Hepatitis C virus among blood donors in Lubumbashi, DRC: Seroprevalence and molecular characterisation.

OBJECTIVES: To date, no study has been done yet on the distribution of Hepatitis C virus genotypes in Lubumbashi, Democratic Republic of Congo. The objective of this work was to determine the seroprevalence and study the distribution of hepatitis C virus (HCV) genotypes among blood donors in Lubumbashi, DRC. METHODS: This was a cross-sectional descriptive study among blood donors. The detection of anti-HCV antibodies was carried out by rapid diagnostic test (RDT) then confirmed by Chemiluminescent immuno-assay (CLIA). Viral load was determined by Nucleic Acid Amplification test (NAT) on Panther system and genotyping by Next Generation Sequencing (NGS) on Sentosa platform. RESULTS: The obtained seroprevalence was 4.8%. Genotypes 3a (5.0%), 4 (90.0%) and 7 (5.0%) and a few drug resistance mutations were identified in the study population. Significant disturbances of some studied biochemical parameters (HDL-cholesterol, direct bilirubin, transaminases, ALP, GGT and albumin) have been observed in positive HCV blood donors. Irregular family and volunteer donors have been found as the socio-demographic characteristics associated with hepatitis C. CONCLUSION: With a seroprevalence of 4.8% obtained among blood donors, Lubumbashi is in an area with medium endemicity for HCV, highlighting the need to implement strategies aiming to improve transfusion safety among blood recipients in Lubumbashi. This study reports for the first time the presence of HCV strains of genotypes 3a, 4 and 7. These results might allow better therapeutic management of HCV infections and contribute to the development of the mapping of HCV genotypes in Lubumbashi and DRC as well.

Inhibition by EGTA of the formation of a biofilm by clinical strains of Staphylococcus aureus.

The effect of EGTA on the adhesion and on the formation of a biofilm by two reference and eight clinical strains of Staphylococcus aureus was studied. All the clinical strains were isolated from patients from Kinshasa. Spa typing confirmed that these clinical strains were distinct. The Biofilm Ring Test (BFRT®) showed that EGTA (100 µM-10 mM) inhibited the adhesion of the four clinical methicillin-resistant (MRSA) strains and the crystal violet staining method that it inhibited the formation of a biofilm by all the strains. Divalent cations abolished the effect of EGTA on the formation of a biofilm, specially in the clinical MRSA strains. EGTA had no effect on established biofilms. Only concentrations of EGTA higher than 10 mM were toxic to eukaryotic cells. Our results establish the effectiveness and the safety of lock solutions with EGTA to prevent the formation in vitro of biofilms by S. aureus.

Evaluation de la sensibilite aux antibiotiques des mycobacteries a croissance rapide

La sensibilite aux antibiotiques des Mycobacteries a Croissance Rapide (MCR) par la methode de microdilution en milieu liquide; et la mesure de la repetabilite des CMI ont ete evaluees sur 15 souches cliniques de mycobacteries a croissance rapide (4 Mycobacterium chelonae; 6 Mycobacteriun abscessus; 3 Mycobacteriun fortuitum; et 2 Mycobacterium peregrinum). Les souches de reference Staphylococcus aureus ATCC 29213 et Mycobacterium peregrinum ATCC 700686 ont ete utilisees pour le controle de qualite. Les resultats ont montre que le controle de qualite etaitacceptable car les valeurs obtenues etaient comprises dans la gamme de Concentrations Minimales Inhibitrices (CMI) proposee par le Clinical and Laboratory Standard Institute (CLSI). Une repetabilite des valeurs de CMI a ete observee. L'amikacine et la clarithromycine etaient les antibiotiques les plus actifs sur presque toutes les MCR etudiees. La tobramycine etait active exclusivement sur M. chelonae (100sensibles) et les fluoroquinolones (Ciprofloxacine et moxifloxacine) sur M. fortuitum (100de sensibilite). Il n'a pas ete observe de correlation entre la methode de microdilution en milieu liquide et celle de Canetti

Study of the formation of a biofilm by clinical strains of Staphylococcus aureus.

