James Cohen Stuart

Guideline for phenotypic screening and confirmation of carbapenemases in Enterobacteriaceae

Adequate detection of carbapenemase-producing Enterobacteriaceae is crucial for infection control measures and appropriate choice of antimicrobial therapy. This guideline aims to improve the detection of carbapenemase-producing Enterobacteriaceae in the routine setting of clinical microbiology laboratories. Detection of carbapenemases in Enterobacteriaceae includes a screening step followed by a genotypic and optional phenotypic confirmatory step. For all Enterobacteriaceae, the meropenem screening breakpoint to detect carbapenemases is set at >or=0.5mg/L or a zone diameter of <or=23 mm (10 microg disk loading). For Escherichia coli, Klebsiella spp., Salmonella spp., Enterobacter spp. and Citrobacter spp., the imipenem screening breakpoint is set at >or=2mg/L or a zone diameter <or=21 mm. Ertapenem is not advised as an indicator carbapenem as it has a lower specificity compared with imipenem and meropenem. On the first isolate from a patient with a positive carbapenemase screen test, a polymerase chain reaction (PCR)-based test should be performed to detect carbapenemase genes. However, if genotypic confirmation is not immediately available, phenotypic confirmation tests should be performed to avoid delayed reporting of carbapenemase-producers to the clinic. Recommended phenotypic confirmation tests are the modified Hodge test as well as carbapenemase inhibition tests with boronic acid for Ambler class A carbapenemases and with ethylene diamine tetra-acetic acid (EDTA) or dipicolinic acid for metallo-carbapenemases.

Transient relapses ("blips") of plasma HIV RNA levels during HAART are associated with drug resistance.

Introduction: In a large number of patients on HAART who achieved plasma HIV RNA levels below the limit of detection (50 copies/ml), transient relapses of HIV RNA levels (“blips”) are observed. Objective: To determine whether relapses of plasma HIV RNA during HAART are associated with development of drug resistance. Methods: Plasma samples from 15 patients with a transient viral load relapse during HAART were studied. All regimens contained lamivudine (3TC). We used an ultrasensitive sequence approach to analyze the presence of drug resistance mutations during the relapse. Results: The median plasma HIV RNA load of the relapse was 76 copies/ml (range 50‐1239). In 11 of 15 cases, a genotype of HIV could be obtained. Mutations in the RT and protease gene conferring resistance to one or more drugs were observed in 8 of 11 patients, 6 of whom had the M184V substitution. During a median follow‐up of 27 months after the relapse, plasma HIV RNA levels remained undetectable in 13 of 15 patients. Conclusions: Plasma HIV RNA blips during HAART can be associated with selection of drug‐resistant HIV. This indicates that viral replication may occur during HAART, probably caused by a temporary decrease in active drug concentrations. A blip containing only wild‐type virus is not necessarily caused by viral replication. In this situation the raise of HIV RNA could also originate from release of wild‐type viruses, caused by activation of the latent virus reservoir. Independent of the mechanism, blips did not preclude successful inhibition of viral replication during 2‐year follow‐up in the majority of these cases.

The Dominant Source of CD4+ and CD8+ T-Cell Activation in HIV Infection Is Antigenic Stimulation

&NA;To distinguish between antigenic stimulation and CD4+ T‐cell homeostasis as the cause of T‐cell hyperactivation in HIV infection, we studied T‐cell activation in 47 patients before and during highly active antiretroviral therapy (HAART). We show that expression of human leukocyte antigen (HLA)‐DR, CD38, and Ki67 on T cells decreased during HAART but remained elevated over normal values until week 48 of therapy. We confirm previous reports that T‐cell activation correlates positively with plasma HIV RNA levels (suggesting antigenic stimulation), and negatively with CD4 count (suggesting CD4+ T‐cell homeostasis). However, these correlations may be spurious, because misleading, due to the well‐established negative correlation between CD4 count and plasma HIV RNA levels. To resolve this conflict, we computed partial correlation coefficients. Correcting for CD4 counts, we show that plasma HIV RNA levels contributed to T‐cell hyperactivation. Correcting for plasma HIV RNA levels, we show that CD4+ T‐cell depletion contributed to T‐cell activation. Correcting for both, activation of CD4+ and CD8+ T cells remained positively correlated. Because this suggests that CD4+ and CD8+ T‐cell activation is caused by a common additional factor, we conclude that antigenic stimulation by HIV or other (opportunistic) infections is the most parsimonious explanation for T‐cell activation in HIV infection. Persistence of HIV antigens may explain why T‐cell activation fails to revert to levels found in healthy individuals after 48 weeks of therapy.

