Prevalence, Characterization, and Antimicrobial Resistance of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli from Domestic Free-Range Poultry in Agogo, Ghana.

Poultry has been suggested as an important source for extended-spectrum beta-lactamase (ESBL)-producing bacteria that can lead to difficult-to treat infections in humans. Therefore, this study aims to determine the frequency, the genetics, and antimicrobial resistance profiles of ESBL-producing Escherichia coli in domestic free-range poultry in Agogo, Ghana. The study was set up and piloted from January 2019 until June 2019. Between June and December 2019, fecal samples (N = 144) were collected from free-roaming chickens from domestic farms in the regions of Sukuumu, Bontodiase, and Freetown and cultured on ESBL screening agar. Strain identification and antibiotic susceptibility were performed using the VITEK 2 compact system. ESBL-producing E. coli were confirmed using the double disk synergy test. Molecular characterization of ESBL-associated genes (blaTEM, blaSHV, and blaCTX-M) were performed using conventional polymerase chain reaction (PCR) and further sequencing of obtained PCR amplicons. The result showed that 56.2% (n/N = 81/144) of collected fecal samples were positive for ESBL-producing E. coli. Majority of the isolates showed resistance to tetracycline (93.8%, n/N = 76/81) and trimethoprim-sulfamethoxazole (66.7, n/N = 54/81), whereas resistance to carbapenems was not found. The majority of ESBL-producing E. coli carried the blaCTX-M genes, with blaCTX-M-15 being the dominant (95.1%, n/N = 77/81) genotype. In this study, we report high frequencies of ESBL-producing E. coli in smallholder free-range poultry representing a potential source of infection, highlighting the need for control of antibiotic use and animal hygiene/sanitation measures, both important from a One Health perspective.

Human genetic variant E756del in the ion channel PIEZO1 not associated with protection from severe malaria in a large Ghanaian study.

Recently, a common genetic variant E756del in the human gene PIEZO1 was associated with protection from severe malaria. Here, we performed a genetic association study of this gain-of-function variant in a large case-control study including 4149 children from the Ashanti Region in Ghana, West Africa. The statistical analysis did not indicate an association with protection from severe malaria and, thus, providing evidence against a strong protective effect of the PIEZO1 E756del variant on severe malaria susceptibility.

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.

Travel-associated neurological disease terminated in a postmortem diagnosed atypical HSV-1 encephalitis after high-dose steroid therapy - a case report.

BACKGROUND: Human encephalitis can originate from a variety of different aetiologies, of which infection is the most common one. The diagnostic work-up is specifically challenging in patients with travel history since a broader spectrum of unfamiliar additional infectious agents, e. g. tropical disease pathogens, needs to be considered. Here we present a case of encephalitis of unclear aetiology in a female traveller returning from Africa, who in addition developed an atypical herpes simplex virus (HSV) encephalitis in close temporal relation with high-dose steroid treatment. CASE PRESENTATION: A previously healthy 48-year-old female presented with confusion syndrome and impaired vigilance which had developed during a six-day trip to The Gambia. The condition rapidly worsened to a comatose state. Extensive search for infectious agents including a variety of tropical disease pathogens was unsuccessful. As encephalitic signs persisted despite of calculated antimicrobial and antiviral therapy, high-dose corticosteroids were applied intravenously based on the working diagnosis of an autoimmune encephalitis. The treatment did, however, not improve the patient's condition. Four days later, bihemispheric signal amplification in the insular and frontobasal cortex was observed on magnetic resonance imaging (MRI). The intracranial pressure rapidly increased and could not be controlled by conservative treatment. The patient died due to tonsillar herniation 21 days after onset of symptoms. Histological examination of postmortem brain tissue demonstrated a generalized lymphocytic meningoencephalitis. Immunohistochemical reactions against HSV-1/2 indicated an atypical manifestation of herpesviral encephalitis in brain tissue. Moreover, HSV-1 DNA was detected by a next-generation sequencing (NGS) metagenomics approach. Retrospective analysis of cerebrospinal fluid (CSF) and serum samples revealed HSV-1 DNA only in specimens one day ante mortem. CONCLUSIONS: This case shows that standard high-dose steroid therapy can contribute to or possibly even trigger fulminant cerebral HSV reactivation in a critically ill patient. Thus, even if extensive laboratory diagnostics including wide-ranging search for infectious pathogens has been performed before and remained without results, continuous re-evaluation of potential differential diagnoses especially regarding opportunistic infections or reactivation of latent infections is of utmost importance, particularly if new symptoms occur.

