveillance study, aimed to address these research gaps and contribute to the control and prevention of invasive salmonellosis. METHODS: A prospective healthcare facility-based surveillance with active screening of enteric fever and clinically suspected severe typhoid fever with complications was performed using a standardized protocol across the study sites in Burkina Faso, the Democratic Republic of Congo (DRC), Ethiopia, Ghana, Madagascar, and Nigeria. Defined inclusion criteria were used for screening of eligible patients for enrollment into the study. Enrolled patients with confirmed invasive salmonellosis by blood culture or patients with clinically suspected severe typhoid fever with perforation were eligible for clinical follow-up. Asymptomatic neighborhood controls and immediate household contacts of each case were enrolled as a comparison group to assess the level of Salmonella-specific antibodies and shedding patterns. Healthcare utilization surveys were performed to permit adjustment of incidence estimations. Postmortem questionnaires were conducted in medically underserved areas to assess death attributed to invasive Salmonella infections in selected sites. RESULTS: Research data generated through SETA aimed to address scientific knowledge gaps concerning the severe typhoid fever and mortality, long-term host immune responses, and bacterial shedding and carriage associated with natural infection by invasive salmonellae. CONCLUSIONS: SETA supports public health policy on typhoid immunization strategy in Africa.

Multicountry Distribution and Characterization of Extended-spectrum ß-Lactamase-associated Gram-negative Bacteria From Bloodstream Infections in Sub-Saharan Africa.

BACKGROUND: Antimicrobial resistance (AMR) is a major global health concern, yet, there are noticeable gaps in AMR surveillance data in regions such as sub-Saharan Africa. We aimed to measure the prevalence of extended-spectrum ß-lactamase (ESBL) producing Gram-negative bacteria in bloodstream infections from 12 sentinel sites in sub-Saharan Africa. METHODS: Data were generated during the Typhoid Fever Surveillance in Africa Program (TSAP), in which standardized blood cultures were performed on febrile patients attending 12 health facilities in 9 sub-Saharan African countries between 2010 and 2014. Pathogenic bloodstream isolates were identified at the sites and then subsequently confirmed at a central reference laboratory. Antimicrobial susceptibility testing, detection of ESBL production, and conventional multiplex polymerase chain reaction (PCR) testing for genes encoding for ß-lactamase were performed on all pathogens. RESULTS: Five hundred and five pathogenic Gram-negative bloodstream isolates were isolated during the study period and available for further characterization. This included 423 Enterobacteriaceae. Phenotypically, 61 (12.1%) isolates exhibited ESBL activity, and genotypically, 47 (9.3%) yielded a PCR amplicon for at least one of the screened ESBL genes. Among specific Gram-negative isolates, 40 (45.5%) of 88 Klebsiella spp., 7 (5.7%) of 122 Escherichia coli, 6 (16.2%) of 37 Acinetobacter spp., and 2 (1.3%) of 159 of nontyphoidal Salmonella (NTS) showed phenotypic ESBL activity. CONCLUSIONS: Our findings confirm the presence of ESBL production among pathogens causing bloodstream infections in sub-Saharan Africa. With few alternatives for managing ESBL-producing pathogens in the African setting, measures to control the development and proliferation of AMR organisms are urgently needed.

Vitamin D receptor ApaI polymorphism associated with progression of liver disease in Vietnamese patients chronically infected with hepatitis B virus.

