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.

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 surveillance and temporal monitoring of malaria parasites in focal Vietnamese provinces.

BACKGROUND: While the World Health Organization (WHO) Southeast Asia region has the second highest incidence of malaria worldwide, malaria in Vietnam is focal to few provinces, where delayed parasite clearance to anti-malarial drugs is documented. This study aims to understand Plasmodium species distribution and the genetic diversity of msp1 and msp2 of parasite populations using molecular tools. METHODS: A total of 222 clinical isolates from individuals with uncomplicated malaria were subjected to Plasmodium species identification by nested real-time PCR. 166 isolates positive for Plasmodium falciparum mono infections were further genotyped for msp1 (MAD20, K1, and RO33), and msp2 allelic families (3D7 and FC27). Amplicons were resolved through capillary electrophoresis in the QIAxcel Advanced system. RESULTS: Mono-infections were high and with 75% P. falciparum, 14% Plasmodium vivax and 9% P. falciparum/P. vivax co-infections, with less than 1% Plasmodium malariae identified. For msp1, MAD20 was the most prevalent (99%), followed by K1 (46%) allelic family, with no sample testing positive for RO33 (0%). For msp2, 3D7 allelic family was predominant (97%), followed by FC27 (10%). The multiplicity of infection of msp1 and msp2 was 2.6 and 1.1, respectively, and the mean overall multiplicity of infection was 3.7, with the total number of alleles ranging from 1 to 7. CONCLUSIONS: Given the increasing importance of antimalarial drugs in the region, the genetic diversity of P. falciparum msp1 and msp2 should be regularly monitored with respect to treatment outcomes and/or efficacy studies in regions, where there are ongoing changes in the malaria epidemiology.

Gut microbiota in vaccine naïve Gabonese children with rotavirus A gastroenteritis.

Background: While the gut microbiome modulates the pathogenesis of enteric viruses, how infections caused by rotavirus A (RVA), with or without diarrhoea, alter the gut microbiota has been sparsely studied. Methods: From a cohort of 224 vaccine naïve Gabonese children with and without diarrhoea (n = 177 and n = 67, respectively), 48 stool samples were analysed: (i) RVA with diarrhoea (n = 12); (ii) RVA without diarrhoea (n = 12); (iii) diarrhoea without RVA (n = 12); (iv) healthy controls without diarrhoea and RVA (n = 12). The 16S rRNA metabarcoding using Oxford Nanopore sequencing data was analysed for taxonomic composition, abundance, alpha and beta diversity, and metabolic pathways. Findings: Alpha diversity showed that children with acute diarrhoea (with and without RVA infection), and children with acute diarrhoea without RVA had low microbial diversity compared to healthy children (p = 0.001 and p = 0.006, respectively). No significant differences observed when comparing children with RVA with or without diarrhoea. Beta diversity revealed high microbial heterogeneity in children without diarrhoea. Proteobacteria (68%) and Firmicutes (69%) were most common in the diarrhoea and non-diarrhoea groups, respectively. Proteobacteria (53%) were most common in children without RVA, while Firmicutes (55%) were most common with RVA. At the genus level, Escherichia (21%), Klebsiella (10%) and Salmonella (4%) were abundant in children with diarrhoea, while Blautia (11%), Clostridium (8%), Lachnoclostridium (6%) and Ruminococcus (5%) were abundant in children without diarrhoea. Metabolites involved in amino acid, carbohydrate, lipid, nucleotide, and vitamin metabolism were quantitatively altered. Interpretation: Although host physiology dictates the intestinal milieu, diarrhoea per se can alter a balanced gut microbiota, whereas infectious diarrhoea disrupts the gut microbiome and reduces its diversity.

Tübingen model study: large-scale introduction of rapid antigen testing in the population and the viral dynamics of SARS-CoV-2.