A study on biofilm formation was carried out using five methicillin-sensitive [MSSA] and five methicillin-resistant [MRSA] strains of S. aureus. In each group, there were four strains isolated from patients from Kinshasa (Democratic Republic of Congo, DRC) and one reference strain. All of the strains were hydrophobic. The adherence of the bacteria to an abiotic surface was studied with the Biofilm Ring Test (BFRT®) and the crystal violet staining method (CVSM). Both techniques showed that eight of the strains formed biofilms within 2-3 h. The extent of the biofilm formed by one strain could only be observed with the CVSM. Periodate prevented the formation of biofilms and, in separate experiments, destroyed the biofilm pre-formed by the MSSA reference, but not those pre-formed by the clinical strains. Proteinase K destroyed all pre-formed biofilms. Six of the strains were icaA+; the clinical MSSA strains were not. The results also indicated different mechanisms of biofilm development between MSSA and MRSA clinical strains. The BFRT® and the CVSM are complementary techniques to study the adhesion of bacteria and the development of biofilms.

Epidemiologie moleculaire des betalactamases a spectre elargi produites par des Enterobacteriaceae d'origine fecale isolees chez les habitants de residences estudiantines a l'Universite de Kinshasa (RDC)

Objectif : Determiner l'epidemiologie moleculaire des Enterobacteriaceae productrices des betalactamases a spectre elargi (E-BLSE) chez les habitants de residence estudiantines a l'Universite de Kinshasa. Methodes : Des echantillons de selles preleves chez 516 etudiants ont ete examines pendant la periode du 15 novembre 2005 au 30 avril 2006. A l'aide de la galerie API 20E; nous avons pu identifier les differentes souches d'enterobacteries. La production de BLSE a ete recherchee par le test de synergie en double disque; puis confirmee et caracterisee par la focalisation isoelectrique; la PCR et le sequencage des genes de resistance. Un questionnaire a permis de recueillir les informations sur la demographie et les antecedents d'antibiotherapie des sujets inclus dans l'etude. Resultats : La frequence des E- BLSE etait de 17;8chez ces etudiants. Aucune correlation n'a ete notee entre un antecedent d'antibiotherapie et la presence d'E-BLSE. Parmi les E-BLSE isolees; Escherichia coli etait l'espece majoritaire (65); suivi de Klebsiella pneumoniae (26) etn d'Enterobacter cloaceae (5;4). CTX-M-15 etait l'ESBL predominante (29); suivie de CTX-M-28 (19;6); TEM- 68 (16;8); TEM-104 (9;3); CTX-M-3 (9;3); CTX-M-n 22 (4;7) ; SHV-12 (4;7); TEM-168 (1;9); TEM-144 (0;9); SHV-5 (0;9); SHV-2 (0;9); CTX-M-34 (0;9); CTX-M-62 (0;9). CTX-M-15 etait presente dans toutes les souches d'Escherichia coli isolees. Conclusion : Cette etude est; a notre connaissance; la premiere sur l'epidemiologie et la caracterisation des BLSE en RDC. La frequence des E-BLSE dans les residences estudiantines de l'Universite de Kinshasa; ainsi que la presence d'une grande variete de BLSE; justifieraient l'extension de ce type d'enquete dans la communaute et en milieu hospitalier; afin d'evaluer l'ampleur reelle du probleme et de definir des strategies adequates de pharmacovigilance et de lutte contre les bacteries multiresistantes aux antibiotiques

Antibacterial activity of the essential oil of Cymbopogon densiflorus.

The essential oil of Cymbopogon densiflorus showed a wide spectrum of activity against Gram-positive and Gram-negative bacteria.

Immunochemical localization of CAMP factor (protein B) in Streptococcus agalactiae.

