Genomic Characterization of Multidrug-Resistant Extended Spectrum ß-Lactamase-Producing Klebsiella pneumoniae from Clinical Samples of a Tertiary Hospital in South Kivu Province, Eastern Democratic Republic of Congo.

Multidrug-resistant (MDR) and extended spectrum ß-lactamase (ESBL)-producing extra-intestinal K. pneumoniae are associated with increased morbidity and mortality. This study aimed to characterize the resistance and virulence profiles of extra-intestinal MDR ESBL-producing K. pneumoniae associated with infections at a tertiary hospital in South-Kivu province, DRC. Whole-genome sequencing (WGS) was carried out on 37 K. pneumoniae isolates displaying MDR and ESBL-producing phenotype. The assembled genomes were analysed for phylogeny, virulence factors and antimicrobial resistance genes (ARG) determinants. These isolates were compared to sub-Saharan counterparts. K. pneumoniae isolates displayed a high genetic variability with up to 16 sequence types (ST). AMR was widespread against ß-lactamases (including third and fourth-generation cephalosporins, but not carbapenems), aminoglycosides, ciprofloxacin, tetracycline, erythromycin, nitrofurantoin, and cotrimoxazole. The blaCTX-M-15 gene was the most common ß-lactamase gene among K. pneumoniae isolates. No carbapenemase gene was found. ARG for aminoglycosides, quinolones, phenicols, tetracyclines, sulfonamides, nitrofurantoin were widely distributed among the isolates. Nine isolates had the colistin-resistant R256G substitution in the pmrB efflux pump gene without displaying reduced susceptibility to colistin. Despite carrying virulence genes, none had hypervirulence genes. Our results highlight the genetic diversity of MDR ESBL-producing K. pneumoniae isolates and underscore the importance of monitoring simultaneously the evolution of phenotypic and genotypic AMR in Bukavu and DRC, while calling for caution in administering colistin and carbapenem to patients.

Optimized DNA-based identification of Toxocara spp. eggs in soil and sand samples.

BACKGROUND: Toxocara canis and Toxocara cati are globally distributed roundworms and causative agents of human toxocariasis, via ingestion of Toxocara eggs. Control of Toxocara infections is constrained by a lack of sensitive methods for screening of animal faeces and environmental samples potentially contaminated by Toxocara eggs. In this work, a pre-analytical method for efficient extraction of DNA from Toxocara eggs in environmental samples was set up using our previously validated T. canis- and T. cati-specific quantitative real-time polymerase chain reaction (qPCR). For this purpose, the influence of different methods for egg lysis, DNA extraction and purification for removal of PCR inhibitors were assessed on environmental samples. METHODS: To select the best egg disruption method, six protocols were compared on pure T. canis egg suspensions, including enzymatic lysis and thermal or mechanical disruption. Based on the selected best method, an analytical workflow was set up to compare two DNA extraction methods (FastDNA™ SPIN Kit for Soil versus DNeasy® PowerMax® Soil Kit) with an optional dilution and/or clean-up (Agencourt® AMPure®) step. This workflow was evaluated on 10-g soil and 10-g sand samples spiked with egg suspensions of T. canis (tenfold dilutions of 104 eggs in triplicate). The capacity of the different methods, used alone or in combination, to increase the ratio of positive tests was assessed. The resulting optimal workflow for processing spiked soil samples was then tested on environmental soil samples and compared with the conventional flotation-centrifugation and microscopic examination of Toxocara eggs. RESULTS: The most effective DNA extraction method for Toxocara eggs in soil samples consisted in the combination of mechanical lysis of eggs using beads, followed by DNA extraction with the DNeasy® PowerMax® Soil Kit, and completed with an additional DNA clean-up step with AMPure® beads and a sample DNA dilution (1:10). This workflow exhibited a limit of detection of 4 and 46 T. canis eggs in 10-g sand and 10-g soil samples, respectively. CONCLUSIONS: The pre-analytical flow process developed here combined with qPCR represents an improved, potentially automatable, and cost-effective method for the surveillance of Toxocara contamination in the environment.

