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.

Predictive Factors of HIV-1 Drug Resistance and Its Distribution among Female Sex Workers in the Democratic Republic of the Congo (DRC).

The predictive factors of HIV-1 drug resistance and its distribution are poorly documented in female sex workers (FSWs) in the Democratic Republic of the Congo (DRC). However, the identification of predictive factors can lead to the development of improved and effective antiretroviral therapy (ART). The objective of the current study was to determine the predictive factors of HIV-1 drug resistance and its distribution based on FSWs in the studied regions in the Democratic Republic of the Congo (DRC). HIV-positive FSWs who were diagnosed as part of the DRC Integrated Biological and Behavioral Surveillance Survey (IBBS) were included in this study. A total of 325 FSWs participated. The HIV-1 viral load (VL) was measured according to the Abbott m2000sp and m2000rt protocols. The homogeneity chi-square test was conducted to determine the homogeneity of HIV-1 drug resistance distribution. Using a significance level of 0.05, multivariate analyses were performed to identify factors associated with HIV-1 drug resistance to ART. HIV drug resistance mutation (HIVDRM) distribution was homogeneous in the three study regions (p = 0.554) but differed based on the HIV-1 VLs of the FSWs. FSWs with high HIV-1 VLs harbored more HIVDRMs (p = 0.028) of predominantly pure HIV-1 strains compared with those that had low HIV-1 VLs. Sexually transmitted infection (STI) history (aOR [95%CI] = 8.51 [1.62, 44.74]), high HIV-1 VLs (aOR [95%CI] = 5.39 [1.09, 26.74]), and HIV-1-syphilis coinfection (aOR [95%CI] = 9.71 [1.84, 51.27]) were associated with HIV drug resistance among FSWs in the DRC. A history of STIs (e.g., abnormal fluid) in the 12 months prior to the survey, a high HIV-1 VL, and HIV-1-syphilis coinfection were associated with HIV-1 drug resistance among FSWs in the DRC. Efforts should be made to systematically test for other infections which increase the HIV-1 VL, in the case of HIV-1 coinfection, in order to maintain ART effectiveness across the DRC.

Spirulina Supplements Improved the Nutritional Status of Undernourished Children Quickly and Significantly: Experience from Kisantu, the Democratic Republic of the Congo.

Aim. Despite high levels of malnutrition, there is still very little information on the nutritional benefits of Spirulina, a natural alga that provides essential amino acids, rare essential lipids, and numerous minerals and vitamins, to undernourished children in the world. Methods. We carried out a prospective study of 50 children aged between six and 60 months. The intervention group consisted of 16 children who received 10 g of Spirulina daily, as well as the local diet administered by the nutritional centre, and the control group of 34 children who just received the local diet. Both groups of children were assessed on day zero, day 15, and day 30. Results. After treatment, the weight-for-age Z scores and weight-for-height Z scores increased significantly in the intervention group. At day 15, there was a statistically significant difference between the mean corpuscular volume, total proteins, and albumin (p < 0.05) in both groups, in favour of the intervention group, and at day 30, this difference extended to all of the studied parameters (p < 0.05). Conclusion. This study found that the nutritional status of undernourished children who received Spirulina supplements as well as the local diet administered by the nutritional centre improved quickly and significantly.

High prevalence of methicillin resistant staphylococci strains isolated from surgical site infections in Kinshasa.

INTRODUCTION: Surgical site infections (SSIs) after surgery are usually caused by Staphylococcus aureus and coagulase-negative staphylococci (CNS). In low income countries, methicillin resistant Staphylococcus aureus (MRSA) and methicillin resistant coagulase-negative staphylococci (MR-CNS) surgical site infections are particularly associated with high treatment cost and remain a source of mortality and morbidity. This study aimed to determine the prevalence and the sensitivity to antibiotics of MRSA and MR-CNS isolated from SSIs. METHODS: Wound swabs were collected from 130 hospitalized surgical patients in two major hospitals of Kinshasa. S. aureus and CNS strains were identified by standard microbiological methods and latex agglutination test (Pastorex Staph-Plus). The antibiotic susceptibility of all staphylococcal strains was carried out using disk-diffusion method. RESULTS: Eighty nine staphylococcal strains were isolated. Out of 74 S. aureus and 15 CNS isolated, 47 (63.5%) and 9 (60%) were identified as MRSA and MR-CNS respectively. Among the MRSA strains, 47 strains (100%) were sensitive to imipenem, 39 strains (89%) to amoxycillin-clavulanic acid and 38 strains (81%) to vancomycin. All MR-CNS were sensitive to imipenem, amoxycillin-clavulanic acid and vancomycin. The isolated MRSA and MR-CNS strains showed multidrug resistance. They were both resistant to ampicillin, cotrimoxazole, erythromycin, clindamycin, ciprofloxacin, cefotaxime and ceftazidime. CONCLUSION: The results of the present study showed a high prevalence of MRSA and MR-CNS. Imipenem, amoxycillin-clavulanic acid and vancomycin were the most active antibiotics. This study suggests that antibiotic surveillance policy should become national priority as MRSA and MR-CNS were found to be multidrug resistant.