BACKGROUND: Vitamin D derivatives and their receptor (VDR) are potent modulators of immune responses in various diseases including malignancies as well as in metabolic and infectious disorders. The impact of vitamin D receptor polymorphisms on clinical outcomes of hepatitis B virus (HBV) infection is not well understood. This study aims to investigate the potential role of VDR polymorphisms (TaqI, FokI, ApaI, and BsmI) in Vietnamese HBV infected patients and to correlate these polymorphisms with the progression of HBV-related liver disease. METHODS: Four hundred forty-three HBV infected patients of the three clinically well-defined subgroups chronic hepatitis B (CHB, n = 183), liver cirrhosis (LC, n = 89) and hepatocellular carcinoma (HCC, n = 171) and 238 healthy individuals (HC) were enrolled. VDR polymorphisms were genotyped by DNA sequencing and in-house validated ARMS assays. Logistic regression models were applied in order to determine the association of VDR polymorphisms with manifest HBV infection as well as with progression of related liver diseases mulin different genetic models. RESULTS: The VDR ApaI CA genotype was less frequent in HCC than in CHB patients in different genetic models (codominant model, OR = 0.5, 95%CI = 0.3-0.84, P = 0.004; dominant model, OR = 0.46, 95%CI = 0.27-0.76, P = 0.0023). In the recessive model, the genotype ApaI AA was found more frequently among HCC compared to CHB patients (OR = 2.56, 95%CI = 1.01-6.48, P = 0.04). Similarly, the ApaI CA genotype was less frequent in HCC than in non-HCC group codominant model, OR = 0.6, 95%CI = 0.4-0.98, dominant model, P = 0.04 and OR = 0.6, 95%CI = 0.38-0.90, P = 0.017). The ApaI genotypes CA and AA was significantly associated with higher levels of liver enzymes, bilirubin, and HBV DNA (P < 0.05). No association between TaqI, FokI and BsmI polymorphisms and any clinical outcome as well as liver disease progression was found. CONCLUSIONS: Among the four investigated VDR polymorphisms, ApaI is associated with clinical outcome and liver disease progression in Vietnamese HBV infected patients.

Genetic diversity and population structure of Plasmodium falciparum in Kenyan-Ugandan border areas.

Kenya has, in the last decade, made tremendous progress in the fight against malaria. Nevertheless, continued surveillance of the genetic diversity and population structure of Plasmodium falciparum is required to refine malaria control and to adapt and improve elimination strategies. Twelve neutral microsatellite loci were genotyped in 201 P. falciparum isolates obtained from the Kenyan-Ugandan border (Busia) and from two inland malaria-endemic sites situated in western (Nyando) and coastal (Msambweni) Kenya. Analyses were done to assess the genetic diversity (allelic richness and expected heterozygosity, [He ]), multilocus linkage disequilibrium ( ISA ) and population structure. A similarly high degree of genetic diversity was observed among the three parasite populations surveyed (mean He  = 0.76; P > 0.05). Except in Msambweni, random association of microsatellite loci was observed, indicating high parasite out-breeding. Low to moderate genetic structure (FST  = 0.022-0.076; P < 0.0001) was observed with only 5% variance in allele frequencies observed among the populations. This study shows that the genetic diversity of P. falciparum populations at the Kenyan-Ugandan border is comparable to the parasite populations from inland Kenya. In addition, high genetic diversity, panmixia and weak population structure in this study highlight the fitness of Kenyan P. falciparum populations to successfully withstand malaria control interventions.

Le Kenya a réalisé d'énormes progrès au cours de la dernière décennie dans la lutte contre le paludisme. Néanmoins, une surveillance continue de la diversité génétique et de la structure de la population de P. falciparum est nécessaire pour affiner la lutte contre le paludisme et pour adapter et améliorer les stratégies d'élimination. Douze loci microsatellites neutres ont été génotypés chez 201 isolats de P. falciparum provenant de la frontière entre le Kenya et l'Ouganda (Busia) et de deux sites d'endémie palustre situés dans l'ouest (Nyando) et sur la côte (Msambweni), au Kenya. Des analyses ont été effectuées pour évaluer la diversité génétique (richesse allélique et hétérozygotie attendue, ([He]), déséquilibre de parenté des multiple loci ( ISA ) et structure de la population. Un degré hautement similaire de diversité génétique a été observé parmi les trois populations de parasites étudiées (He = 0,76; P > 0,05). A l'exception de Msambweni, une association aléatoire entre les microsatellites a été observée, indiquant une forte reproduction des parasites. Une structure génétique faible à modérée (FST  = 0,022-0,076; P < 0,0001) a été observée avec seulement 5% de variance dans la fréquence des allèles observée parmi les populations. Cette étude montre que la diversité génétique des populations de P. falciparum à la frontière entre le Kenya et l'Ouganda est comparable à celle des populations de parasites à l'intérieur du Kenya. De plus, la diversité génétique élevée, la panmixia et la structure démographique faible dans cette étude soulignent l'aptitude des populations de P. falciparum du Kenya à résister aux interventions de lutte contre le paludisme.