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.

Differentiation of Staphylococcus argenteus (formerly: Staphylococcus aureus clonal complex 75) by mass spectrometry from S. aureus using the first strain isolated from a wild African great ape.

The species Staphylococcus argenteus was separated recently from Staphylococcus aureus (Tong S.Y., F. Schaumburg, M.J. Ellington, J. Corander, B. Pichon, F. Leendertz, S.D. Bentley, J. Parkhill, D.C. Holt, G. Peters, and P.M. Giffard, 2015). The objective of this work was to characterise the genome of a non-human S. argenteus strain, which had been isolated from the faeces of a wild-living western lowland gorilla in Gabon, and analyse the spectrum of this species in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The full genome sequence revealed a scarcity of virulence genes and absence of resistance genes, indicating a decreased virulence potential compared to S. aureus and the human methicillin-resistant S. argenteus isolate MSHR1132T. Spectra obtained by MALDI-TOF MS and the analysis of available sequences in the genome databases identified several MALDI-TOF MS signals that clearly differentiate S. argenteus, the closely related Staphylococcus schweitzeri and S. aureus. In conclusion, in the absence of biochemical tests that identify the three species, mass spectrometry should be employed as method of choice.

Human genetic factors in tuberculosis: an update.

Tuberculosis (TB) is a major threat to human health, especially in many developing countries. Human genetic variability has been recognised to be of great relevance in host responses to Mycobacterium tuberculosis infection and in regulating both the establishment and the progression of the disease. An increasing number of candidate gene and genome-wide association studies (GWAS) have focused on human genetic factors contributing to susceptibility or resistance to TB. To update previous reviews on human genetic factors in TB we searched the MEDLINE database and PubMed for articles from 1 January 2014 through 31 March 2017 and reviewed the role of human genetic variability in TB. Search terms applied in various combinations were 'tuberculosis', 'human genetics', 'candidate gene studies', 'genome-wide association studies' and 'Mycobacterium tuberculosis'. Articles in English retrieved and relevant references cited in these articles were reviewed. Abstracts and reports from meetings were also included. This review provides a recent summary of associations of polymorphisms of human genes with susceptibility/resistance to TB.

A Mutation in IL4RA Is Associated with the Degree of Pathology in Human TB Patients.

The contribution of interleukin- (IL-) 4 receptor-alpha- (Rα-) dependent events in the pathogenesis of tuberculosis (TB) is controversial. We have recently shown IL-13 overexpression in mice to cause recrudescent Mtb replication and centrally necrotizing granulomas strongly resembling pathology of human TB. A deletion of IL-4Rα completely abrogates TB tissue pathology in these mice. To validate our results in human TB patients, we here determined the association of distinct variants of the IL4, IL13, IL4RA, IL13RA1, and IL13RA2 genes with cavity formation in a large Ghanaian cohort of HIV-negative individuals with newly diagnosed pulmonary TB. In fact, the structural variant of the IL4RA I50V, previously shown to result in enhanced signal transduction, was significantly associated with greater cavity size, and a variant of IL13RA2 was associated with disease in females. To evaluate whether the human-like TB pathology in IL-13-overexpressing mice is specifically mediated through the IL-4Rα subunit, we analyzed IL-13 transgenic mice with a genetic ablation of the IL-4Rα. In these mice, the IL-13-mediated increased susceptibility, human-like pathology of collagen deposition around centrally necrotizing granulomas, and alternative macrophage activation were abolished. Together, our genetic association study in human TB patients further supports the assumption that IL-13/IL-4Rα-dependent mechanisms are involved in mediating tissue pathology of human TB.

Molecular characterization of antibiotic resistant Salmonella enterica across the poultry production chain in Costa Rica: A cross-sectional study.