Comparison of ESBL contamination in organic and conventional retail chicken meat

Contamination of retail chicken meat by Extended Spectrum Beta-Lactamase (ESBL) producing bacteria likely contributes to the increasing incidence of infections with these bacteria in humans. This study aimed to compare the prevalence and load of ESBL positive isolates between organic and conventional retail chicken meat samples, and to compare the distribution of ESBL genes, strain genotypes and co-resistance. In 2010, 98 raw chicken breasts (n=60 conventional; n=38 organic) were collected from 12 local stores in the Netherlands. Prevalence of ESBL producing micro-organisms was 100% on conventional and 84% on organic samples (p<0.001). Median loads of ESBL producing micro-organisms were 80 (range <20-1360) in conventional, and <20 (range 0-260) CFU/25 g in organic samples (p=0.001). The distribution of ESBL genes in conventional samples and organic samples was 42% versus 56%, respectively (N.S.), for CTX-M-1, 20% versus 42% (N.S.) for TEM-52, and 23% versus 3% (p<0.001) for SHV-12. CTX-M-2 (7%), SHV-2 (5%) and TEM-20 (3%) were exclusively found in conventional samples. Co-resistance rates of ESBL positive isolates were not different between conventional and organic samples (co-trimoxazole 56%, ciprofloxacin 14%, and tobramycin 2%), except for tetracycline, 73% and 46%, respectively, p<0.001). Six of 14 conventional meat samples harbored 4 MLST types also reported in humans and 5 of 10 organic samples harbored 3 MLST types also reported in humans (2 ST10, 2 ST23, ST354). In conclusion, the majority of organic chicken meat samples were also contaminated with ESBL producing E. coli, and the ESBL genes and strain types were largely the same as in conventional meat samples.

Occult hepatitis B in persons infected with HIV is associated with low CD4 counts and resolves during antiretroviral therapy.

Occult hepatitis B virus (HBV) is defined by the presence of plasma HBV DNA in individuals with HBV core antibodies (anti‐HBc), but without HBV surface antigen (HBsAg). The prevalence of occult HBV in HIV‐infected patients remains controversial, and the risk factors, clinical significance and effect of highly active antiretroviral therapy (HAART) are unknown. The aim of this study was to determine prevalence, risk factors, and clinical significance of occult HBV in HIV‐infected patients and to evaluate the effect of HAART. Plasma HBV DNA levels were determined in 191 HIV positive, antiretroviral naïve patients, who were anti‐HBc positive and HBsAg negative. Quantitative HBV DNA was determined using a Taqman real‐time nested PCR. Additionally, plasma HIV RNA levels, CD4 cell counts, anti‐HBs‐antibodies, anti‐HCV‐antibodies, ALT, AST, and γGT were determined. Occult HBV (a plasma HBV DNA level >50 copies/ml) was detected in 9/191 (4.7%) of the patients. Among 45 anti‐HBs‐negative patients (isolated anti‐HBc positive), the prevalence was 11.1%. Patients with occult HBV had significantly lower CD4 count compared to anti‐HBc‐positive/HBsAg negative/HBV DNA‐negative patients (105 ± 157 (median ± SD) vs. 323 ± 299 cells/mm3, P = 0.019). When HAART (including lamivudine) was initiated in the patients with occult HBV, HBV DNA was no longer detectable in any of the patients during 3 years of follow‐up. In conclusion, occult HBV was associated with low CD4 counts and may be viewed as opportunistic reactivation of HBV that resolves as a consequence of HAART induced immune reconstitution and/or the effect of lamivudine. J. Med. Virol. 81:441–445, 2009.

A set of multiplex PCRs for genotypic detection of extended-spectrum β-lactamases, carbapenemases, plasmid-mediated AmpC β-lactamases and OXA β-lactamases

Worldwide, resistance of Gram-negative micro-organisms to third-generation cephalosporins and carbapenems owing to β-lactamases is an increasing problem. Although the CTX-M, TEM and SHV extended-spectrum β-lactamases (ESBLs) are most widely disseminated, other β-lactamase families have also recently emerged, such as plasmid-mediated AmpC β-lactamases and carbapenemases. Here we describe a new set of multiplex polymerase chain reactions (PCRs) with one amplification protocol enabling detection of 25 prevalent β-lactamase families, including ESBLs, carbapenemases, plasmid-mediated AmpC β-lactamases and OXA β-lactamases.

Reconstitution of naive T cells during antiretroviral treatment of HIV-infected adults is dependent on age.

Objective: To determine the influence of age on the regeneration rate of naive and memory T cells in the blood of 45 adults on highly active antiretroviral therapy (HAART). Methods: The age of the patients ranged from 25 to 57 years. Naive cells were defined as CD45RA+CD27+. Cells negative for CD45RA and/or CD27 were considered memory type cells. Results: The recovery rates of naive CD4 and CD8 T cells were similar, were negatively correlated with age and were decreasing 5% and 3.6% per year, respectively. In a multivariate regression analysis, only age was significantly correlated with the naive T cell recovery rates. The recovery rate of memory T cells showed no relation to age. The average regeneration rate of naive CD4 T cells during HAART, i.e., 0.34 × 106 cells/l per day, is not lower than regeneration rates in HIV-negative adults following cytotoxic chemotherapy or CD4 monoclonal antibody therapy. Conclusion: These observations suggest that the thymus contributes considerably to the regeneration of naive T cells in adults on HAART, and that the impact of HIV infection on naive T cell production is small, or rapidly reversible.

