Claude-James Soussy

EUCAST Expert Rules in Antimicrobial Susceptibility Testing

EUCAST expert rules have been developed to assist clinical microbiologists and describe actions to be taken in response to specific antimicrobial susceptibility test results. They include recommendations on reporting, such as inferring susceptibility to other agents from results with one, suppression of results that may be inappropriate, and editing of results from susceptible to intermediate or resistant or from intermediate to resistant on the basis of an inferred resistance mechanism. They are based on current clinical and/or microbiological evidence. EUCAST expert rules also include intrinsic resistance phenotypes and exceptional resistance phenotypes, which have not yet been reported or are very rare. The applicability of EUCAST expert rules depends on the MIC breakpoints used to define the rules. Setting appropriate clinical breakpoints, based on treating patients and not on the detection of resistance mechanisms, may lead to modification of some expert rules in the future.

Unexpected Occurrence of Plasmid-Mediated Quinolone Resistance Determinants in Environmental Aeromonas spp.

Identification of plasmid-mediated qnr genes outside Enterobacteriaceae underlines a possible diffusion of those resistance determinants within gram-negative rods.

Curbing methicillin-resistant Staphylococcus aureus in 38 French hospitals through a 15-year institutional control program.

BACKGROUND The Assistance Publique-Hôpitaux de Paris (AP-HP) institution administers 38 teaching hospitals (23 acute care and 15 rehabilitation and long-term care hospitals; total, 23 000 beds) scattered across Paris and surrounding suburbs in France. In the late 1980s, the proportion of methicillin resistance among clinical strains of Staphylococcus aureus (MRSA) reached approximately 40% at AP-HP. METHODS A program aimed at curbing the MRSA burden was launched in 1993, based on passive and active surveillance, barrier precautions, training, and feedback. This program, supported by the strong commitment of the institution, was reinforced in 2001 by a campaign promoting the use of alcohol-based hand-rub solutions. An observational study on MRSA rate was prospectively carried out from 1993 onwards. RESULTS There was a significant progressive decrease in MRSA burden (-35%) from 1993 to 2007, whether recorded as the proportion (expressed as percentage) of MRSA among S aureus strains (41.0% down to 26.6% overall; 45.3% to 24.2% in blood cultures) or incidence of MRSA cases (0.86 down to 0.56 per 1000 hospital days). The MRSA burden decreased more markedly in intensive care units (-59%) than in surgical (-44%) and medical (-32%) wards. The use of ABHR solutions (in liters per 1000 hospital days) increased steadily from 2 L to 21 L (to 26 L in acute care hospitals and to 10 L in rehabilitation and long-term care hospitals) following the campaign. CONCLUSION A sustained reduction of MRSA burden can be obtained at the scale of a large hospital institution with high endemic MRSA rates, providing that an intensive program is maintained for a long period.

Single and Double Mutations in gyrA but Not in gyrB Are Associated with Low- and High-Level Fluoroquinolone Resistance in Helicobacter pylori

ABSTRACT In one French hospital the rate of resistance to ciprofloxacin in Helicobacter pylori was 3.3% (2 of 60 strains) in 1999. The six resistant clinical strains (four from 1996 and two from 1999) and three ciprofloxacin-selected single-step mutants studied carried one gyrA mutation but none in gyrB. Clinafloxacin and garenoxacin were the most active fluoroquinolones against these mutants. Occurrence of a second gyrA mutation was associated with high MICs of all fluoroquinolones tested.

Genetic Complexity of the Hypervariable Region 1 (HVR1) of Hepatitis C Virus (HCV): Influence on the Characteristics of the Infection and Responses to Interferon Alfa Therapy in Patients With Chronic Hepatitis C