Despite of contact restrictions, population mobility remains the main reason for the spread of SARS-CoV-2. The state of Baden-Württemberg (BW), Germany, approved a model study in Tübingen (TÜMOD) to evaluate how mandatory rapid diagnostic tests (RDT) could reduce transmission. Between 16 March and 24 April 2021, approximately 165,000 residents and visitors to the city were screened for SARS CoV-2 infection using Abbott Panbio™ COVID-19 Antigen rapid test device. We assessed incidences and recorded epidemiological characteristics in a subset of 4,118 participants recruited at three of the nine testing stations. PCR tests were performed in RDT-positives to determine the positive predictive value (PPV), and circulating variants of SARS-CoV-2 were identified by whole-genome sequencing. 2,282 RDT-negative samples were tested by pooled PCR to calculate the false negative rate (FNR). Viral load was compared between variants. 116 (3%) participants were positive by RDT, and of these, 57 (49%) were positive by PCR, 55 (47%) were negative. This resulted in a PPV of 51%. Of the 57 positives, 52 SARS-CoV-2 genomes were successfully sequenced. Of these, 50 belonged to the B.1.1.7 lineage, which had a high viral load (average Ct = 19). Of the 2,282 RDT negatives tested, all were PCR negative (FNR 0%). At the end of TÜMOD, the incidence in Tübingen, which was initially lower, had reached the incidence in the state of BW. While it is difficult to assess the impact of TÜMOD on incidence independent of confounding factors, further studies are needed to identify the effect of close-meshed testing on infection rates.

High Hepatitis E Virus (HEV) Seroprevalence and No Evidence of HEV Viraemia in Vietnamese Blood Donors.

The prevalence of hepatitis E virus (HEV) in the Vietnamese population remains underestimated. The aim of the present study was to investigate the seroprevalence of HEV IgG/IgM antibodies and the presence of HEV RNA in blood donors as a part of epidemiological surveillance for transfusion-transmitted viruses. Serum samples from blood donors (n = 553) were analysed for markers of past (anti-HEV IgG) and recent/ongoing (anti-HEV IgM) HEV infections. In addition, all serum samples were subsequently tested for HEV RNA positivity. The overall prevalence of anti-HEV IgG was 26.8% (n = 148/553), while the seroprevalence of anti-HEV IgM was 0.5% (n = 3/553). Anti-HEV IgG seroprevalence in male and female donors was similar (27.1% and 25.5%, respectively). A higher risk of hepatitis E exposure was observed with increasing age. None of the blood donors were HEV RNA positive, and there was no evidence of HEV viraemia. Although the absence of HEV viraemia in blood donors from Northern Vietnam is encouraging, further epidemiological surveillance in other geographical regions is warranted to rule out transfusion-transmitted HEV.

Host genetic loci LZTFL1 and CCL2 associated with SARS-CoV-2 infection and severity of COVID-19.

OBJECTIVES: Host genetic factors contribute to the variable severity of COVID-19. We examined genetic variants from genome-wide association studies and candidate gene association studies in a cohort of patients with COVID-19 and investigated the role of early SARS-CoV-2 strains in COVID-19 severity. METHODS: This case-control study included 123 COVID-19 cases (hospitalized or ambulatory) and healthy controls from the state of Baden-Wuerttemberg, Germany. We genotyped 30 single nucleotide polymorphisms, using a custom-designed panel. Cases were also compared with the 1000 genomes project. Polygenic risk scores were constructed. SARS-CoV-2 genomes from 26 patients with COVID-19 were sequenced and compared between ambulatory and hospitalized cases, and phylogeny was reconstructed. RESULTS: Eight variants reached nominal significance and two were significantly associated with at least one of the phenotypes "susceptibility to infection", "hospitalization", or "severity": rs73064425 in LZTFL1 (hospitalization and severity, P <0.001) and rs1024611 near CCL2 (susceptibility, including 1000 genomes project, P = 0.001). The polygenic risk score could predict hospitalization. Most (23/26, 89%) of the SARS-CoV-2 genomes were classified as B.1 lineage. No associations of SARS-CoV-2 mutations or lineages with severity were observed. CONCLUSION: These host genetic markers provide insights into pathogenesis and enable risk classification. Variants which reached nominal significance should be included in larger studies.