Socioeconomic Status and Temporal Urban Environmental Change in Accra: a Comparative Analysis of Area-based Socioeconomic and Urban Environmental Quality Conditions Between Two Time Points.

The influence of area-based socioeconomic (SE) conditions on environmental quality conditions has recently been reported showing the precise spatial relationship between area-based SE conditions and neighborhood environmental quality in an urban area in a low-income setting. Nonetheless, there is still a lack of understanding of the nature of the relationship on a temporal scale. This study aimed to investigate the dynamic temporal relationship between area-based SE conditions and urban environmental quality conditions over a decadal period in Accra, Ghana. The results showed that there were differences in environmental quality across the SE quintiles in space (with regard to per capita waste generation (p < 0.012), waste collection/clearing (p < 0.01), and waste deposition (p < 0.001) and that the urban environmental quality conditions had changed dramatically over the decade for most of the environmental variables (p < 0.001). Despite the enormous urban development initiatives, some of the environmental quality indicators (e.g., proportion of households without flush toilet/Water Closet, connection to central sewer p < 0.001) appeared to have worsened in the high class quintile, suggesting that a high proportion of households were without acceptable sanitation facilities. The study concludes that urban development in low-income countries will need to follow strictly international best practice by observing standardized building codes and guidelines, if progress should be made in meeting the Millennium Development Goals targets.

Determining the Best Immunization Strategy for Protecting African Children Against Invasive Salmonella Disease.

Background: The World Health Organization recently prequalified a typhoid conjugate vaccine (TCV), recommending its use in persons ≥6 months to 45 years residing in typhoid fever (TF)-endemic areas. We now need to consider how TCVs can have the greatest impact in the most vulnerable populations. Methods: The Typhoid Fever Surveillance in Africa Program (TSAP) was a blood culture-based surveillance of febrile patients from defined populations presenting at healthcare facilities in 10 African countries. TF and invasive non-typhoidal Salmonella (iNTS) disease incidences were estimated for 0-10 year-olds in one-year age increments. Results: Salmonella Typhi and iNTS were the most frequently isolated pathogens; 135 and 94 cases were identified, respectively. Analysis from three countries was excluded (incomplete person-years of observation (PYO) data). Thirty-seven of 123 TF cases (30.1%) and 71/90 iNTS disease cases (78.9%) occurred in children aged <5 years. No TF and 8/90 iNTS infections (8.9%) were observed in infants aged <9 months. The TF incidences (/100 000 PYO) for children aged <1 year and 1 to <2 years were 5 and 39, respectively; the highest incidence was 304 per 100 000 PYO in 4 to <5 year-olds. The iNTS disease incidence in the defined age groups ranged between 81 and 233 per 100 000 PYO, highest in 1 to <2 year-olds. TF and iNTS disease incidences were higher in West Africa. Conclusions: High burden of TF detected in young children strengthens the need for TCV introduction. Given the concurrent iNTS disease burden, development of a trivalent vaccine against S. Typhi, S. Typhimurium, and S. Enteritidis may be timely in this region.

Differential contribution of interleukin-10 promoter variants in malaria and schistosomiasis mono- and co-infections among Nigerian children.

