Raphaël Boreux

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

AbstractBackgroundThere 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.MethodsFaecal 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.ResultsDuring 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%).ConclusionsThe 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.

Assessment of pfcrt 72-76 haplotypes eight years after chloroquine withdrawal in Kinshasa, Democratic Republic of Congo

BackgroundIn 2001, the World Health Organization (WHO) has recommended the use of artemisinin-based combination therapy (ACT) as the first-line treatment of uncomplicated malaria cases, as monotherapies had become ineffective in many parts of the world. As a result, the Democratic Republic of Congo (DRC) withdrew chloroquine (CQ) from its malaria treatment policy in 2002 and an artesunate (AS)-amodiaquine (AQ) combination became the ACT of choice in DRC in 2005. AQ-resistance (AQR) has been reported in several parts of the world and mutations in codons 72-76 of the Plasmodium falciparum chloroquine-resistance transporter (pfcrt) gene have been strongly correlated with resistance, especially mutations encoding the SVMNT haplotype. This haplotype was first identified in Southeast Asia and South America but was recently reported in two African countries neighbouring DRC. These facts raised two questions: the first about the evolution of CQ resistance (CQR) in DRC and the second about the presence of the SVMNT haplotype, which would compromise the use of AQ as a partner drug for ACT.MethodsA total of 213 thick blood films were randomly collected in 2010 from a paediatric clinic in Kinshasa, DRC. Microscopy controls and real-time polymerase chain reaction (RT-PCR) were performed for Plasmodium species identification. Haplotypes of the pfcrt gene were determined by sequencing.ResultsThe K76T mutation was detected in 145 out of 198 P. falciparum-positive samples (73.2%). In these 145 resistant strains, only the CVIET haplotype was detected.ConclusionsThis study is the first to assess the molecular markers of resistance to CQ and AQ after the introduction of ACT in DRC. The results suggest first that CQR is decreasing, as wild-type pfcrt haplotypes were found in only 26.8% of the samples and secondly that the SVMNT haplotype is not yet present in Kinshasa, suggesting that AQ remains valid as a partner drug for ACT in this region.

Human cytomegalovirus and primary intracranial tumours: frequency of tumour infection and lack of correlation with systemic immune anti-viral responses

Human cytomegalovirus (HCMV) is a ubiquitous beta human herpesvirus able to influence infected cell survival and proliferation and to modulate the host immune response. As there is accumulating evidence that HCMV is detected in primary intracranial astrocytic tumours, in this study we looked for the presence of HCMV in intracranial tumours and tried to correlate this eventual presence with the anti‐HCMV systemic immunoreactivity and with the detection of HCMV in peripheral blood.

Prevalence and spread of extended-spectrum β-lactamase-producing Enterobacteriaceae in Ngaoundere, Cameroon

During April 2010 and June 2010, 334 Enterobacteriaceae isolates from 590 participants (outpatients, inpatients, inpatient carers, hospital workers and members of their households) were collected from faecal samples. Based on β-lactamase pattern, origin of strains and the relationship between participants, 44 isolates of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae were selected from 44 participants (in Ngaoundere Protestant Hospital and Ngaoundere Regional Hospital, Cameroon). To determine the relatedness of bacterial strains, these isolates were fingerprinted using the automated, repetitive-sequenced-based PCR-based DiversiLab system. Subsequently, E. coli isolates that had undergone DiversiLab analysis were examined with respect to their phylogenetic group and detection of the ST131 clone to shed light on the epidemiology of these isolates in the Ngaoundere hospitals. The prevalence of faecal carriage of ESBL-producing Enterobacteriaceae among the study participants was 54.06%. According to participant groups, the prevalence of faecal carriage was also high (outpatients 45%; inpatients 67%; inpatient carers 57%; hospital workers 44%; and members of their households 46%). Analysis of the molecular epidemiology of ESBL-producing E. coli and K. pneumoniae showed a close relationship of the isolates between related and non-related individuals. In addition, DiversiLab results of E. coli identified four related isolates (4/22) from cluster III belonging to the epidemiologically important clone ST131. Our results highlight the importance of outpatients, inpatients, their carers, hospital workers and their families as reservoirs of ESBL-producing Enterobacteriaceae.

Prevalence of Campylobacter among goats and retail goat meat in Congo

BACKGROUND The prevalence of Campylobacter jejuni and Campylobacter coli was determined in goat and goat meat sold at retail outlets in Lubumbashi, Democratic Republic of Congo (DR Congo). METHODOLOGY A total of 644 samples, including 177 goat meat, 86 goat stomachs, 139 ready to eat (RTE) goat skewers, and 242 goat faecal samples were examined for the presence of Campylobacter jejuni and Campylobacter coli using polymerase chain reaction. RESULTS Overall, Campylobacter spp. were found in 34.6% of the examined samples. C. jejuni was isolated in 10.1% and C. coli in 26.7% of samples. Only 2.2% of all samples were positive for both species. There was a significant association between the prevalence of C. coli and the type of sample (p < 0.05). The overall prevalence of Campylobacter in different sample groups was 41.2%, 37.2%, 23.7%, and 35.1% for goat meat, goat stomachs, RTE goat skewers, and goat faecal samples, respectively. There was no significant difference (p > 0.05) between the prevalence observed in the rainy season (16.7%) and the dry season (20.0%). Moreover, the overall prevalence of Campylobacter in slaughter sites, open-air markets, warehouses, and semi-open-air markets was 28.2%, 34.2%, 35.4%, and 42.9%, respectively. Statistically, there was no influence of the sample collection site on the frequency of isolation of Campylobacter (p > 0.05). CONCLUSION This study shows that, considering the relatively high prevalence of this pathogen, live goat and goat meat are major sources of human and environmental contamination by Campylobacter spp. in Lubumbashi.

Clinical evaluation of the DermaGenius Nail real-time PCR assay for the detection of dermatophytes and Candida albicans in nails

Onychomycosis represents one of the most frequent mycoses in the world. Causative agents are mainly dermatophytes, but yeasts and nondermatophyte moulds can also be involved. Conventional diagnostic methods include direct microscopy (or histology) and culturing. However, molecular methods are becoming increasingly popular in this field. The DermaGenius® (DG) Nail multiplex assay (PathoNostics, The Netherlands) is a new commercial real-time polymerase chain reaction (PCR) kit, which can detect Trichophyton rubrum, Trichophyton interdigitale and Candida albicans directly in nails. The present study is a retrospective evaluation of the kit applied to 138 finger and toenail clippings in comparison to histology and culture methods. The sensitivity and specificity of the PCR assay are 80% (76/95) and 74.4% (32/43), respectively, when histology and culture are used as reference to define onychomycosis. DG performance is not different from histology combined with culture (P = .11) but the best diagnostic efficacy (88.4%, 122/138) is obtained by the combination of histology and DG. In conclusion, this study emphasizes the clinical usefulness of the DG in diagnostics. The high specificity of this test guarantees a better identification compared to culture that can lead to dermatophyte misidentifications. It is a reliable PCR assay that shortens the time to diagnosis and can unmask the presence of nongrowing fungal pathogens in nails.

P1869 Evaluation of a new commercial real-time PCR for the de-tection of Aspergillus spp. in serum and respiratory samples

1. Ascioglu S. et coll. 2002.Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus. Clin. lnfect. dis.34:7-14. 2. Yamakami Y. A. Hashimoto, I. Tokymatsu and M. Nasu. 1996. PCR detection for Aspergillus species in serum of patients with nvasive aspergillosis. J. Clin. Microbiol. 36:3619-3623. 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 87% 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.