Antibiotic resistant Salmonella enterica are on the increase, worldwide. Given the scarcity of data, this study aimed to investigate its occurrence, virulence, and antibiotic resistance in Costa Rica's food chain. In total, 65 chicken meat- and 171 chicken caecal samples were collected and examined for Salmonella. High frequencies of Salmonella were found in chicken meat (58.5 %, n/N = 38/65) and poultry farms (38.0 %, n/N = 65/171). The majority of Salmonella from chicken meat (89.5 %, n/N = 34/38) and caecum samples (93.6 %, n/N = 59/63) exhibited multidrug resistance (MDR). Serovar Infantis was the most prevalent (94 %, n/N = 67/71), followed by serovars Anatum and Kentucky (3 %, n/N = 2/71). A pESI-like plasmid (92 %, n/N = 65/71) containing virulence and resistance markers was found in S. Infantis. Given the high prevalence of MDR Salmonella, this study emphasizes the need to enhance surveillance systems for foodborne pathogens and antimicrobial resistance in Costa Rica's food production chain.

Molecular Characterization and Phylogenetic Analysis of Dengue Fever Viruses in Three Outbreaks in Tanzania Between 2017 and 2019.

BACKGROUND: Dengue is a disease of public health interest, and Tanzania experienced major outbreaks in 2014 and 2019. Here, we report our findings on the molecular characterization of dengue viruses (DENV) that circulated during two smaller outbreaks (2017 and 2018) and one major epidemic (2019) in Tanzania. METHODOLOGY/PRINCIPAL FINDINGS: We tested archived serum samples from 1,381 suspected dengue fever patients, with a median age of 29 (IQR:22-40) years, referred to the National Public Health Laboratory for confirmation of DENV infection. DENV serotypes were identified by reverse transcription polymerase chain reaction (RT-PCR), and specific genotypes were identified by sequencing the envelope glycoprotein gene and phylogenetic inference methods. DENV was confirmed in 823 (59.6%) cases. More than half (54.7%) of patients with dengue fever infection were males, and nearly three-quarters (73%) of the infected individuals were living in Kinondoni district, Dar es Salaam. DENV-3 Genotype III caused the two smaller outbreaks in 2017 and 2018, while DENV-1 Genotype V caused the 2019 epidemic. DENV-1 Genotype I was also detected in one patient in 2019. CONCLUSION/SIGNIFICANCE: This study has demonstrated the molecular diversity of dengue viruses circulating in Tanzania. We found that contemporary circulating serotypes did not cause the major epidemic of 2019 but rather due to a serotype shift from DENV-3 (2017/2018) to DENV-1 in 2019. Such a change increases the risk for patients previously infected with a particular serotype to develop severe symptoms upon potential re-infection with a heterologous serotype due to antibody-dependent enhancement of infection. Therefore, the circulation of serotypes emphasizes the need to strengthen the country's dengue surveillance system for better management of patients, early detection of outbreaks, and vaccine development.

Prediction of Antibiotic Susceptibility Profiles of Vibrio cholerae Isolates From Whole Genome Illumina and Nanopore Sequencing Data: CholerAegon.

During the last decades, antimicrobial resistance (AMR) has become a global public health concern. Nowadays multi-drug resistance is commonly observed in strains of Vibrio cholerae, the etiological agent of cholera. In order to limit the spread of pathogenic drug-resistant bacteria and to maintain treatment options the analysis of clinical samples and their AMR profiles are essential. Particularly, in low-resource settings a timely analysis of AMR profiles is often impaired due to lengthy culturing procedures for antibiotic susceptibility testing or lack of laboratory capacity. In this study, we explore the applicability of whole genome sequencing for the prediction of AMR profiles of V. cholerae. We developed the pipeline CholerAegon for the in silico prediction of AMR profiles of 82 V. cholerae genomes assembled from long and short sequencing reads. By correlating the predicted profiles with results from phenotypic antibiotic susceptibility testing we show that the prediction can replace in vitro susceptibility testing for five of seven antibiotics. Because of the relatively low costs, possibility for real-time data analyses, and portability, the Oxford Nanopore Technologies MinION sequencing platform-especially in light of an upcoming less error-prone technology for the platform-appears to be well suited for pathogen genomic analyses such as the one described here. Together with CholerAegon, it can leverage pathogen genomics to improve disease surveillance and to control further spread of antimicrobial resistance.

Epidemiology of Plasmids in Escherichia coli and Klebsiella pneumoniae with Acquired Extended Spectrum Beta-Lactamase Genes Isolated from Chronic Wounds in Ghana.

