Abdoulaye Seck

Class D OXA-48 Carbapenemase in Multidrug-Resistant Enterobacteria, Senegal

To the Editor: Class D OXA β-lactamases are characterized as penicillinases that can hydrolyze oxacillin and cloxacillin and are poorly inhibited by clavulanic acid and EDTA. OXA-48 is one of the few members of this family to possess notable carbapenem-hydrolyzing activity (1). First described in 2004 in Turkey, OXA-48 has recently started to spread in Europe and the Middle East (2). We report the recent emergence of the plasmid-encoded blaOXA-48 gene in multidrug-resistant Enterobacteriaceae recovered from patients in Dakar, Senegal, in hospitals and in the community. From November 2008 through October 2009, 11 Enterobacteriaceae isolates (8 Klebsiella pneumoniae, 1 Escherichia coli, 1 Enterobacter cloacae, and 1 Enterobacter sakazakii) with reduced susceptibility to imipenem were identified at the Institut Pasteur (Dakar, Senegal). Antibacterial drug susceptibility was determined by the disk diffusion method and interpreted according to the European Committee on Antimicrobial Susceptibility Testing guidelines (www.eucast.org). Nine isolates were resistant to expanded-spectrum cephalosporins and also to other antibacterial drug classes. The isolates were recovered from 6 patients with urinary tract infections, 4 patients with surgical infections, and 1 patient with omphalitis. Nine infections were hospital acquired (Le Dantec and Principal Hospitals). Because the patients died before antibacterial drug susceptibility testing could be completed, all 5 patients with surgical infections or omphalitis received only empirical therapy with amoxicillin/clavulanate. One patient with a nosocomial urinary tract infection caused by a co-trimoxazole–susceptible strain was successfully treated with this antibacterial agent. The antibacterial drug regimens of the remaining 4 patients were not known, and they were lost to follow-up. We determined the MICs of imipenem, meropenem, and ertapenem by using the Etest method (AB Biodisk, Solna, Sweden), which showed that 9 isolates were susceptible to imipenem and meropenem but either intermediately susceptible or resistant to ertapenem (Table). The 2 imipenem-nonsusceptible isolates were susceptible or intermediately susceptible to meropenem, and both were resistant to ertapenem. We used previously described PCRs (1,3–7) to screen for carbapenem-hydrolyzing β-lactamase genes (blaVIM, blaIMP, blaKPC, and blaOXA-48), as well as plasmid-encoded blaCTX-M, blaAmpC, blaOXA-1, and blaTEM β-lactamase genes; the aac(6′)-Ib aminoglycoside resistance gene; the quinolone resistance genes qnrA,B,S; the tetracycline resistance genes tetA,B,D; and class 1 integron. The blaOXA-48, blaCTX-M, blaAmpC, and aac(6′)-Ib genes and the variable region of class 1 integron were then characterized by direct DNA sequencing of the PCR products. blaOXA-48 was present in all 11 isolates. blaVIM, blaIMP, and blaKPC were not detected. The qnr genes were present in 7 isolates resistant to ciprofloxacin. The aac(6′)-Ib-cr variant was present in 7 isolates resistant to gentamicin, tobramycin, and ciprofloxacin. The 9 isolates resistant to expanded-spectrum cephalosporins all harbored the blaCTX-M-15 gene. The E. coli isolate also harbored the plasmid-encoded blaAmpC gene ACT-1; blaCTX-M-15, blaOXA-1, blaTEM, and aac(6′)lb-cr were associated in 6 isolates. Long-range PCRs showed that these latter 4 genes were located in the same “multidrug resistance region,” as described in Senegal (6). Positive conjugation experiments with sodium azide–resistant E. coli J53 showed through PCR results, plasmid DNA extraction, and antibiogram patterns of the obtained transconjugants that blaOXA-48 was located on a 70-kb self-conjugative plasmid. The genetic environment of blaOXA-48 was then investigated by PCR with primers specific for insertion sequence IS1999 and for the 5′ part of blaOXA-48 (1). blaOXA-48 was found to be part of a Tn1999 composite transposon composed of 2 copies of the insertion sequence IS1999, as reported (2). Further sequencing of the IS1999 located upstream of blaOXA-48 showed that it was consistently truncated by the insertion sequence IS1R, as initially described in Turkey and more recently in Lebanon and Egypt (2,8). XbaI pulsed-field gel electrophoresis was then used to study the genetic relatedness of the 8 K. pneumoniae isolates. Three isolates had similar restriction profiles and had been recovered from 3 patients concurrently hospitalized at Le Dantec Hospital, suggesting nosocomial transmission. A class 1 integron harboring the dfrA1 trimethoprim-resistance gene was detected in the 3 clonal isolates. Together, these findings show the recent emergence of blaOXA-48 in Senegal in community and hospital settings. They may also suggest the spread of the same major carrying plasmid between the Middle East and Africa. Although 9 of the 11 isolates were found to be susceptible to imipenem on the basis of their MICs, their MICs were nonetheless higher than those of blaOXA-48–negative isolates. This raises 2 issues. First, these strains might go undetected during routine antibacterial drug susceptibility testing, a problem that could be overcome by using ertapenem, a compound more susceptible to carbapenemases. Second, the clinical efficacy of imipenem on such strains is uncertain. The frequency of acquired carbapenemases, which emerged early after imipenem introduction in Senegal (2008), is probably strongly underestimated, partly owing to the limited availability of reliable clinical laboratories (9). Because multidrug resistance is prevalent among Enterobacteriaceae isolated in Dakar hospitals (B. Garin, unpub. data) and in rural communities (6), the emergence of blaOXA-48 is a clear cause for concern. Table Resistance genes and carbapenem MICs of 11 Enterbacteriaceae isolates, Senegal, 2008–2009*