OBJECTIVE: Interleukin-10 (IL-10) is an anti-inflammatory cytokine produced by Th1 cells and macrophages. The rationale of this study was to examine and validate possible contributions of IL-10 promoter polymorphisms in sub-Saharan Africa in children infected with either Plasmodium falciparum or Schistosoma haematobium and in children co-infected with both parasites. MATERIALS AND METHODS: A total of 309 Nigerian children aged 4-15 years were recruited. The study group consisted of individuals infected either with P. falciparum (n = 76) or S. haematobium (n = 94) in mono-infections, a group of children co-infected with both P. falciparum and S. haematobium (n = 62) and matched healthy controls (n = 77). The IL-10 promoter polymorphisms -1082G/A, -819C/T and -592C/A were genotyped by direct sequencing. RESULTS: The frequencies of the IL-10 -1082GG genotype, the -1082G allele and haplotype GCC (positions -1082, -819 and -592) were higher in children infected with P. falciparum than in healthy controls, indicating that the -1082GG genotype and the -1082G allele and the GCC haplotype are associated with increased susceptibility to malaria infection (OR = 3.4, 95% CI = 1.2-10.8, P = 0.02; OR = 2.5, 95% CI = 1.1-3.4, P = 0.02; OR = 3.8, 95% CI = 2.0-7.2, P = 0.0001, respectively). Children with the -1082GG genotype had a higher parasitaemia than children with the -1082AA or -1082AG genotypes (P = 0.0017). Haplotype GCC occurred more frequently in children infected with S. haematobium, while haplotype GTA was less frequent than in controls (OR = 2.2, 95% CI = 1.2-4.4, P = 0.017 and OR = 0.1, 95% CI = 0.02-0.5, P = 0.0004, respectively). No differences in the frequencies of IL-10 promoter polymorphisms were observed between children with P. falciparum-S. haematobium co-infections and healthy controls. CONCLUSION: Although IL-10 promoter polymorphisms are not associated with P. falciparum and S. haematobium co-infection, variant -1082G/A and haplotype GCC are associated with malaria, whereas the IL-10 haplotypes GCC and GTA are associated with schistosomiasis.

Soluble fibrinogen-like protein 2 levels in patients with hepatitis B virus-related liver diseases.

BACKGROUND: Clinical progression of HBV-related liver diseases is largely associated with the activity of HBV-specific T cells. Soluble fibrinogen-like protein 2 (sFGL2), mainly secreted by T cells, is an important effector molecule of the immune system. METHODS: sFGL2 levels were determined by ELISA assays in sera of 296 HBV patients clinically classified into the subgroups of acute hepatitis B (AHB), chronic hepatitis B (CHB), liver cirrhosis (LC), hepatocellular carcinoma (HCC) and patients with LC plus HCC. As control group, 158 healthy individuals were included. FGL2 mRNA was quantified by qRT-PCR in 32 pairs of tumor and adjacent non-tumor liver tissues. RESULTS: sFGL2 levels were elevated in HBV patients compared to healthy controls (P <  0.0001). In the patient group, sFGL2 levels were increased in AHB compared to CHB patients (P = 0.017). sFGL2 levels were higher in LC patients compared to those without LC (P = 0.006) and were increased according to the development of cirrhosis as staged by Child-Pugh scores (P = 0.024). Similarly, HCC patients had increased sFGL2 levels compared to CHB patients (P = 0.033) and FGL2 mRNA was up-regulated in tumor tissues compared to adjacent non-tumor tissues (P = 0.043). In addition, sFGL2 levels were positively correlated with HBV-DNA loads and AST (Spearman's rho = 0.21, 0.25 and P = 0.006, 0.023, respectively), but reversely correlated with platelet counts and albumin levels (Spearman's rho = - 0.27, - 0.24 and P = 0.014, 0.033, respectively). CONCLUSIONS: sFGL2 levels are induced by HBV infection and correlated with the progression and clinical outcome of HBV-related liver diseases. Thus, sFGL2 may serve as a potential indicator for HBV-related liver diseases.

Genome-wide association study indicates two novel resistance loci for severe malaria.

Malaria causes approximately one million fatalities per year, mostly among African children. Although highlighted by the strong protective effect of the sickle-cell trait, the full impact of human genetics on resistance to the disease remains largely unexplored. Genome-wide association (GWA) studies are designed to unravel relevant genetic variants comprehensively; however, in malaria, as in other infectious diseases, these studies have been only partly successful. Here we identify two previously unknown loci associated with severe falciparum malaria in patients and controls from Ghana, West Africa. We applied the GWA approach to the diverse clinical syndromes of severe falciparum malaria, thereby targeting human genetic variants influencing any step in the complex pathogenesis of the disease. One of the loci was identified on chromosome 1q32 within the ATP2B4 gene, which encodes the main calcium pump of erythrocytes, the host cells of the pathogenic stage of malaria parasites. The second was indicated by an intergenic single nucleotide polymorphism on chromosome 16q22.2, possibly linked to a neighbouring gene encoding the tight-junction protein MARVELD3. The protein is expressed on endothelial cells and might therefore have a role in microvascular damage caused by endothelial adherence of parasitized erythrocytes. We also confirmed previous reports on protective effects of the sickle-cell trait and blood group O. Our findings underline the potential of the GWA approach to provide candidates for the development of control measures against infectious diseases in humans.