HCV exists within its host as pools of related genetic variants referred to as quasispecies. The hypervariable region 1 (HVR1) of the E2 envelope gene is subjected to strong selective pressure from neutralizing antibodies. The genetic complexity of this region is defined as the total number of genetic variants within the quasispecies population. The genetic complexity of the HVR1 region was examined in patients with chronic hepatitis C and its relationship with the epidemiology of HCV infection, and its influence on liver disease and the response to interferon treatment were determined in 114 patients with chronic hepatitis C. The genetic complexity of the HVR1 major variants was measured before treatment by using a polymerase chain reaction (PCR)‐single‐strand conformation polymorphism (SSCP) technique, and was compared with epidemiological, clinical, virological and histological features. The patients were treated with 3 megaunits of interferon (IFN) alfa for 3 to 6 months and the response to treatment was assessed at 3, 6 and 12 months. The HVR1 could be studied in 101 of the 114 patients (89%). Genetic complexity was significantly higher in patients infected through blood transfusion than intravenous drug use (mean complexity index: 5.7 ± 2.3 vs. 4.7 ± 1.5, respectively; P = 0.04). This relationship was independent of age and the estimated time since infection. No significant relationship was found with other parameters of infection or liver disease. In univariate analysis, the genetic complexity of HVR1 major variants did not affect the rates of ALT normalization at months 3 and 6 of IFN treatment. HVR1 genetic complexity was lower in patients with a sustained virological response than in non‐responders (4.0 ± 1.7 vs. 5.4 ± 2.0, respectively; P = 0.07). In multivariate analysis of pretreatment parameters associated with a sustained virological response to treatment, three parameters appeared to be independent predictors of such a response: a low viral load (P < 0.04), a low anti‐HCV core IgM titer (P = 0.03) and a low genetic complexity of HVR1 major variants (P < 0.04). In conclusion, the HVR1 of HCV has a quasispecies distribution in infected individuals. Its genetic complexity is significantly higher in transfusion recipients than in intravenous drug users, suggesting that the size of the initial inoculum affects the later emergence and development of viral quasispecies. The genetic complexity of HVR1, together with viral load and the anti‐HCV IgM titer, are independent predictors of a sustained virological response to IFN alfa in patients with chronic hepatitis C. J. Med. Virol. 54:256–264, 1998.

Evaluation of a New Test, GenoType HelicoDR, for Molecular Detection of Antibiotic Resistance in Helicobacter pylori

ABSTRACT The eradication rate of Helicobacter pylori by standard therapy is decreasing due to antibiotic resistance, mainly to clarithromycin. Our aim was to provide a new molecular test to guide the treatment of new and relapsed cases. We first studied 126 H. pylori strains for phenotypic (MIC) and genotypic resistance to clarithromycin (rrl mutation) and levofloxacin (gyrA mutation) and then developed a DNA strip genotyping test on the basis of the correlation results and literature data. Clinical strains (n = 92) and gastric biopsy specimens containing H. pylori (n = 105) were tested blindly with the new molecular test GenoType HelicoDR. The presence of mutations or the absence of hybridization with wild-type sequences was predictive, in rrl for clarithromycin resistance in 91 cases (mostly the A2147G mutation) and in gyrA for levofloxacin resistance in 58 cases (mutations at codon 87 or 91). Genotyping revealed a mix of genotypes in 33% of the cases, reflecting a coinfection or selection for resistant mutants. The sensitivity and specificity of detecting resistance were 94% and 99% for clarithromycin and 87% and 98.5% for levofloxacin, respectively. The concordance scores were 0.96 for clarithromycin and 0.94 for levofloxacin. With global resistance rates of 46% for clarithromycin and 25% for levofloxacin, which were observed for consecutive positive biopsy specimens from 2007 and 2008, the positive and negative predictive values for detecting resistance were 99% and 94% for clarithromycin and 96% and 96% for fluoroquinolone. GenoType HelicoDR is efficient at detecting mutations predictive of antibiotic resistance in H. pylori when applied to strains or directly to gastric biopsy specimens.

Reemergence of Gentamicin-Susceptible Strains of Methicillin-Resistant Staphylococcus aureus: Roles of an Infection Control Program and Changes in Aminoglycoside Use

The spread of methicillin-resistant Staphylococcus aureus (MRSA) in our hospital in the 1980s correlated with increasing acquisition of resistance to antibiotics including gentamicin, rifampin, and fluoroquinolones. During the period 1993-1995, there was a major change in clinical MRSA isolates: the percentage of aminoglycoside-resistant MRSA isolates decreased from 75% to 52%, while the proportion of heterogeneous MRSA strains susceptible to gentamicin, rifampin, and tetracycline increased gradually from 4.9% to 27.5%. We used five epidemiological markers (i.e., antibiotyping, phage typing, pulsed-field gel electrophoresis, and restriction analysis of PCR amplified coagulase and protein A genes) to characterize recent isolates. With use of these techniques, we confirmed the persistence of the aminoglycoside-resistant MRSA clone and identified a clone of erythromycin-susceptible strains among the gentamicin-susceptible isolates and found that the remaining strains were diverse. These changes were due to the introduction of various MRSA strains from outside the hospital, while implementation of infection control measures in 1991 could have led to reduced transmission of the aminoglycoside-resistant MRSA strain. Changes in antibiotic prescribing patterns that resulted in reduced selective pressure from gentamicin may have contributed to the spread of gentamicin-susceptible MRSA strains.

