Profiles of opportunistic infections in people living with HIV followed at the Military Hospital of Kinshasa Reference (Camp Kokolo), DRC

peer reviewedContext: The in-house techniques or experimental methods are increasingly recommended for their low-cost reagents for the determination of the Viral Load (VL) in resource-limited settings. The objective of this study was to compare the determination of VL from HIV-1 non-B samples by an in-house technique with the COBAS AmpliPrep/TaqMan version 2.0. Method: In this cross-sectional study, 39 plasma samples from patients infected with HIV type 1 non-B from N’Djamena and Kinshasa were used to determine the VL using the two techniques. Results: The mean values of VL are respectively 4.68 ± 1.26 and 4.58 ± 1.33 log10 RNA copies/ml for the COBAS AmpliPrep/TaqMan assays and the in-house assays. A good correlation (Spearman Correlation) was obtained, with a coefficient (R2) of 0.9452. Conclusion: This study demonstrates that there is no significant difference between the results of VL determined by the COBAS AmpliPrep/TaqMan assays and the in-house assays used

Biennial surveillance of Plasmodium falciparum anti-malarial drug resistance markers in Democratic Republic of Congo, 2017 and 2019

peer reviewedBackground: Because of the loss of chloroquine (CQ) effectiveness, the Democratic Republic of Congo (DRC)’s malaria treatment policy replaced CQ by sulfadoxine–pyrimethamine (SP) as first-line treatment of uncomplicated malaria in 2003, which in turn was replaced by artemisinin-based combination therapies (ACT) in 2005. The World Health Organization (WHO) recommends monitoring of anti-malarial drug resistance every 2 years. The study aimed to provide baseline data for biennial molecular surveillance of anti-malarial drug resistance by comparing data from a study conducted in 2019 to previously published data from a similar study conducted in 2017 in the DRC. Methods: From July to November 2019, a cross-sectional study was conducted in ten sites which were previously selected for a similar study conducted in 2017 across the DRC. P. falciparum malaria was diagnosed by a rapid diagnostic test (RDT) or by microscopy and dried blood samples (DBS) were taken from patients who had a positive test. Segments of interest in pfcrt and pfk13 genes were amplified by conventional PCR before sequencing. Results: Out of 1087 enrolled patients, 906 (83.3%) were PCR-confirmed for P. falciparum. Like in the 2017-study, none of the mutations known to be associated with Artemisinine (ART) resistance in Southeast Asia was detected. However, non-synonymous (NS) mutations with unknown functions were observed among which, A578S was detected in both 2017 and 2019-studies. The overall prevalence of pfcrt-K76T mutation that confers CQ-resistance was 22.7% in 2019-study compared to 28.5% in 2017-study (p-value = 0.069), but there was high variability between sites in the two studies. Like in 2017-study, the pfcrt 72–76 SVMNT haplotype associated with resistance to amodiaquine was not detected. Conclusion: The study reported, within 2 years, the non-presence of molecular markers currently known to be associated with resistance to ART and to AQ in P. falciparum isolated in the DRC. However, the presence of polymorphisms with as-yet unknown functions was observed, requiring further characterization. Moreover, an overall decrease in the prevalence of CQ-resistance marker was observed in the DRC, but this prevalence remained highly variable from region to region. © 2022, The Author(s)

Épidémiologie clinique et grande diversité génétique parmi les isolats de Cryptococcus spp. infectant les personnes vivant avec le VIH à Kinshasa, République démocratique du Congo