Appropriateness of Empirical Treatment and Outcome in Bacteremia Caused by Extended-Spectrum-β-Lactamase-Producing Bacteria

ABSTRACT We studied clinical characteristics, appropriateness of initial antibiotic treatment, and other factors associated with day 30 mortality in patients with bacteremia caused by extended-spectrum-β-lactamase (ESBL)-producing bacteria in eight Dutch hospitals. Retrospectively, information was collected from 232 consecutive patients with ESBL bacteremia (due to Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae) between 2008 and 2010. In this cohort (median age of 65 years; 24 patients were <18 years of age), many had comorbidities, such as malignancy (34%) or recurrent urinary tract infection (UTI) (15%). One hundred forty episodes (60%) were nosocomial, 54 (23%) were otherwise health care associated, and 38 (16%) were community acquired. The most frequent sources of infection were UTI (42%) and intra-abdominal infection (28%). Appropriate therapy within 24 h after bacteremia onset was prescribed to 37% of all patients and to 54% of known ESBL carriers. The day 30 mortality rate was 20%. In a multivariable analysis, a Charlson comorbidity index of ≥3, an age of ≥75 years, intensive care unit (ICU) stay at bacteremia onset, a non-UTI bacteremia source, and presentation with severe sepsis, but not inappropriate therapy within <24 h (adjusted odds ratio [OR], 1.53; 95% confidence interval [CI], 0.68 to 3.45), were associated with day 30 mortality. Further assessment of confounding and a stratified analysis for patients with UTI and non-UTI origins of infection did not reveal a statistically significant effect of inappropriate therapy on day 30 mortality, and these results were insensitive to the possible misclassification of patients who had received β-lactam–β-lactamase inhibitor combinations or ceftazidime as initial treatment. In conclusion, ESBL bacteremia occurs mostly in patients with comorbidities requiring frequent hospitalization, and 84% of episodes were health care associated. Factors other than inappropriate therapy within <24 h determined day 30 mortality.

Multi-centre evaluation of real-time multiplex PCR for detection of carbapenemase genes OXA-48, VIM, IMP, NDM and KPC

BackgroundResistance to carbapenem antibiotics is emerging worldwide among Enterobacteriaceae. To prevent hospital transmission due to unnoticed carriage of carbapenemase producing micro-organisms in newly admitted patients, or follow-up of patients in an outbreak setting, a molecular screening method was developed for detection of the most prevalent carbapenemase genes; blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC.MethodsA real-time multiplex PCR assay was evaluated using a collection of 86 Gram negative isolates, including 62 carbapenemase producers. Seven different laboratories carried out this method and used the assay for detection of the carbapenemase genes on a selection of 20 isolates.ResultsBoth sensitivity and specificity of the multiplex PCR assay was 100%, as established by results on the strain collection and the inter-laboratory comparisons.ConclusionsIn this study, we present a multiplex real-time PCR that is a robust, reliable and rapid method for the detection of the most prevalent carbapenemases blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC, and is suitable for screening of broth cultured rectal swabs and for identification of carbapenemase genes in cultures.

Population Distribution of Beta-Lactamase Conferring Resistance to Third-Generation Cephalosporins in Human Clinical Enterobacteriaceae in The Netherlands

There is a global increase in infections caused by Enterobacteriaceae with plasmid-borne β-lactamases that confer resistance to third-generation cephalosporins. The epidemiology of these bacteria is not well understood, and was, therefore, investigated in a selection of 636 clinical Enterobacteriaceae with a minimal inhibitory concentration >1 mg/L for ceftazidime/ceftriaxone from a national survey (75% E. coli, 11% E. cloacae, 11% K. pneumoniae, 2% K. oxytoca, 2% P. mirabilis). Isolates were investigated for extended-spectrum β-lactamases (ESBLs) and ampC genes using microarray, PCR, gene sequencing and molecular straintyping (Diversilab and multi-locus sequence typing (MLST)). ESBL genes were demonstrated in 512 isolates (81%); of which 446 (87%) belonged to the CTX-M family. Among 314 randomly selected and sequenced isolates, bla CTX-M-15 was most prevalent (n = 124, 39%), followed by bla CTX-M-1 (n = 47, 15%), bla CTX-M-14 (n = 15, 5%), bla SHV-12 (n = 24, 8%) and bla TEM-52 (n = 13, 4%). Among 181 isolates with MIC ≥16 mg/L for cefoxitin plasmid encoded AmpCs were detected in 32 and 27 were of the CMY-2 group. Among 102 E. coli isolates with MIC ≥16 mg/L for cefoxitin ampC promoter mutations were identified in 29 (28%). Based on Diversilab genotyping of 608 isolates (similarity cut-off >98%) discriminatory indices of bacteria with ESBL and/or ampC genes were 0.994, 0.985 and 0.994 for E. coli, K. pneumoniae and E. cloacae, respectively. Based on similarity cut-off >95% two large clusters of E. coli were apparent (of 43 and 30 isolates) and 21 of 21 that were typed by belonged to ST131 of which 13 contained bla CTX-M-15. Our findings demonstrate that bla CTX-M-15 is the most prevalent ESBL and we report a larger than previously reported prevalence of ampC genes among Enterobacteriaceae responsible for resistance to third-generation cephalosporins.