Host genetic studies in adult pulmonary tuberculosis.

Early observations, candidate gene studies and, more recently, genome-wide association studies have shown that susceptibility to tuberculosis has a host genetic component. Because the value of candidate gene studies has been doubted due to major limitations such as lack of sufficient power and small study groups, lack of reproducibility in independent groups and, often, ambiguous or even contrasting results in attempts of replication, much hope and expectancy has been put on the progress the genome-wide association approach has created. However, much less than initially expected became clear by the results obtained in genome-wide studies, emphasizing the need of increasing sample sizes, e.g. through meta-analyses, and of increasing the density of genetic variants studied across the human genome. A further reason why a rather low number of associated genetic variants were identified to date in infectious diseases in general and tuberculosis in particular might be the fact that selection acts strongly in diseases that affect the reproductive success. As in most genome-wide association studies performed so far, significant signals, often most likely surrogate marker only, have been found in non-coding regions of genomes, the identification of truly causative genetic variation and of the functionality of associated factors needs urgent attention. In the following we briefly discuss genetic studies in tuberculosis and describe new technologies that are currently employed in the search for responsible genetic elements involved in tuberculosis susceptibility.

Pyruvate Kinase and Fcγ Receptor Gene Copy Numbers Associated With Malaria Phenotypes.

Genetic factors are associated with susceptibility to many infectious diseases and may be determinants of clinical progression. Gene copy number variation (CNV) has been shown to be associated with phenotypes of numerous diseases, including malaria. We quantified gene copy numbers of the pyruvate kinase, liver, and red blood cell (PKLR) gene as well as of the Fcγ receptor 2A and Fcγ receptor 2C (FCGR2A, FCGR2C) and Fcγ receptor 3 (FCGR3) genes using real-time quantitative polymerase chain reaction (RT-qPCR) assays in Gabonese children with severe (n = 184) or and mild (n = 189) malaria and in healthy Gabonese and white individuals (n = 76 each). The means of PKLR, FCGR2A, FCGR2C, and FCGR3 copy numbers were significantly higher among children with severe malaria compared to those with mild malaria (P < .002), indicating that a surplus of copies of those genes is significantly associated with malaria severity. Copy numbers of the FCGR2A and FCGR2C genes were significantly lower (P = .005) in Gabonese individuals compared with white individuals. In conclusion, CNV of the PKLR, FCGR2A, FCGR2C, and FCGR3 genes is associated with malaria severity, and our results provide evidence for a role of CNV in host responses to malaria.

Current perspectives on invasive nontyphoidal Salmonella disease.

PURPOSE OF REVIEW: We searched PubMed for scientific literature published in the past 2 years for relevant information regarding the burden of invasive nontyphoidal Salmonella disease and host factors associated with nontyphoidal Salmonella infection and discuss current knowledge on vaccine development. The following search terms were used: Salmonella, non typhoidal/nontyphoidal, NTS, disease, bloodstream infection, invasive, sepsis/septicaemia/septicemia, bacteraemia/bacteremia, gastroenteritis, incidence, prevalence, morbidity, mortality, case fatality, host/risk factor, vaccination, and prevention/control. RECENT FINDINGS: Estimates of the global invasive nontyphoidal Salmonella disease burden have been recently updated; additional data from Africa, Asia, and Latin America are now available. New data bridge various knowledge gaps, particularly with respect to host risk factors and the geographical distribution of iNTS serovars. It has also been observed that Salmonella Typhimurium sequence type 313 is emergent in several African countries. Available data suggest that genetic variation in the sequence type 313 strain has led to increased pathogenicity and human host adaptation. A bivalent efficacious vaccine, targeting Salmonella serovars Typhimurium and Enteritidis, would significantly lower the disease burden in high-risk populations. SUMMARY: The mobilization of surveillance networks, especially in Asia and Latin America, may provide missing data regarding the invasive nontyphoidal Salmonella disease burden and their corresponding antimicrobial susceptibility profiles. Efforts and resources should be directed toward invasive nontyphoidal Salmonella disease vaccine development.