Neuromeningeal cryptococcosis (NMC) is a life-threatening opportunistic infection in advanced HIV disease patients (AHDP). It is caused by Cryptococcus spp. complexes and mainly occurs in sub-Saharan Africa. In this study, we performed molecular characterization and antifungal susceptibility profiling of Cryptococcus isolates from AHDP in Kinshasa (DRC). Additionally, we investigated a possible association between NMC severity factors and the Cryptococcus neoformans (Cn) multilocus sequence typing (MLST) profiles. We characterized the isolates using PCR serotyping, MALDI-TOF MS, internal transcribed spacer (ITS) sequencing, and MLST. Susceptibility testing for the major antifungal drugs was performed according to the EUCAST guidelines. Parameters associated with NMC severity, such as hypoglycorrhachia ( 30 cm H2O), and poor therapeutic outcome were compared with the Cn MLST sequences type (ST). Twenty-three out of 29 Cryptococcus isolates were identified as serotype A using PCR serotyping (79.3%; 95% IC: 65.5-93.1), while six (20.7%; 95% IC: 6.9-34.5) were not serotypable. The 29 isolates were identified by ITS sequencing as follows: Cryptococcus neoformans (23/29, 79.3%), Cutaneotrichosporon curvatus (previously called Cryptococcus curvatus) (5/29, 17.2%), and Papiliotrema laurentii (Cryptococcus laurentii) (1/29, 3.5%). Using the ISHAM MLST scheme, all Cn isolates were identified as molecular type VNI. These comprised seven different STs: ST93 (n = 15), ST5 (n = 2), ST53 (n = 1), ST31 (n = 1), ST4 (n = 1), ST69 (n = 1), and one novel ST that has not yet been reported from other parts of the world and was subsequently assigned as ST659 (n = 2). Of the included strains, only Papiliotrema laurentii was resistant to amphoterin B (1/29, 3.5%), 6.8% (2/29) were resistant to 5-flucytosine (the single Papiliotrema laurentii strain and one Cryptococcus neoformans isolate), and 13.8% (4/29) to fluconazole, including two of five (40%) Cutaneotrichosporon curvatus and two of 23 (8.7%) C. neoformans strains. We found a significative association between poor therapeutic outcome and a non-ST93 sequence type of causative strains (these concerned the less common sequence types: ST53, ST31, ST5, ST4, ST659, and ST69) (87.5% versus 40%, p = 0.02). Molecular analysis of Cryptococcus spp. isolates showed a wide species diversity and genetic heterogenicity of Cn within the VNI molecular type. Furthermore, it is worrying that among included strains we found resistances to several of the commonly used antifungals.Cryptococcose chez les personnes vivant avec le VIH à Kinshasa : étude épidémiologique et moléculaire3. Good health and well-bein

Proportion of extended-spectrum ß-lactamase-producing Enterobacteriaceae in community setting in Ngaoundere, Cameroon

BACKGROUND: There is no information regarding the resistance mechanisms of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae in community setting in Cameroon. The current study aimed to determine the proportion of ESBLs in Enterobacteriaceae isolated in the community and to analyse some risk factors associated with ESBL carriage. METHODS: Faecal samples were collected from 208 different outpatients and 150 healthy student volunteers between 3 January and 3 April 2009. Enterobacterial isolates resistant to third-generation cephalosporins were screened for ESBL production by the double-disk synergy test. Presumptive ESBL-producing isolates with positive synergy test were identified by Mass Spectrometry using the BioTyper MALDI-TOF. For such ESBL positive isolates, antibiotic susceptibility was determined by the Vitek 2 system. PCR and sequencing were performed for the detection of different types of ESBL genes in presumptive ESBL-producing isolates. Statistical methods were used for the univariate calculation of risk factors. RESULTS: During the study period, a total of 358 faecal samples were analysed; 58 of such samples (16%) showed an ESBL phenotype and were confirmed by PCR. The proportion of ESBL producers in faecal carriage was statistically different between outpatients and student volunteers (23.1% vs. 6.7%: p < 0.000). According to a univariate analysis, previous use of antibiotics (ciprofloxacin) appeared to be a risk factor for ESBL carriage (p < 0.05).Escherichia coli was the species most frequently isolated among the ESBL producers in outpatients (66.7%) and student volunteers (90%). Isolates showed additional resistance to gentamicin, ciprofloxacin and trimethoprim/sulfamethoxazole but none of them was resistant to temocillin, amikacin or meropenem. Most of the strains (97%) produced a CTX-M group 1 enzymes [CTX-M-15 (98%) or CTX-M-1 (2%)] and the remaining strains produced SHV-12 enzyme (3%). CONCLUSIONS: The use of drugs such as amoxicillin, ciprofloxacin and trimethoprim/sulfamethoxazole does not seem appropriate for empirical treatment because of emerging resistance. The implementation in Cameroon or in other African countries of methods of screening ESBL-producing organisms in routine laboratories is of great importance in order for us to offer patients appropriate treatment and for infection control efforts to succeed

DISTRIBUTION OF CAPSULAR POLYSACCHARIDES OF STREPTOCOCCUS AGALACTIAE STRAINS FROM URUGUAY. MULTIPLEX PCR VERSUS CONVENTIONAL CAPSULAR SEROTYPING

peer reviewe

Evaluation of a new commercial real time PCR for the detection of Aspergillus spp. in serum and respiratory samples

