Molecular analysis and the toxin, MSCRAMM, and biofilm genes of methicillin-resistant Staphylococcus aureus strains isolated from pemphigus wounds: A study based on SCCmec and dru typing.

INTRODUCTION: Pemphigus is a chronic autoimmune blistering disease. Pemphigus blisters can damage the natural skin barrier and increase the risk of life-threatening conditions. Colonization of pemphigus wounds with methicillin-resistant Staphylococcus aureus (MRSA) prolongs wound healing and increases mortality rate. Assessing MRSA prevalence, types, and toxin and adhesion genes can facilitate the detection of MRSA strains which cause infections, selection of appropriate treatments, and healing of pemphigus wounds. This study aimed to determine the SCCmec, the direct repeat unit (dru) types (dts), and the toxin, MSCRAMM, and biofilm genes of MRSA strains isolated from pemphigus wounds. METHODS: In this cross-sectional study, 118 S. aureus isolates were gathered from 118 patients with pemphigus. MRSA detection was performed using the mecA gene. Using the polymerase chain reaction method, all MRSA isolates were assessed for the presence of the sea, seb, sec, tst, eta, pvl, hla, hlb, MSCRAMM, and ica genes. Typing and subtyping were performed through respectively SCCmec typing and dru typing methods. The Bionumerics software was used for analyzing the data and drawing the minimum spanning tree. FINDINGS: From 118 S. aureus isolates, 51 were MRSA. SCCmec typing revealed the prevalence of SCCmec II with a prevalence of 64.7% (33 out of 51 isolates) and SCCmec III with a prevalence of 35.3% (18 out of 51 isolates). Dru typing indicated seven dts, namely dts 10a, 10g, 10m, 13i, 8h, 8i, and 9ca in two main clusters. The dt9ca was a new dru type and was registered in the dru-typing database (www.dru-typing.org). The prevalence rates of the hla, sea, and sec genes in MRSA isolates were respectively 54.9%, 27.4%, and 1.9%, while the hlb, seb, eta, and pvl genes were not detected at all. Only one MRSA with SCCmec III and dt10a carried the tst encoding gene. MSCRAMM gene analysis revealed the high prevalence of the eno (31.3%) and the fib (21.5%) genes. The prevalence rates of the icaA and icaD biofilm formation genes were 3.9% and 5.8%, respectively. There were no significant differences between the two detected SCCmec types and between the two detected dts clusters respecting the prevalence of the encoding genes of virulence factors and MSCRAMMs. CONCLUSION: The toxin genes hla and sea are prevalent among MRSA strains with SCCmec II and III isolated from pemphigus wounds. The most prevalent dts are dt10a and dt10g among MRSA with SCCmec III and dt8h and dt8i among MRSA with SCCmec II.

Prevalence of active tuberculosis infection in transplant recipients: A systematic review and meta-analysis.

INTRODUCTION: Tuberculosis (TB) is considered as a serious complication of organ transplant; therefore, the detection and appropriate treatment of active TB infection is highly recommended for the reduction of mortality in the future. The aim of this review was to conduct a systematic review and meta-analysis assessing the prevalence of active TB infection in transplant recipients (TRs). MATERIAL AND METHODS: Electronic databases, including MEDLINE (via PubMed), SCOPUS and Web of Science were searched up to December 24, 2017. The prevalence of active TB was estimated using the random effects meta-analysis. Heterogeneity was evaluated by subgroup analysis. Data were analyzed by STATA version 14. RESULTS: The pooled prevalence of post-transplant active TB was estimated 3% [95% CI: 2-3]. The pooled prevalence of active TB in different transplant forms was as follows: renal,3% [95% CI: 2-4]; stem cell transplant (SCT), 1% [95% CI: 0-3]; lung, 4% [95% CI: 2-6]; heart, 3% [95% CI: 2-4]; liver, 1% [95% CI: 1], and hematopoietic stem cell transplant (HSCT), 2% [95% CI: 1-3]. The prevalence of different clinical presentations of TB was as follows: pulmonary TB (59%; 95% CI: 54-65), extra pulmonary TB (27%; 95% CI: 21-33), disseminated TB (15%; 95% CI: 12-19) and miliary TB (8%; 95% CI: 4-13). The pooled prevalence of different diagnostic tests was as follows: chest X-ray, 57% [95% CI, 46-67]; culture, 56% [95% CI, 45-68]; smear, 49% [95% CI, 40-58]; PCR, 43% [95% CI, 40-58]; histology, 26% [95% CI, 20-32], and tuberculin skin test, 19% [95% CI, 10-28]. CONCLUSION: A high suspicion level for TB, the early diagnosis and the prompt initiation of therapy could increase the survival rates among SOT patients. Overall, renal and lung TRs appear to have a higher predisposition for acquiring TB than other type of recipients. Monitoring of the high-risk recipients, prompt diagnosis, and appropriate treatment are required to manage TB infection among TRs especially in endemic areas.