Primary antibiotic resistance and associated mechanisms in Helicobacter pylori isolates from Senegalese patients

BackgroundAntibiotic combination therapy for Helicobacter pylori eradication must be adapted to local resistance patterns, but the epidemiology of H. pylori resistance to antibiotics is poorly documented in Africa. The aim was to determine the antibiotic resistance rates, as well as the associated molecular mechanisms, of strains isolated in Dakar, Senegal.MethodsOne hundred and eight H. pylori strains were isolated between 2007 and 2009 from 108 patients presenting with upper abdominal pain to the Gastroenterology Department of Le Dantec Hospital. Antimicrobial susceptibility testing was performed for amoxicillin, clarithromycin, metronidazole, levofloxacin and tetracyclin using the E-test method. Mutations in the 23S rRNA gene of clarithromycin-resistant strains and in gyrA and gyrB of levofloxacin-resistant strains were investigated.ResultsIsolates were characterized by no resistance to amoxicillin (0%), tetracycline (0%), and very low rate of resistance to clarithromycin (1%), but a high rate of resistance to metronidazole (85%). The clarithromycin-resistant strain displayed the A2143G mutation. A worrying rate of levofloxacin resistance was detected (15%). N87I and D91N were the most common mutations in the quinolone-resistance-determining region of gyrA.ConclusionsThe first-line empirical regimen for H. pylori eradication in Senegal should include clarithromycin. Increasing rates of fluoroquinolone resistance detected should discourage the use of levofloxacin-containing regimens without prior antimicrobial susceptibility testing.

Epidemiology of Staphylococcus aureus in pigs and farmers in the largest farm in Dakar, Senegal.

Between December 2009 and November 2011, we collected 57 (12.3%) Staphylococcus aureus isolates from 464 pigs and 16 (30.8%) isolates from 52 farmers in the largest farm in Dakar. Fifty-one isolates (70%) belonged to four major multilocus sequence typing clonal complexes (CCs): CC152 (26.0%), CC15 (19.2%), CC5 (13.7%), and CC97 (10.9%). The CC variability among the pigs was similar to that observed among the farmers. Six isolates that were recovered only among pigs were resistant to methicillin (10.5%). They were assigned to the ST5-staphylococcal cassette chromosome mec type (SCCmec) IV (n = 5) and ST88-SCCmec IV (n = 1) clones. The luk-PV genes encoding Panton-Valentine leukocidin (PVL), present in 43 (58.9%) isolates overall, including all major CCs and the MRSA ST5-SCCmec IV clone, were highly prevalent compared to data from industrialized countries. This finding is of major concern with regard to the potential virulence of these strains.