Genomic characterisation of extended-spectrum ß-lactamase-producing multidrug-resistant Escherichia coli in Rabat, Morocco.

OBJECTIVES: Extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli are an increasingly significant cause of hospital- and community-acquired infections worldwide. Whereas several reports have highlighted their increased prevalence also in North African countries, genomic data on isolates associated with these infections are still scarce. This study aimed to provide data on ESBL-producing E. coli isolates from patients with extraintestinal infections at the Military Teaching Hospital Mohamed V of Rabat, Morocco. METHODS: Whole-genome sequencing was carried out on 18 ESBL-producing extraintestinal pathogenic E. coli (ExPEC) isolates for analysis of phylogenomic evolution, virulence factors and antimicrobial resistance genes. Data were compared with ExPEC lineages from several surrounding countries using multilocus sequence typing (MLST) and single nucleotide polymorphism-based phylogenetic approaches. RESULTS: The majority of E. coli isolates were ST131 (n = 15), followed by ST617 (n = 2) and a novel sequence type (ST10703) that is closely related to the pandemic ST405 clone. All ST131 isolates belonged to the O25b-ST131 pandemic clone. They harboured more virulence genes than their non-ST131 counterparts. IncF plasmid replicons and the blaCTX-M-15 ß-lactamase gene were identified in all isolates. No ESBL-producing E. coli isolates carried any known carbapenemase gene. CONCLUSION: Our findings underscore the pre-eminence of ST131 as the major factor driving the expansion of ExPEC in the Rabat region while highlighting the potential links with isolates circulating in other neighbouring countries.

Diagnostic Value of IgM and IgG Detection in COVID-19 Diagnosis by the Mobile Laboratory B-LiFE: A Massive Testing Strategy in the Piedmont Region.

Coronavirus disease 2019 (COVID-19) is an acute infectious disease caused by the novel coronavirus (SARS-CoV-2) identified in 2019. The COVID-19 outbreak continues to have devastating consequences for human lives and the global economy. The B-LiFe mobile laboratory in Piedmont, Italy, was deployed for the surveillance of COVID-19 cases by large-scale testing of first responders. The objective was to assess the seroconversion among the regional civil protection (CP), police, health care professionals, and volunteers. The secondary objective was to detect asymptomatic individuals within this cohort in the light of age, sex, and residence. In this paper, we report the results of serological testing performed by the B-LiFe mobile laboratory deployed from 10 June to 23 July 2020. The tests included whole blood finger-prick and serum sampling for detection of SARS-CoV-2 spike receptor-binding domain (S-RBD) antibodies. The prevalence of SARS-CoV-2 antibodies was approximately 5% (294/6013). The results of the finger-prick tests and serum sample analyses showed moderate agreement (kappa = 0.77). Furthermore, the detection rates of serum antibodies to the SARS-CoV-2 nucleocapsid protein (NP) and S-RBD among the seroconverted individuals were positively correlated (kappa = 0.60), at least at the IgG level. Seroprevalence studies based on serological testing for the S-RBD protein or SARS-CoV-2 NP antibodies are not sufficient for diagnosis but might help in screening the population to be vaccinated and in determining the duration of seroconversion.

Genome Sequence of a Pathogenic Vibrio cholerae O1 El Tor Strain Defective for the Entire Vibrio Pathogenicity Island 1, Isolated in Eastern Democratic Republic of the Congo.

We report here a complete genome sequence of a Vibrio cholerae O1 El Tor (Inaba; sequence type 515 [ST515]) strain isolated from a cholera patient in North Kivu Province, Democratic Republic of the Congo (DRC), which showed a complete deletion (∼80 kb) of the Vibrio pathogenicity island 1.

Genomic analysis of pathogenic isolates of Vibrio cholerae from eastern Democratic Republic of the Congo (2014-2017).