Fast and Accurate Quantitative Detection of Helicobacter pylori and Identification of Clarithromycin Resistance Mutations in H. pylori Isolates from Gastric Biopsy Specimens by Real-Time PCR

ABSTRACT Rapid identification of patients infected with clarithromycin-resistant Helicobacter pylori without the need for culture can help to avoid useless prescriptions of clarithromycin. We developed and tested a routine real-time quantitative PCR assay dedicated to that purpose. One hundred ninety-six consecutive gastric biopsy specimens were examined by culture, histology performed by a trained physician, and rapid PCR with the LightCycler apparatus. Infection was defined as (i) positivity of culture, (ii) positivity of histology, or (iii) positivity of PCR if confirmed by positivity of a concomitant indirect test (serology or urea breath test). Susceptibility to clarithromycin was tested by E-test and PCR. The prevalence of infection was 33.7% (66 of 196 samples). The sensitivities of culture, histology, and PCR were 90.9% (60 of 66 samples), 87.9% (58 of 66 samples), and 97.0% (64 of 66 samples), respectively. The specificity of PCR was 94.6% (123 of 130 samples). The linearity of the PCR results was achieved over a 6-log range of input DNA, and we were able to accurately quantify as few as 300 bacteria and to qualitatively detect as few as 30 bacteria per DNA sample. For clarithromycin susceptibility testing, there was 98.2% (55 of 56 samples) concordance between E-test and PCR. Forty-eight strains were clarithromycin susceptible, and 9 strains were clarithromycin resistant. The single discrepancy concerned a culture which was a mixture of mutant and wild type, with a susceptible-to-resistant ratio of 11.5: the resistant population was detected by E-test but not by PCR. Our PCR assay is accurate for fast detection of H. pylori as well as of clarithromycin resistance and is also able to objectively determine bacterial density.

Impairment of polymorphonuclear neutrophil functions precedes nosocomial infections in critically ill patients

Objective A postinjury immunodepression involving neutrophil functions has been described in critically ill patients. The aim of this prospective study was to search for a relationship between an impairment of neutrophil functions and the subsequent development of nosocomial infection. Design Twenty-one severely ill (simplified acute physiology score II >20 on admission), nonimmunosuppressed patients who were receiving no antibiotics active against methicillin-resistant Staphylococcus aureus and highly resistant Pseudomonas aeruginosa were included. Twelve healthy subjects constituted a control group. Measurements Neutrophil functions (phagocytosis and bactericidal activity toward S. aureus and P. aeruginosa in homologous plasma, reactive oxygen species secretion) were studied at day 4 ± 1 after admission, and occurrence of nosocomial infection was prospectively recorded over the following 5 days. Interleukin-10 concentration was assessed by enzyme-linked immunosorbent assay. Results are expressed as median (25th–75th percentiles). Main Results Six out of the 21 patients acquired a nosocomial infection during the 5 days after blood sampling (infected group). Compared with the patients who did not acquire nosocomial infection (noninfected group, n = 15), the neutrophils of the infected group demonstrated a higher percentage of intracellular bacterial survival (17% [2% to 67%] vs. infected: 62% [22% to 100%], p < .05), leading to an impairment of S. aureus killing in homologous plasma (killed bacteria: 4.93 log10 colony forming units/mL [4.24–5.29] vs. infected: 3.62 log10 colony forming units/mL [0.00–4.58], p < .05). Interleukin-10 plasma concentration was higher in infected patients (78 pg/mL [60–83]) compared with noninfected patients (22 pg/mL [14–58], p < .05). By contrast, P. aeruginosa killing was similar in patients whether or not they acquired a nosocomial infection. Conclusion A decrease in S. aureus killing capabilities of neutrophils can be evidenced within the days before occurrence of a nosocomial infection.

Frequent Association between Alteration of the rdxA Gene and Metronidazole Resistance in French and North African Isolates of Helicobacter pylori

ABSTRACT Mutations in the rdxA gene have been associated with the acquisition of resistance to metronidazole in Helicobacter pylori. This gene encodes an NADPH nitroreductase whose expression is necessary for intracellular activation of the drug. We wished to examine whether mutations in rdxA were present in resistant H. pylori isolates infecting either French or North African patients. We determined the complete nucleotide sequences of the rdxA genes from seven French and six North African patients infected with paired resistant and sensitive strains. Genotyping by random amplified polymorphic DNA analysis confirmed the close genetic relatedness of the susceptible and resistant isolates from individual biopsies. Eight French and five North African individual resistant strains were also studied. For the French strains, an alteration in rdxA most probably implicated in resistance was found in 10 cases (seven frameshift mutations, two missense mutations, and one deletion of 211 bp). One to three putative missense mutations were identified in four cases, and a missense mutation possibly not implicated in resistance was discovered in the last case. For the North African strains, an alteration inrdxA was found in eight cases (three frameshift mutations, three missense mutations, one deletion of 6 bp, and one insertion of a variant of IS605). Two strains contained putative missense mutations, and no change was observed in rdxA of the last strain. Thus, inactivation of the rdxA gene is frequently, but not always, associated with resistance to metronidazole in French and North African clinical isolates of H. pylori. In addition, a variety of alterations of rdxA are associated with the resistant phenotype.