Objectives. Diagnosis of invasive aspergillosis is still disappointing and often delayed because of the lack of sensitivity of diagnostic tools. DNA detection based-methods have been developed, but differ widely and comparisons are difficult to assess. The objective of the study is to compare a new commercial real-time PCR kit, affigene® Aspergillus tracer assay, with an in house nested PCR targeting 18S rRNA Aspergillus sp. gene. Methods. Twelve patients at risk for invasive aspergillosis were included in the study. They were classified to have possible (5 cases), probable (1 case) or proven (6 cases) invasive aspergillosis following E.O.R.T.C. criteria. Fifteen serum and respiratory paired samples were collected. The DNA extraction was performed by using the QIAmp DNA mini kit® (Qiagen, Germany). All samples were tested by both PCR assays and respiratory samples were cultured. Results. Respiratory samples. A. fumigatus, A. niger and A. flavus were isolated from 10/15 samples; both PCR methods were positive for these samples except one that was positive for affigene® and equivocal for the nested PCR. The real-time PCR assay reported cycle thresholds ranging from 25 to 38. Three of the five culture-negative samples were negative by both PCR methods; one of three was negative in affigene® assay and equivocal by nested PCR; the last sample was positive in affigene® assay and negative by nested PCR. Serum. Thirteen of fifteen blood samples were negative by both PCR methods. One sample was equivocal by nested PCR and was inhibited in affigene® assay despite a culture-positive paired respiratory sample. The last case was inhibited by the real-time PCR assay and negative by nested PCR. Nor the nested PCR, nor affigene® assay could detect any Aspergillus DNA in serum. In total, there was 93% of agreement between the two PCR assays. Conclusion. Both methods are in good agreement and can detect at least three different species of Aspergillus. However, the sensitivity of both assays does not permit the detection of Aspergillus DNA in serum. affigene® assay can easy replace the “in house” assay: it allows a fast and standardized detection of Aspergillus sp. DNA in respiratory samples without inconvenient due to the handling of PCR products

Typage des souches de Norovirus circulant dans les populations symptomatiques et asymptomatiques au Burkina Faso

Appartenant à la famille des Caliciviridae, genre Norovirus, les norovirus (NoV) sont des virus non enveloppés dont le génome est composé d’un ARN monocaténaire de polarité positive d’approximativement 7,5 kb. Les NoV infectent l’homme chez qui ils représentent au niveaumondial un agent majeur de gastroentérites épidémiques, d’origine souvent alimentaire mais aussi sporadique, et ce, toutes classes d'âges confondues. Les souches humaines sont classées génétiquement dans différents génotypes au sein de trois des cinq génogroupes, nommés (G) I, II et IV, composant le genre Norovirus. La voie de transmission des NoV est féco-orale. Les NoV sont très résistants dans l’environnement et la dose infectieuse est faible. Dans la population humaine, une grande diversité de souches appartenant principalement aux G I et II co-circulent. Parmi ces souches, le génotype Lordsdale (GII-4) est prédominant dans les épidémies actuelles, notamment lorsqu'une transmission de personne à personne est incriminée, alors que les souches du G I semblent plus fréquemment rapportées au cours des épidémies d’origine environnementale, comme celles liées à la consommation de fruits de mer. Si de nombreuses études d'épidémiologie moléculaire concernant ces virus ont été réalisées dans les pays industrialisés, les données sont par contre manquantes ou ténues pour bien des pays non industrialisés, et en particulier africains. Au cours d'une étude épidémiologique réalisée à Bobo Dioulasso au Burkina Faso et portant sur la prévalence des NoV dans les échantillons de selles de patients présentant ou non des symptômes de gastro-entérite, les souches détectées ont été quantifiées, leur génogroupe a été déterminé et pour certaines d'entre elles le génotype a été précisé. Quatre cent cinquante trois patients ont été prélevés, dont 319 présentant des symptômes diarrhéiques et 134 sujets témoins ne présentant pas de symptomatologie digestive. La détection des NoV et la quantification des charges virales excrétées ont été effectuées sur tous les échantillons par RT-PCR en temps réel permettant de discriminer les souches appartenant aux G I ou II. Une RT-PCR conventionnelle visant les régions de la polymérase (ORF1 du virus) ou de la capside (ORF2) a ensuite été réalisée sur une partie des échantillons détectés positifs en vue du séquençage de ces régions. Les relations phylogénétiques des souches circulant dans la population du Burkina Faso aux souches de référence ont aussi été inférées. Les résultats de RT-PCR en temps réel ont permis de mettre en évidence que les prévalences apparentes de l'infection par les NoV sont similaires dans les populations symptomatique et asymptomatique : une détection moléculaire de NoV chez 67 patients présentant de la diarrhée (21,0 %) et chez 31 des sujets témoins (23,1 %) a pu être observée. Les génotypes circulant détectés sont très variés dans les deux génogroupes, avec une proportion assez surprenante de NoV appartenant au G I. Université polytechnique de Bobo-Dioulasso, Institut supérieur des Sciences de la Santé (INSSA), Bobo-Dioulasso, Burkina Faso. Cette étude a permis de préciser l'épidémiologie moléculaire des souches de NoV circulant dans un pays représentatif de l'Afrique de l'Ouest. Elle a également montré que des individus asymptomatiques pourraient jouer un rôle assez important de réservoir du virus. Elle souligne enfin que, malgré le fait que les souches GII, et en particulier celles de génotype GII.4, soient à l'heure actuelle rapportées majoritairement au niveau mondial, les souches G I doivent être excrétées en égale proportion dans l'environnement. L'origine épidémiologique de la différence entre les prévalences apparentes des infections par les souches de GI et de GII, bien que partiellement expliquée par les différences de sensibilité génétique et d'immunité de population, reste donc à élucider. Remerciements: à la fondation A. Seghers, au Centre de Coopération au Développement de l'Université de Liège, à R. Boreux (assistance technique), aux membres du laboratoire du CMA de Dô et aux agents de santé de Bobo-Dioulasso (Burkina-Faso)