Community-acquired infectious diarrhoea in children under 5 years of age in Dakar, Senegal

Abstract Background: In sub-Saharan Africa, infectious diarrhoea is a major cause of childhood morbidity and mortality. A cross-sectional study was undertaken to document the pathogens potentially involved in community-acquired childhood diarrhoea in Dakar, the capital of Senegal. Methods: Between September 2007 and March 2008, 176 children aged 1 month to 5 years were recruited consecutively from a primary health care institution in an urban area. Clinical data were recorded and stool samples were collected. Bacterial pathogens were identified using conventional methods and/or PCR assays. Rotaviruses and adenoviruses were detected by a rapid immunochromatographic test. Intestinal parasites were diagnosed by microscopy. Results: Rotavirus was the most common enteric pathogen, detected in 27% of patients, followed by Shigella (12%), diarrhoeagenic Escherichia coli (8%), enteric adenovirus (8%), Salmonella (4%), Campylobacter jejuni (3%) and Plesiomonas shigelloides (2%). Mixed bacterial/viral infections were detected in 6% of cases. Parasites, mostly protozoa, were detected in 14% of children. Using ipaH PCR, 30% of samples were positive for Shigella/entero-invasive E. coli. Detection of rotavirus was more frequently associated with younger age groups (<24 months), whereas bacterial diarrhoea was isolated more often in children over 1 year of age. Detection of bacterial pathogens was significantly associated with malnutrition. Antibiotics were prescribed for 77% of children who attended for consultation. No pathogen was found in 36% of them, whereas a virus was detected without any other associated bacterial or parasitic pathogen in 23% of patients. Conclusion: In developing countries, there is a need to develop reliable, easy-to-use, inexpensive rapid diagnostic tests to guide the management of diarrhoea in infants and children and thereby prevent over-use of antimicrobial agents.

Low Immune Response to Hepatitis B Vaccine among Children in Dakar, Senegal

HBV vaccine was introduced into the Expanded Programme on Immunization (EPI) in Senegal and Cameroon in 2005. We conducted a cross-sectional study in both countries to assess the HBV immune protection among children. All consecutive children under 4 years old, hospitalized for any reason between May 2009 and May 2010, with an immunisation card and a complete HBV vaccination, were tested for anti-HBs and anti-HBc. A total of 242 anti-HBc-negative children (128 in Cameroon and 114 in Senegal) were considered in the analysis. The prevalence of children with anti-HBs ≥10 IU/L was higher in Cameroon with 92% (95% CI: 87%–97%) compared to Senegal with 58% (95% CI: 49%–67%), (p<0.001). The response to vaccination in Senegal was lower in 2006–2007 (43%) than in 2008–2009 (65%), (p = 0.028). Our results, although not based on a representative sample of Senegalese or Cameroonian child populations, reveal a significant problem in vaccine response in Senegal. This response problem extends well beyond hepatitis B: the same children who have not developed an immune response to the HBV vaccine are also at risk for diphtheria, tetanus, pertussis (DTwP) and Haemophilus influenzae type b (Hib). Field biological monitoring should be carried out regularly in resource-poor countries to check quality of the vaccine administered.

Hepatitis B virus exposure during childhood in Cameroon, Central African Republic and Senegal after the integration of HBV vaccine in the expanded program on immunization.

Background: More than 2 billion people worldwide have been exposed to hepatitis B virus (HBV). To prevent these infections, Senegal and Cameroon integrated the HBV vaccine into their Expanded Program on Immunization (EPI) in 2005, as did the Central African Republic (CAR) in 2008. We evaluated the prevalence of HBV exposure and infection after the integration of the HBV vaccine in the EPI. Methods: An observational cross-sectional study was conducted among the hospitalized children 3 months to 6 years of age in Cameroon, CAR and Senegal. Plasma was collected for the detection of anti-HBc, anti-HBs and hepatitis B surface antigen in children with anti-HBc and anti-HBs. Results: Between April 2009 and May 2010, 1783 children were enrolled, 19.4% of whom were anti-HBc positive. The percentage of children with anti-HBc was 44.4% among the children younger than 6 months, decreasing after 6 months to reach 18.8% at 12 months. This decline was followed by a rapid increase in anti-HBc positivity rate in CAR observed as early as 12 months of age compared with Cameroon and Senegal, where the anti-HBc increased between 18 and 36 months of age, respectively. The prevalence of hepatitis B surface antigen–positive children was significantly higher in CAR than that in Cameroon and Senegal (5.1% versus 0.7% and 0.2%; P < 0.001). Socioeconomic level, age and country were factors associated with the presence of anti-HBc. Conclusions: Passive transfer of anti-HBc maternal antibodies versus HBV exposure could be differentiated as early as 12 months of age. The low prevalence of anti-HBc and hepatitis B surface antigen among children born after the integration of HBV vaccine in the EPI in Cameroon and Senegal suggests a positive impact of HBV vaccination.

HIV-1 genetic diversity and drug resistance among Senegalese patients in the public health system.