Antimicrobial susceptibility patterns of enterobacteriaceae isolated from HIV-infected patients in Kinshasa.

INTRODUCTION: People infected by Human Immunodeficiency Virus (HIV) are susceptible to develop severe bacterial infections. We set out to determine the frequency and the sensitivity to antibiotics of enterobaceriaceae isolated from urine and feces of HIV-infected persons. METHODS: Urine and feces samples were collected from HIV-infected patients of the Centre de Traitement Ambulatoire de Kabinda (CTA/Kabinda, Kinshasa) and analyzed at the Reference National Laboratory for HIV/AIDS and Sexually Transmitted Infections. The isolated enterobacteriaceae strains were identified by conventional microbiological methods. Antibiotic sensitivity pattern was carried out by disc diffusion method. RESULTS: THE FOLLOWING BACTERIA PATHOGENS WERE ISOLATED: Escherichia coli, Klebsiella, Enterobacter, Proteus, and Providencia. Most species were sensitive to cefotaxim, ceftriaxon, and gentamicin and resistant to chloramphenicol, cotrimoxazole, tetracycline, and norfloxacin. CONCLUSION: The results of the present study show that the most frequently bacteria isolated were Esherichia coli and cefotaxim, ceftriaxon, and gentamicin were the most active antibiotics.

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

Production and characterization of Escherichia coli enterohemolysin and its effects on the structure of erythrocyte membranes.

Hemolysins are cell-damaging protein toxins produced by pathogenic bacteria, which are usually released into the extracellular medium. Escherichia coli enterohemolysin is an intracellular toxin produced during the log phase of growth, with a maximal intracellular accumulation in the late log phase. In the present study, we have employed electron microscopy and SDS-PAGE to assess the effects of enterohemolysin on erythocyte membranes from different species. The erythrocyte cell damage began immediately after exposure to enterohemolysin with chemically detectable changes in cell membrane permeability, and the formation of surface lesions which increased rapidly in size. This process resulted in complete cell destruction. Ring-shaped structures with a diameter of 10nm were observed by electron microscopy after treatment of horse erythrocyte membranes with enterohemolysin. The ring structures were found clustered and irregularly distributed on the surface of the membranes. Following incubation of the toxin with horse erythrocyte ghosts and detergent-solubilization, the enterohemolysin was isolated from the cytoplasm in its membrane-bound form by sucrose density gradient. SDS-PAGE and silver staining of deoxycholate-solubilized target membranes revealed heterogeneous forms of the toxin. By using SDS-PAGE and gel filtration, the molecular weight of the toxin was estimated to be 35 kDa. With respect to species specificity, horse erythrocytes showed the highest sensitivity to the enterohemolysin, followed by human and guinea pig erythrocytes. The hemolytic sensitivity correlated with the toxin binding capacity of erythrocyte membranes of different animal species. The degree of hemolysis was unaffected by temperature in the range of 4 degrees C-37 degrees C and was optimal at pH 9.0. In contrast to pore-forming cytolysins, the hemolytic activity of enterohemolysin was enhanced continuously in the presence of increasing concentrations of dextran 4 and dextran 8 within the range of 5 to 30 mM. Trypsin sensitivity of membrane-bound enterohemolysin indicates that the cell surface is the most likely target site for this toxin. Additionally, the fact that proteinase and phosphatase inhibitors failed to inhibit lysis suggests that enterohemolysin alters and disrupts cell membranes by a detergent-like mechanism.

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.