Evaluation d'un test commercial semi-automatique PCR-SERRS pour la détection de Candida sp. dans le sang

Objectives Microbiological diagnosis of invasive candidiasis is still dependent on culture-based methods. The use of beta-D-glucan antigen detection is included in the EORTC microbiological diagnostic criteria but is rarely available in the clinical labs. On the other hand, PCR-based methods lack standardization. The RenDx Fungiplex® is a new commercially available semi-automated PCR SERS assay designed for the detection of Aspergillus sp. and Candida sp. including the differentiation of resistant strains as C. glabrata, C. krusei and A. terreus. This study was performed for sensitivity and reproducibility testing of the method on 8 different Candida species. Methods The study was conducted on EDTA-blood collected from a healthy donor. Blood samples were spiked with 10 Candida reference strains: C. albicans ATCC 10231, C. albicans NEQAS 1206 and C. albicans NEQAS 2359; C. glabrata ATCC 90030 ; C. krusei ATCC 6258 ; C. tropicalis NEQAS 1036 ; C. guillermondii NEQAS 1035 ; C. parapsilosis ATCC 22019 ; C. lusitaniae NEQAS 1511 and C. dubliniensis IHEM 14280. Spiked samples were diluted at final concentrations ranging from 1 CFU/mL to 1000 CFU/mL. Cultures on Sabouraud dextrose agar were performed in parallel to control yeasts dilutions. DNA extraction was performed by using proteinase K-based method followed by purification on QIAcube automate. The RenDx Fungiplex®kit (Renishaw) was used for the amplification process and the final detection was processed on the SP-1000 sample analyzer. Reproducibility testing was performed on the three C. albicans reference strains by repeating each test 5 times. Results A total of 142 samples were included in the study. A sensitivity of 10 CFU/mL was reached for C. glabrata, C. krusei, C. tropicalis, C. dubliniensis spiked samples while C. lusitaniae and C. tropicalis performed better at 1 CFU/mL. The three tested reference C. albicans strains and C. guillermondii gave the lowest sensitivity (100 CFU/mL). The reproducibility of the assay was 96% Conclusion RenDx Fungiplex®kit allows the detection of the most frequent Candida species responsible for invasive candidiasis in spiked blood samples. The sensitivity of the test is comprised between 10 and 100 CFU/mL for most Candida sp. and reproducibility is very high. This evaluation allows us to consider this commercial kit for inclusion in a clinical study on invasive candidiasis in comparison with non-molecular diagnostic assays