Little information is available on the local epidemiology of mobile genetic elements such as plasmids harboring acquired beta-lactamase genes in Western African Ghana. In the present study, we screened for plasmids in three Escherichia coli and four Klebsiella pneumoniae isolates expressing extended spectrum beta-lactamases (ESBL) mediated by the blaCTX-M-15 gene from chronically infected wounds of Ghanaian patients. Bacterial isolates were subjected to combined short-read and long-read sequencing to obtain the sequences of their respective plasmids. In the blaCTX-M-15-gene-carrying plasmids of the four ESBL-positive K. pneumoniae isolates, IncFIB/IncFII (n = 3) and FIA (n = 1) sequences were detected, while in the blaCTX-M-15-gene-carrying plasmids of the three ESBL-positive E. coli isolates, IncFIA/IncFIB (n = 2) and IncFIB (n = 1) sequences were found. The three IncFIB/IncFII sequence-containing plasmids were almost identical to a K. pneumoniae plasmid reported from France. They belonged to the clonal lineages ST17, ST36 and ST39 of K. pneumoniae, suggesting transversal spread of this obviously evolutionary successful plasmid in Ghana. Other resistance gene-encoding plasmids observed in the assessed Enterobacterales harbored IncFIA/IncR and IncFII sequences. International spread was confirmed by the high genetic similarity to resistance-mediating plasmids published from Asia, Australia, Europe and Northern America, including a blaCTX-M-15-gene-carrying plasmid isolated from a wild bird in Germany. In conclusion, the study contributed to the scarcely available information on the epidemiology of third-generation cephalosporine resistance-mediating plasmids in Ghana. Furthermore, the global spread of resistance-mediating plasmids provided hints on the evolutionary success of individual resistance-harboring plasmids by transversal spread among K. pneumoniae lineages in Ghana.

MCP-1 promoter variant -362C associated with protection from pulmonary tuberculosis in Ghana, West Africa.

Current endeavour focuses on human genetic factors that contribute to susceptibility to or protection from tuberculosis (TB). Monocytes are crucial in containing Mycobacterium tuberculosis infection, and the monocyte chemoattractant protein-1 (MCP-1) cytokine plays a role in their recruitment to the site of infection. The G allele of the MCP-1 promoter polymorphism at position -2581 relative to the ATG transcription start codon has been described to be associated in Mexican and Korean TB patients with increased susceptibility to TB. We genotyped this and additional MCP-1 variants in sample collections comprising more than 2000 cases with pulmonary TB and more than 2300 healthy controls and 332 affected nuclear families from Ghana, West Africa, and more than 1400 TB patients and more than 1500 controls from Russia. In striking contrast to previous reports, MCP-1 -2581G was significantly associated with resistance to TB in cases versus controls [odds ratio (OR) 0.81, corrected P-value (P(corr)) = 0.0012] and nuclear families (OR 0.72, P(corr) = 0.04) and not with disease susceptibility, whereas in the Russian sample no evidence of association was found (P = 0.86). Our and other results do not support an association of MCP-1 -2581 with TB. In the Ghanaian population, eight additional MCP-1 polymorphisms were genotyped. MCP-1 -362C was associated with resistance to TB in the case-control collection (OR 0.83, P(corr) = 0.00017) and in the affected families (OR 0.7, P(corr) = 0.004). Linkage disequilibrium (LD) and logistic regression analyses indicate that, in Ghanaians, the effect results exclusively from the MCP-1 -362 variant, whereas the effect of -2581 may in part be explained by its LD with -362.

Autophagy gene variant IRGM -261T contributes to protection from tuberculosis caused by Mycobacterium tuberculosis but not by M. africanum strains.

The human immunity-related GTPase M (IRGM) has been shown to be critically involved in regulating autophagy as a means of disposing cytosolic cellular structures and of reducing the growth of intracellular pathogens in vitro. This includes Mycobacterium tuberculosis, which is in agreement with findings indicating that M. tuberculosis translocates from the phagolysosome into the cytosol of infected cells, where it becomes exposed to autophagy. To test whether IRGM plays a role in human infection, we studied IRGM gene variants in 2010 patients with pulmonary tuberculosis (TB) and 2346 unaffected controls. Mycobacterial clades were classified by spoligotyping, IS6110 fingerprinting and genotyping of the pks1/15 deletion. The IRGM genotype -261TT was negatively associated with TB caused by M. tuberculosis (OR 0.66, CI 0.52-0.84, P(nominal) 0.0009, P(corrected) 0.0045) and not with TB caused by M. africanum or M. bovis (OR 0.95, CI 0.70-1.30. P 0.8). Further stratification for mycobacterial clades revealed that the protective effect applied only to M. tuberculosis strains with a damaged pks1/15 gene which is characteristic for the Euro-American (EUAM) subgroup of M. tuberculosis (OR 0.63, CI 0.49-0.81, P(nominal) 0.0004, P(corrected) 0.0019). Our results, including those of luciferase reporter gene assays with the IRGM variants -261C and -261T, suggest a role for IRGM and autophagy in protection of humans against natural infection with M. tuberculosis EUAM clades. Moreover, they support in vitro findings indicating that TB lineages capable of producing a distinct mycobacterial phenolic glycolipid that occurs exclusively in strains with an intact pks1/15 gene inhibit innate immune responses in which IRGM contributes to the control of autophagy. Finally, they raise the possibility that the increased frequency of the IRGM -261TT genotype may have contributed to the establishment of M. africanum as a pathogen in the West African population.