BACKGROUND: Over the past recent years, Vibrio cholerae has been associated with outbreaks in sub-Saharan Africa, notably in Democratic Republic of the Congo (DRC). This study aimed to determine the genetic relatedness of isolates responsible for cholera outbreaks in eastern DRC between 2014 and 2017, and their potential spread to bordering countries. METHODS/PRINCIPAL FINDINGS: Phenotypic analysis and whole genome sequencing (WGS) were carried out on 78 clinical isolates of V. cholerae associated with cholera in eastern provinces of DRC between 2014 and 2017. SNP-based phylogenomic data show that most isolates (73/78) were V. cholerae O1 biotype El Tor with CTX-3 type prophage. They fell within the third transmission wave of the current seventh pandemic El Tor (7PET) lineage and were contained in the introduction event (T)10 in East Africa. These isolates clustered in two sub-clades corresponding to Multiple Locus Sequence Types (MLST) profiles ST69 and the newly assigned ST515, the latter displaying a higher genetic diversity. Both sub-clades showed a distinct geographic clustering, with ST69 isolates mostly restricted to Lake Tanganyika basin and phylogenetically related to V. cholerae isolates associated with cholera outbreaks in western Tanzania, whereas ST515 isolates were disseminated along the Albertine Rift and closely related to isolates in South Sudan, Uganda, Tanzania and Zambia. Other V. cholerae isolates (5/78) were non-O1/non-O139 without any CTX prophage and no phylogenetic relationship with already characterized non-O1/non-O139 isolates. CONCLUSIONS/SIGNIFICANCE: Current data confirm the association of both DRC O1 7PET (T)10 sub-clades ST69 and ST515 with recurrent outbreaks in eastern DRC and at regional level over the past 10 years. Interestingly, while ST69 is predominantly a locally endemic sequence type, ST515 became adaptable enough to expand across DRC neighboring countries.

Backward compatibility of whole genome sequencing data with MLVA typing using a new MLVAtype shiny application for Vibrio cholerae.

BACKGROUND: Multiple-Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) is widely used by laboratory-based surveillance networks for subtyping pathogens causing foodborne and water-borne disease outbreaks. However, Whole Genome Sequencing (WGS) has recently emerged as the new more powerful reference for pathogen subtyping, making a data conversion method necessary which enables the users to compare the MLVA identified by either method. The MLVAType shiny application was designed to extract MLVA profiles of Vibrio cholerae isolates from WGS data while ensuring backward compatibility with traditional MLVA typing methods. METHODS: To test and validate the MLVAType algorithm, WGS-derived MLVA profiles of nineteen Vibrio cholerae isolates from Democratic Republic of the Congo (n = 9) and Uganda (n = 10) were compared to MLVA profiles generated by an in silico PCR approach and Sanger sequencing, the latter being used as the reference method. RESULTS: Results obtained by Sanger sequencing and MLVAType were totally concordant. However, the latter were affected by censored estimations whose percentage was inversely proportional to the k-mer parameter used during genome assembly. With a k-mer of 127, less than 15% estimation of V. cholerae VNTR was censored. Preventing censored estimation was only achievable when using a longer k-mer size (i.e. 175), which is not proposed in the SPAdes v.3.13.0 software. CONCLUSION: As NGS read lengths and qualities tend to increase with time, one may expect the increase of k-mer size in a near future. Using MLVAType application with a longer k-mer size will then efficiently retrieve MLVA profiles from WGS data while avoiding censored estimation.

Whole-genome sequences of multidrug-resistant Escherichia coli in South-Kivu Province, Democratic Republic of Congo: characterization of phylogenomic changes, virulence and resistance genes.