Custom gene expression panel for evaluation of potential molecular markers in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide. It is a highly heterogeneous disease with poor prognosis and limited treatment options, which highlights the need for reliable biomarkers. This study aims to explore molecular markers that allow stratification of HCC and may lead to better prognosis and treatment prediction. MATERIALS AND METHODS: We studied 20 candidate genes (HCC hub genes, potential drug target genes, predominant somatic mutant genes) retrieved from literature and public databases with potential to be used as the molecular markers. We analysed expression of the genes by RT-qPCR in 30 HCC tumour and adjacent non-tumour paired samples from Vietnamese patients. Fold changes in expression were then determined using the 2-∆∆CT method, and unsupervised hierarchical clustering was generated using Cluster v3.0 software. RESULTS: Clustering of expression data revealed two subtypes of tumours (proliferative and normal-like) and four clusters for genes. The expression profiles of the genes TOP2A, CDK1, BIRC5, GPC3, IGF2, and AFP were strongly correlated. Proliferative tumours were characterized by high expression of the c-MET, ARID1A, CTNNB1, RAF1, LGR5, and GLUL1 genes. TOP2A, CDK1, and BIRC5 HCC hub genes were highly expressed (> twofold) in 90% (27/30), 83% (25/30), and 83% (24/30) in the tissue samples, respectively. Among the drug target genes, high expression was observed in the GPC3, IGF2 and c-MET genes in 77% (23/30), 63% (19/30), and 37% (11/30), respectively. The somatic mutant Wnt/ß-catenin genes (CTNNB1, GLUL and LGR5) and TERT were highly expressed in 40% and 33% of HCCs, respectively. Among the HCC marker genes, a higher percentage of tumours showed GPC3 expression compared to AFP expression [73% (23/30) vs. 43% (13/30)]. CONCLUSION: The custom panel and molecular markers from this study may be useful for diagnosis, prognosis, biomarker-guided clinical trial design, and prediction of treatment outcomes.

SARS-CoV-2 B.1.214.1, B.1.214.2 and B.1.620 are predominant lineages between December 2020 and July 2021 in the Republic of Congo.

Background: : SARS-CoV-2 variants have been emerging and are shown to increase transmissibility, pathogenicity, and decreased vaccine efficacies. The objective of this study was to determine the distribution, prevalence, and dynamics of SARS-CoV-2 variants circulating in Brazzaville, the Republic of Congo (ROC). Methods: : Between December 2020 and July 2021, a total of n=600 oropharyngeal specimens collected in the community were tested for COVID-19. Of the samples tested, 317 (53%) were SARS-CoV-2 positive. All samples that had a threshold of Ct <30 (n=182) were sequenced by next-generation sequencing (NGS), and all complete sequenced genomes were submitted to GISAID; lineages were assigned using pangolin nomenclature and a phylogenetic tree was reconstructed. In addition, the global prevalence of the predominant lineages was analysed using data from GISAID and Outbreak databases. Results: : A total of 15 lineages circulated with B.1.214.2 (26%), B.1.214.1 (19%) and B.1.620 (18%) being predominant. The variants of concern (VOC) alpha (B.1.1.7) (6%) and for the first time in June delta (B.1.617.2) (4%) were observed. In addition, the B.1.214.1 lineage first reported from ROC was observed to be spreading locally and regionally. Phylogenetic analysis suggests that the B.1.620 variant (VUM) under observation may have originated from either Cameroon or the Central African Republic. SARS-CoV-2 lineages were heterogeneous, with the densely populated districts of Poto-Poto and Moungali likely the epicenter of spread. Conclusion: : Longitudinal monitoring and molecular surveillance across time and space are critical to understanding viral phylodynamics, which could have important implications for transmissibility and impact infection prevention and control measures.

Low Risk of Occult Hepatitis B Infection among Vietnamese Blood Donors.