MCP1 haplotypes associated with protection from pulmonary tuberculosis.

BACKGROUND: The monocyte chemoattractant protein 1 (MCP-1) is involved in the recruitment of lymphocytes and monocytes and their migration to sites of injury and cellular immune reactions. In a Ghanaian tuberculosis (TB) case-control study group, associations of the MCP1 -362C and the MCP1 -2581G alleles with resistance to TB were recently described. The latter association was in contrast to genetic effects previously described in study groups originating from Mexico, Korea, Peru and Zambia. This inconsistency prompted us to further investigate the MCP1 gene in order to determine causal variants or haplotypes genetically and functionally. RESULTS: A 14 base-pair deletion in the first MCP1 intron, int1del554-567, was strongly associated with protection against pulmonary TB (OR=0.84, CI 0.77-0.92, Pcorrected=0.00098). Compared to the wildtype combination, a haplotype comprising the -2581G and -362C promoter variants and the intronic deletion conferred an even stronger protection than did the -362C variant alone (OR=0.78, CI 0.69-0.87, Pnominal=0.00002; adjusted Pglobal=0.0028). In a luciferase reporter gene assay, a significant reduction of luciferase gene expression was observed in the two constructs carrying the MCP1 mutations -2581 A or G plus the combination -362C and int1del554-567 compared to the wildtype haplotype (P=0.02 and P=0.006). The associated variants, in particular the haplotypes composed of these latter variants, result in decreased MCP-1 expression and a decreased risk of pulmonary TB. CONCLUSIONS: In addition to the results of the previous study of the Ghanaian TB case-control sample, we have now identified the haplotype combination -2581G/-362C/int1del554-567 that mediates considerably stronger protection than does the MCP1 -362C allele alone (OR=0.78, CI 0.69-0.87 vs OR=0.83, CI 0.76-0.91). Our findings in both the genetic analysis and the reporter gene study further indicate a largely negligible role of the variant at position -2581 in the Ghanaian population studied.

A functional haplotype in the 3'untranslated region of TNFRSF1B is associated with tuberculosis in two African populations.

RATIONALE: Susceptibility to tuberculosis is not only determined by Mycobacterium tuberculosis infection, but also by the genetic component of the host. The pleiotropic cytokine tumor necrosis factor-alpha is essential to control tuberculosis infection, and various tumor necrosis factor family members and their respective receptors may contribute to tuberculosis risk. OBJECTIVES: To investigate four functionally relevant polymorphisms in the tumor necrosis factor receptor 2-encoding gene, tumor necrosis factor receptor superfamily member 1B, for association with tuberculosis susceptibility. METHODS: Genotyping of four polymorphisms was performed in independent populations from South Africa (429 cases and 482 control subjects) and Ghana (640 cases and 1,158 control subjects), and the association of the variants with tuberculosis was tested using two case-control association studies. MEASUREMENTS AND MAIN RESULTS: Single-point and haplotype analysis in South Africans revealed an association in the 3'untranslated region of the investigated gene. The T allele of rs3397 alone and/or the 3' untranslated region haplotype GTT may confer protection against tuberculosis insofar as both allele and haplotype frequencies were significantly lower in case subjects than in controls. The GTT genotype had previously been shown to increase the decay of tumor necrosis factor receptor 2 messenger ribonucleic acid, and messenger ribonucleic acid destabilization may represent a key molecular mechanism for disease susceptibility. Interestingly, the association signal appeared to be restricted to women. The genetic finding was validated in female participants from Ghana. The combined P value in the haplotype analysis was P = 0.00011. CONCLUSIONS: Our finding emphasizes the importance of tumor necrosis factor/tumor necrosis factor receptor-mediated immune responses in the pathogenesis of tuberculosis.