BACKGROUND: Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli are responsible for severe infections worldwide. Whereas their genotypic and pathogenic characteristics are not documented in Democratic Republic of Congo (DRC), recent studies conducted at the Bukavu General Hospital in the South Kivu province highlighted their high prevalence in extra-intestinal infections. Here we provide data on molecular characterization of ESBL producing-Escherichia coli isolates from patients with extra-intestinal infections at this provincial hospital. METHODS: Whole-genome sequencing was carried out on 21 of these ESBL-producing Extra-intestinal Pathogenic Escherichia coli (ExPEC) for analysis of phylogenomic evolution, virulence factor and antimicrobial resistance (AMR) genes. Data were compared to phylogenetically close genomes using Multi-Locus Sequence Typing and Single Nucleotide Polymorphism-based phylogenetic approaches. RESULTS: The distribution of E. coli sequence types (ST) was as follows: ST 131 (n = 7), ST405 (n = 4), ST410 (n = 2), and other STs (ST10, ST58, ST95, ST393, ST443, S617, ST648, and ST2450). All ST131 belonged to the O25b-ST131 pandemic clone. Unexpectedly, they harbored more virulence genes than their GenBank counterparts. IncF plasmid replicons included novel FIB 69, FII 105 and FII 107 alleles. ESBL-genes included the plasmid-mediated CTX-M-15 in all isolates, and the SHV-12 allele. Other AMR genes included blaOXA-1, blaTEM-1, as well as genes encoding resistance against aminoglycosides, quinolones, chloramphenicol, rifampicin, tetracyclines, sulfonamides and trimethoprim. CONCLUSION: Current data confirm the clonal spread of ESBL-producing ST131 and ST405 clones in patients from South Kivu, and the acquisition of resistance and virulence genes. A closer survey of AMR and virulence should therefore be prompted in this high-risk area.

Comparative pharmacogenetic analysis of risk polymorphisms in Caucasian and Vietnamese children with acute lymphoblastic leukemia: prediction of therapeutic outcome?

AIMS: Acute lymphoblastic leukemia (ALL) is the most common of all paediatric cancers. Aside from predisposing to ALL, polymorphisms could also be associated with poor outcome. Indeed, genetic variations involved in drug metabolism could, at least partially, be responsible for heterogeneous responses to standardized leukemia treatments, hence requiring more personalized therapy. The aims of this study were to (a) to determine the prevalence of seven common genetic polymorphisms including those that affect the folate and/or thiopurine metabolic pathways, i.e. cyclin D1 (CCND1-G870A), γ-glutamyl hydrolase (GGH-C452T), methylenetetrahydrofolate reductase (MTHFR-C677T and MTHFR-A1298C), thymidylate synthase promoter (TYMS-TSER), thiopurine methyltransferase (TPMT*3A and TPMT*3C) and inosine triphosphate pyrophosphatase (ITPA-C94A), in Caucasian (n = 94, age < 20) and Vietnamese (n = 141, age < 16 years) childhood ALL and (b) to assess the impact of a multilocus genetic risk score (MGRS) on relapse-free survival (RFS) using a Cox proportional-hazards regression model. RESULTS: The prevalence of MTHFR-677TT genotype was significantly higher in Caucasians (P = 0.008), in contrast to the prevalence of TYMS-TSER*3R/3R and ITPA-94AA/AC genotypes which were significantly higher in Vietnamese (P < 0.001 and P = 0.02, respectively). Compared with children with a low MGRS (≤ 3), those with a high MGRS (≥ 4) were 2.06 (95% CI = 1.01, 4.22; P = 0.04) times more likely to relapse. Adding MGRS into a multivariate Cox regression model with race/ethnicity and four clinical variables improved the predictive accuracy of the model (AUC from 0.682 to 0.709 at 24 months). CONCLUSION: Including MGRS into a clinical model improved the predictive accuracy of short and medium term prognosis, hence confirming the association between well determined pharmacogenotypes and outcome of paediatric ALL. Whether variants on other genes associated with folate metabolism can substantially improve the predictive value of current MGRS is not known but deserves further evaluation.

Unambiguous detection of multiple TP53 gene mutations in AAN-associated urothelial cancer in Belgium using laser capture microdissection.