ABSTRACT In this study, we investigated the prevalence of human immunodeficiency virus type 1 (HIV-1) drug resistance mutations and genetic variability among Senegalese patients undergoing highly active antiretroviral therapy (ART) in the public health system. We conducted a cross-sectional study of 72 patients with suspected therapeutic failure. HIV-1 genotyping was performed with Viroseq HIV-1 Genotyping System v2.0 or the procedure developed by the ANRS AC11 resistance study group, and a phylogenetic analysis was performed. The median follow-up visit was at 40 (range, 12 to 123) months, and the median viral load was 4.67 (range, 3.13 to 6.94) log10 copies/ml. The first-line therapeutic regimen was nucleoside reverse transcriptase inhibitors (NRTIs) plus efavirenz (EFV) or NRTIs plus nevirapine (NVP) (54/72 patients; 75%), and the second-line therapy was NRTIs plus a protease inhibitor (PI/r) (18/72; 25%). Fifty-five patients (55/72; 76.39%) had at least one drug resistance mutation. The drug resistance rates were 72.22 and 88.89% for the first-line and second-line ARTs, respectively. In NRTI mutations, thymidine analog mutations (TAMs) were found in 50.79% and the M184V mutation was found in 34.92% of the samples. For non-NRTI resistance, we noted a predominance of the K103N mutation (46.27%). For PI/r, several cases of mutations were found with a predominance of M46I and L76V/F at 24% each. The phylogenetic analysis revealed CRF02_AG as the predominant circulating recombinant form (43/72; 59.72%). We found a high prevalence of resistance mutations and a high rate of TAMs among Senegalese patients in the public health system. These findings emphasize the need to improve virological monitoring in resource-limited settings.

Staphylococcus aureus nasal and pharyngeal carriage in Senegal

Nasal and pharyngeal swabs were collected from 132 patients admitted to the Principal Hospital in Dakar (Senegal), in January and February 2012. The prevalence of Staphylococcus aureus carriage was 56.1% (n = 74): 40.2% for pharyngeal samples and 36.4% for nasal samples. None of the isolates was methicillin-resistant. Carriage was independently associated with being female (p <0.01) and large households (≥15 members) (p 0.04). The luk-PV genes encoding Panton-Valentine leukocidin (PVL) were present in 26.2% of the isolates. These data highlight the importance of the oropharynx as a site of colonization, and the high prevalence of PVL-positive isolates in Senegal as compared with industrialized countries.

Population Genetic Structure and Isolation by Distance of Helicobacter pylori in Senegal and Madagascar

Helicobacter pylori has probably infected the human stomach since our origins and subsequently diversified in parallel with their human hosts. The genetic population history of H. pylori can therefore be used as a marker for human migration. We analysed seven housekeeping gene sequences of H. pylori strains isolated from 78 Senegalese and 24 Malagasy patients and compared them with the sequences of strains from other geographical locations. H. pylori from Senegal and Madagascar can be placed in the previously described HpAfrica1 genetic population, subpopulations hspWAfrica and hspSAfrica, respectively. These 2 subpopulations correspond to the distribution of Niger-Congo speakers in West and most of subequatorial Africa (due to Bantu migrations), respectively. H. pylori appears as a single population in Senegal, indicating a long common history between ethnicities as well as frequent local admixtures. The lack of differentiation between these isolates and an increasing genetic differentiation with geographical distance between sampling locations in Africa was evidence for genetic isolation by distance. The Austronesian expansion that started from Taiwan 5000 years ago dispersed one of the 10 subgroups of the Austronesian language family via insular Southeast Asia into the Pacific and Madagascar, and hspMaori is a marker for the entire Austronesian expansion. Strain competition and replacement of hspMaori by hpAfrica1 strains from Bantu migrants are the probable reasons for the presence of hspSAfrica strains in Malagasy of Southeast Asian descent. hpAfrica1 strains appear to be generalist strains that have the necessary genetic diversity to efficiently colonise a wide host spectrum.

Combating Global Antibiotic Resistance: Emerging One Health Concerns in Lower- and Middle-Income Countries

Antibiotic misuse in lower- and middle-income countries (LMICs) contributes to the development of antibiotic resistance that can disseminate globally. Strategies specific to LMICs that seek to reduce antibiotic misuse by humans, but simultaneously improve antibiotic access, have been proposed. However, most approaches to date have not considered the growing impact of animal and environmental reservoirs of antibiotic resistance, which threaten to exacerbate the antibiotic resistance crisis in LMICs. In particular, current strategies do not prioritize the impacts of increased antibiotic use for terrestrial food-animal and aquaculture production, inadequate food safety, and widespread environmental pollution. Here, we propose new approaches that address emerging, One Health challenges.