Occult hepatitis B infection (OBI) is characterized by the presence of low levels of hepatitis B virus (HBV) DNA and undetectable HBsAg in the blood. The prevalence of OBI in blood donors in Asia ranges from 0.013% (China) to 10.9% (Laos), with no data available from Vietnam so far. We aimed to investigate the prevalence of OBI among Vietnamese blood donors. A total of 623 (114 women and 509 men) HBsAg-negative blood donors were screened for anti-HBc and anti-HBs by ELISA assays. In addition, DNA from sera was isolated and nested PCR was performed for the HBV surface gene (S); a fragment of the S gene was then sequenced in positive samples. The results revealed that 39% (n = 242) of blood donors were positive for anti-HBc, and 70% (n = 434) were positive for anti-HBs, with 36% (n = 223) being positive for both anti-HBc and anti-HBs. In addition, 3% of blood donors (n = 19) were positive for anti-HBc only, and 34% (n = 211) had only anti-HBs as serological marker. A total of 27% (n = 170) were seronegative for any marker. Two of the blood donors (0.3%) were OBI-positive and sequencing revealed that HBV sequences belonged to HBV genotype B, which is the predominant genotype in Vietnam.

Genomic surveillance of SARS-CoV-2 in the Republic of Congo.

OBJECTIVE: The aim of this study was to carry out whole-genome sequencing (WGS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), using samples collected from Congolese individuals between April and July 2020. METHODS: Ninety-six samples were screened for SARS-CoV-2 using RT-PCR, and 19 samples with Ct values <30 were sequenced using Illumina Next-Generation Sequencing (NGS). The genomes were annotated and screened for mutations using the web tool 'coronapp'. Subsequently, different SARS-CoV-2 lineages were assigned using PANGOLIN and Nextclade. RESULTS: Eleven SARS-CoV-2 genomes were successfully sequenced and submitted to the GSAID database. All genomes carried the spike mutation D614G and were classified as part of the GH clade. The Congolese SARS-CoV-2 sequences were shown to belong to lineage B1 and Nextclade 20A and 20C, which split them into distinct clusters, indicating two separate introductions of the virus into the Republic of Congo. CONCLUSION: This first study provides valuable information on SARS CoV-2 transmission in the central African region, contributing to SARS CoV-2 surveillance on a temporal and spatial scale.

Predominance of HBV Genotype B and HDV Genotype 1 in Vietnamese Patients with Chronic Hepatitis.

Hepatitis delta virus (HDV) coinfection will additionally aggravate the hepatitis B virus (HBV) burden in the coming decades, with an increase in HBV-related liver diseases. Between 2018 and 2019, a total of 205 HBV patients clinically characterized as chronic hepatitis B (CHB; n = 115), liver cirrhosis (LC; n = 21), and hepatocellular carcinoma (HCC; n = 69) were recruited. HBV surface antigen (HBsAg), antibodies against surface antigens (anti-HBs), and core antigens (anti-HBc) were determined by ELISA. The presence of hepatitis B viral DNA and hepatitis delta RNA was determined. Distinct HBV and HDV genotypes were phylogenetically reconstructed and vaccine escape mutations in the "a" determinant region of HBV were elucidated. All HBV patients were HbsAg positive, with 99% (n = 204) and 7% (n = 15) of them being positive for anti-HBc and anti-HBs, respectively. Anti-HBs positivity was higher among HCC (15%; n = 9) compared to CHB patients. The HBV-B genotype was predominant (65%; n = 134), followed by HBV-C (31%; n = 64), HBV-D, and HBV-G (3%; n = 7). HCC was observed frequently among young individuals with HBV-C genotypes. A low frequency (2%; n = 4) of vaccine escape mutations was observed. HBV-HDV coinfection was observed in 16% (n = 33) of patients with the predominant occurrence of the HDV-1 genotype. A significant association of genotypes with alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme levels was observed in HBV monoinfections. The prevalence of the HDV-1 genotype is high in Vietnam. No correlation was observed between HDV-HBV coinfections and disease progression when compared to HBV monoinfections.

Pharmacogenetic considerations in the treatment of co-infections with HIV/AIDS, tuberculosis and malaria in Congolese populations of Central Africa.