In the Balkan and Taiwan, the relationship between exposure to aristolochic acid and risk of urothelial neoplasms was inferred from the A>T genetic hallmark in TP53 gene from malignant cells. This study aimed to characterize the TP53 mutational spectrum in urothelial cancers consecutive to Aristolochic Acid Nephropathy in Belgium. Serial frozen tumor sections from female patients (n=5) exposed to aristolochic acid during weight-loss regimen were alternatively used either for p53 immunostaining or laser microdissection. Tissue areas with at least 60% p53-positive nuclei were selected for microdissecting sections according to p53-positive matching areas. All areas appeared to be carcinoma in situ. After DNA extraction, mutations in the TP53 hot spot region (exons 5-8) were identified using nested-PCR and sequencing. False-negative controls consisted in microdissecting fresh-frozen tumor tissues both from a patient with a Li-Fraumeni syndrome who carried a p53 constitutional mutation, and from KRas mutated adenocarcinomas. To rule out false-positive results potentially generated by microdissection and nested-PCR, a phenacetin-associated urothelial carcinoma and normal fresh ureteral tissues (n=4) were processed with high laser power. No unexpected results being identified, molecular analysis was pursued on malignant tissues, showing at least one mutation in all (six different mutations in two) patients, with 13/16 exonic (nonsense, 2; missense, 11) and 3/16 intronic (one splice site) mutations. They were distributed as transitions (n=7) or transversions (n=9), with an equal prevalence of A>T and G>T (3/16 each). While current results are in line with A>T prevalence previously reported in Balkan and Taiwan studies, they also demonstrate that multiple mutations in the TP53 hot spot region and a high frequency of G>T transversion appear as a complementary signature reflecting the toxicity of a cumulative dose of aristolochic acid ingested over a short period of time.

Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea) in soil and fecal samples.

BACKGROUND: Toxocarosis is a zoonotic disease caused by Toxocara canis (T. canis) and/or Toxocara cati (T. cati), two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of T. canis or T. cati, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount. METHODS: A duplex quantitative real-time PCR (2qPCR) targeting the ribosomal RNA gene internal transcribed spacer (ITS2) has been developed and used for rapid and specific identification of T. canis and T. cati eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including T. canis, T. cati, T. leonina, Ascaris suum (A. suum) and Parascaris equorum (P. equorum). The assay was used to assess the presence of T. cati eggs in several samples, including 12 clean soil samples spiked with eggs of either T. cati or A. suum, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination. RESULTS: 2qPCR assay allowed specific detection of T. canis and T. cati, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD) in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces. CONCLUSION: The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of T.canis and/or T. cati eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits.

Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples.

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

Real-time PCR and DNA sequencing for detection and identification of Trichophyton rubrum as a cause of culture negative chronic granulomatous dermatophytosis.

A 31-year-old patient presented with a diagnosis of granulomatous dermatophytosis based on the clinical aspect of the lesions and the rare presence of hyphae on direct microscopic examination of clinical material. A chronic evolution and progression of the disease, its resistance to a wide range of antifungal agents, the occasional presence of hyphae on direct examination but consistently negative cultures over a 5-year period prompted the use of amplification-based DNA analyses of several successive swab samples or skin biopsies. DNA was extracted using a combination of two semi-automated DNA isolation methods (FastPrep preparation and NucliSENS lysis magnetic extraction method). Identification relied both on sequence analysis of amplicons after SYBR Green real-time PCR of the panfungal internal transcribed spacer 1 (ITS1) genetic target, as well as the unique amplicon melting curve profile of positive samples. Accordingly, Trichophyton rubrum was unambiguously identified in several clinical samples collected over a 7-month period. This case illustrates the contribution of DNA-based assays applied directly to sample biopsies for identifying causative agents in cases in which fungal pathogens are highly suspected but culture are repeatedly negative. It also pinpoints the benefit of combining semi-automated DNA preparation methods, analysis of ITS1 amplicon melting curve profiles and sequence analysis on repeated skin biopsy samples for unambiguous identification of the causative fungal species.