Clonal Clusters, Molecular Resistance Mechanisms and Virulence Factors of Gram-Negative Bacteria Isolated from Chronic Wounds in Ghana.

Wound infections are common medical problems in sub-Saharan Africa but data on the molecular epidemiology are rare. Within this study we assessed the clonal lineages, resistance genes and virulence factors of Gram-negative bacteria isolated from Ghanaian patients with chronic wounds. From a previous study, 49 Pseudomonas aeruginosa, 21 Klebsiellapneumoniae complex members and 12 Escherichia coli were subjected to whole genome sequencing. Sequence analysis indicated high clonal diversity with only nine P. aeruginosa clusters comprising two strains each and one E. coli cluster comprising three strains with high phylogenetic relationship suggesting nosocomial transmission. Acquired beta-lactamase genes were observed in some isolates next to a broad spectrum of additional genetic resistance determinants. Phenotypical expression of extended-spectrum beta-lactamase activity in the Enterobacterales was associated with blaCTX-M-15 genes, which are frequent in Ghana. Frequently recorded virulence genes comprised genes related to invasion and iron-uptake in E. coli, genes related to adherence, iron-uptake, secretion systems and antiphagocytosis in P. aeruginosa and genes related to adherence, biofilm formation, immune evasion, iron-uptake and secretion systems in K. pneumonia complex. In summary, the study provides a piece in the puzzle of the molecular epidemiology of Gram-negative bacteria in chronic wounds in rural Ghana.

Low Sensitivity of Real Time PCRs Targeting Retrotransposon Sequences for the Detection of Schistosoma japonicum Complex DNA in Human Serum.

While hybridization probe-based real-time PCR assays targeting highly repetitive multi-copy genome sequences for the diagnosis of S. mansoni complex or S. haematobium complex from human serum are well established, reports on the evaluation of respective assays for the identification of S. japonicum complex DNA in human serum are scarce. Here, we assessed the potential use of the retrotransposon sequences SjR2 and SjCHGCS19 from S. japonicum, S. mekongi and S. malayensis for the diagnosis of Asian Schistosoma infections. Based on available S. japonicum sequences and newly provided S. mekongi and S. malayensis sequences, hybridization probe-based real-time PCRs targeting SjR2 and SjCHGCS19 of the S. japonicum complex were designed both as consensus primer assays as well as multi-primer assays for the coverage of multiple variants of the target sequences. The assays were established using plasmids and S. mekongi DNA. While the consensus primer assays failed to detect S. mekongi DNA in human serum samples, the multi-primer assays showed positive or borderline positive results but only in 9.8% (6/61) of serum samples from patients with confirmed S. mekongi infections. Some cross-reactions with samples positive for S. mansoni or S. haematobium were observed but with the SjCHGCS19-PCR only. In spite of the low sensitivity, the presented experience may guide future evaluations of S. japonicum-complex-specific PCRs from human serum.

Common virulence gene expression in adult first-time infected malaria patients and severe cases.

Sequestration of Plasmodium falciparum(P. falciparum)-infected erythrocytes to host endothelium through the parasite-derived P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins is central to the development of malaria pathogenesis. PfEMP1 proteins have diversified and expanded to encompass many sequence variants, conferring each parasite a similar array of human endothelial receptor-binding phenotypes. Here, we analyzed RNA-seq profiles of parasites isolated from 32 P. falciparum-infected adult travellers returning to Germany. Patients were categorized into either malaria naive (n = 15) or pre-exposed (n = 17), and into severe (n = 8) or non-severe (n = 24) cases. For differential expression analysis, PfEMP1-encoding var gene transcripts were de novo assembled from RNA-seq data and, in parallel, var-expressed sequence tags were analyzed and used to predict the encoded domain composition of the transcripts. Both approaches showed in concordance that severe malaria was associated with PfEMP1 containing the endothelial protein C receptor (EPCR)-binding CIDRα1 domain, whereas CD36-binding PfEMP1 was linked to non-severe malaria outcomes. First-time infected adults were more likely to develop severe symptoms and tended to be infected for a longer period. Thus, parasites with more pathogenic PfEMP1 variants are more common in patients with a naive immune status, and/or adverse inflammatory host responses to first infections favor the growth of EPCR-binding parasites.