BACKGROUND: HIV-infection, tuberculosis and malaria are the big three communicable diseases that plague sub-Saharan Africa. If these diseases occur as co-morbidities they require polypharmacy, which may lead to severe drug-drug-gene interactions and variation in adverse drug reactions, but also in treatment outcomes. Polymorphisms in genes encoding drug-metabolizing enzymes are the major cause of these variations, but such polymorphisms may support the prediction of drug efficacy and toxicity. There is little information on allele frequencies of pharmacogenetic variants of enzymes involved in the metabolism of drugs used to treat HIV-infection, TB and malaria in the Republic of Congo (ROC). The aim of this study was therefore to investigate the occurrence and allele frequencies of 32 pharmacogenetic variants localized in absorption distribution, metabolism and excretion (ADME) and non-ADME genes and to compare the frequencies with population data of Africans and non-Africans derived from the 1000 Genomes Project. RESULTS: We found significant differences in the allele frequencies of many of the variants when comparing the findings from ROC with those of non-African populations. On the other hand, only a few variants showed significant differences in their allele frequencies when comparing ROC with other African populations. In addition, considerable differences in the allele frequencies of the pharmacogenetic variants among the African populations were observed. CONCLUSIONS: The findings contribute to the understanding of pharmacogenetic variants involved in the metabolism of drugs used to treat HIV-infection, TB and malaria in ROC and their diversity in different populations. Such knowledge helps to predict drug efficacy, toxicity and ADRs and to inform individual and population-based decisions.

Low Prevalence of HEV Infection and No Associated Risk of HEV Transmission from Mother to Child among Pregnant Women in Vietnam.

Infections with HEV in low- and middle-income countries (LMICs) are associated with increased rates of preterm birth, miscarriage, and stillbirth. The aim of the present study was to investigate HEV infections in pregnant women and the possibility of mother-to-child transmission, and associated outcomes. A total of 183 pregnant women in their third trimester were recruited and followed until delivery. Anti-HEV IgG and IgM were determined via enzyme-linked immunosorbent assay (ELISA), and HEV nucleic acids were detected in stool and cord blood samples. HEV genotypes were identified by Sanger sequencing, and phylogenetic analyses were performed. Mother-to-child transmission and associated adverse outcomes were not observed. Only 2% of patients (n = 4/183) tested positive for anti-HEV IgM, and 8% (n = 14/183) tested positive for anti-HEV IgG antibodies. Cord blood (n = 150) analysis showed that there was no IgM detected, while 4% (n = 6/150) tested positive for anti-HEV IgG, which was consistent with mothers testing positive for anti-HEV IgG. Nucleic acid tests for HEV RNA yielded 2% (n = 4/183) from the serum and stool of pregnant women, and none from cord blood. The HEV isolates belonged to the genotype HEV-3a, with 99% homology with humans and 96% with pigs. No association was found between the risk of HEV infection and pregnancy outcomes or HEV transmission from mother to child. HEV-3 infections of zoonotic origin in pregnancy might have eventually resolved without complications.

Longitudinal monitoring of laboratory markers characterizes hospitalized and ambulatory COVID-19 patients.

Early detection of severe forms of COVID-19 is absolutely essential for timely triage of patients. We longitudinally followed-up two well-characterized patient groups, hospitalized moderate to severe (n = 26), and ambulatory mild COVID-19 patients (n = 16) at home quarantine. Human D-dimer, C-reactive protein (CRP), ferritin, cardiac troponin I, interleukin-6 (IL-6) levels were measured on day 1, day 7, day 14 and day 28. All hospitalized patients were SARS-CoV-2 positive on admission, while all ambulatory patients were SARS-CoV-2 positive at recruitment. Hospitalized patients had higher D-dimer, CRP and ferritin, cardiac troponin I and IL-6 levels than ambulatory patients (p < 0.001, p < 0.001, p = 0.016, p = 0.035, p = 0.002 respectively). Hospitalized patients experienced significant decreases in CRP, ferritin and IL-6 levels from admission to recovery (p < 0.001, p = 0.025, and p = 0.001 respectively). Cardiac troponin I levels were high during the acute phase in both hospitalized and ambulatory patients, indicating a potential myocardial injury. In summary, D-dimer, CRP, ferritin, cardiac troponin I, IL-6 are predictive laboratory markers and can largely determine the clinical course of COVID-19, in particular the prognosis of critically ill COVID-19 patients.