Biochemical and immunochemical investigations were used in order to study the quantitative and qualitative localization of CAMP factor (protein B) in the cell fractions of Streptococcus agalactiae during the logarithmic growth phase. The dynamic quantitative distribution of CAMP factor activity showed that higher concentrations of CAMP factor were found in the cytoplasm than in the cell envelopes. A maximal intracellular accumulation of CAMP factor activity was observed in the late log phase. Immunoblotting analysis using specific anti-CAMP-IgG showed that CAMP factor could be detected in the different cell fractions of S. agalactiae. During the early log phase, CAMP factor was present as a single 25 kD protein band in the cytoplasm; white it was found together with its 26 and 24 kD satellite proteins in the cytoplasmic membrane and the cell wall as well as in all the cell fractions in the mid- and late log phases. Intracellular CAMP factor exhibited the same antigenic and amphiphilic behaviour as the extracellular species. Additionally, a newly discovered amphiphilic protein of approximately 54 kD which exhibited similar antigenicity with the CAMP factor was present in all the cell fractions. Immunoelectron microscopic examinations using ferritin-labelled antibodies revealed that CAMP factor was mainly found in the cytoplasm, whereas it was associated to a minor extent with the cell envelopes. Interestingly, an accumulation of CAMP factor was also localized either at the sites of cross-wall initiation or at the cell surfaces where the cell wall became autolysed.

Microcalorimetric and electron microscopic investigation on the effects of essential oil from Cymbopogon densiflorus on Staphylococcus aureus.

Microcalorimetry, optical density measurements and electron microscopy, were used to assess the influence of various amounts of the essential oil of Cymbopogon densiflorus (lemongrass oil) on the metabolic activity, growth and morphology of Staphylococcus aureus. Relatively high concentrations of the oil impaired staphylococcal growth in a bacteriostatic manner (chloramphenicol type), and in low doses metabolism became ineffective due to energy losses in the form of heat. Ultrastructural data revealed morphological changes characteristic for the influence of bactericidal antibiotics inducing bacteriolysis (penicillin type). The essential oil may have antibacterial activity by influencing bacterial targets involved in cytoplasmic and cell wall metabolism.

Effects of secretion inhibitors on the production of CAMP factor from Streptococcus agalactiae.

Investigations of exopeptide secretion with inhibitors were performed to study the synthesis and release of CAMP factor in drug-treated growing cells of Streptococcus agalactiae. Besides a reduction in cell growth, membrane-active substances including cerulenin and neuroactive drugs, such as procaine, dibucaine and atropine, increased the CAMP factor activity in culture supernatant. Quinacrine and phenylmethylsulphonyl fluoride, inhibitors of exopeptide-releasing proteases, reduced the bacterial growth, but did not affect the differential rate of the CAMP factor release. Polyanethole sulphonic acid, an anticoagulant preventing cell wall autolysis, promoted cell growth, but caused approximately 40% reduction in the production of CAMP factor from growing cells of S. agalactiae.

Electron microscopic and microcalorimetric investigations of the possible mechanism of the antibacterial action of a defined propolis provenance.

Microcalorimetric and electron microscopic studies on the mode of the antibacterial action of propolis were performed on Streptococcus agalactiae. It was shown that propolis inhibits bacterial growth by preventing cell division, thus resulting in the formation of pseudo-multicellular streptococci. In addition, propolis disorganized the cytoplasm, the cytoplasmic membrane, and the cell wall, caused a partial bacteriolysis, and inhibited protein synthesis. It was evident that the mechanism of action of propolis on bacterial cells is complex and a simple analogy cannot be made to the mode of action of any classic antibiotics.

Flow microcalorimetry investigation of the influence of glucose and maltose on the growth of Streptococcus agalactiae and the production of CAMP factor (protein B).

Microcalorimetric investigations of the growth of Streptococcus agalactiae and production of CAMP factor were performed in combination with the photometric estimation of bacterial mass and the bioluminescent measurement of extracellular ATP in trypticase peptone-yeast extract broth supplemented with either 2% maltose or glucose. Maltose was found to be a more efficient energy substrate than glucose, providing more energy and C-atoms to the growing cells. In the presence of maltose, the metabolic activity of S. agalactiae was more efficient and better balanced than in that of glucose. Bacterial cells growing in maltose-containing medium used more substrate energy for their anabolic activities, while cells growing in the presence of glucose lost more substrate energy in the form of heat. The addition of 0.2% NaHCO3 to the carbohydrate-supplemented medium enhanced the efficiency of the anabolic activity of growing cells, but did not promote bacterial growth. Microcalorimetry should be considered as a useful alternative as well as a complementary method for the optimization of the growth media or growth conditions.