The efficacy of lyticase and ß-glucosidase enzymes on biofilm degradation of Pseudomonas aeruginosa strains with different gene profiles.

BACKGROUND: Pseudomonas aeruginosa is a nosocomial pathogen that causes severe infections in immunocompromised patients. Biofilm plays a significant role in the resistance of this bacterium and complicates the treatment of its infections. In this study, the effect of lyticase and ß-glucosidase enzymes on the degradation of biofilms of P. aeruginosa strains isolated from cystic fibrosis and burn wound infections were assessed. Moreover, the decrease of ceftazidime minimum biofilm eliminating concentrations (MBEC) after enzymatic treatment was evaluated. RESULTS: This study demonstrated the effectiveness of both enzymes in degrading the biofilms of P. aeruginosa. In contrast to the lyticase enzyme, ß-glucosidase reduced the ceftazidime MBECs significantly (P < 0.05). Both enzymes had no cytotoxic effect on the A-549 human lung carcinoma epithelial cell lines and A-431 human epidermoid carcinoma cell lines. CONCLUSION: Considering the characteristics of the ß-glucosidase enzyme, which includes the notable degradation of P. aeruginosa biofilms and a significant decrease in the ceftazidime MBECs and non-toxicity for eukaryotic cells, this enzyme can be a promising therapeutic candidate for degradation of biofilms in burn wound patients, but further studies are needed.

High diversity in SCCmec elements among multidrug-resistant Staphylococcus haemolyticus strains originating from paediatric patients; characterization of a new composite island.

PURPOSE: Staphylococcus haemolyticus has emerged as a highly antimicrobial-resistant healthcare-associated pathogen, in particular for patients admitted to neonatal intensive care. The objective of this study was to study the nature of SCCmec types among MDR-SH strains isolated from paediatric patients. METHODOLOGY: S. haemolyticus strains (n=60) were isolated from paediatric patients. Antibiotic resistance patterns were established using the disk agar diffusion and micro-broth dilution methods. SCCmec typing was performed using whole-genome sequencing (WGS) and an additional PCR analysis. RESULTS: All S. haemolyticus isolates demonstrated multidrug resistance. Using WGS, various novel mec types and combinations of SCCmec types were found, including a new composite island [SCCmec type V (Vd)+SCC cad/ars/cop] comprising 30 % of the strains. SCCmec type V was identified in 23 % of the isolates. A combination of the mecA gene enclosed by two copies of IS431 and absence of the mecRI and ccr genes was identified in 11 strains. In total, mecA regulatory genes were absent in all SH isolates used in this study. CONCLUSION: A high diversity of SCCmec elements with the prevalence of a new composite island was determined among MRSH strains. The structure of the composite island represented by MDR-SH strains in this study, in combination with the presence of a restriction-modification system type III, is described for the first time in this study. The presence of an 8 bp direct repeat (DR) and the sequences flanking the DR may support the integration of the mecA gene complex as a composite transposon (IS431-mecA-IS431) independently from recombinase genes.

Prevalence of Group B Streptococcus in Pregnant Women in Iran: A Systematic Review and Meta-analysis.

BACKGROUND: Group B Streptococcus (GBS or S. agalactiae) is a major cause of severe disease in neonates. In perinatal infections or early-onset disease, GBS is transmitted vertically to the newborn from the birth canal during labor and delivery. Limited information is available on the prevalence of GBS recto-vaginal colonization among pregnant women in Iran. METHODS: We performed a systematic search by using different electronic databases including Medline (via Pubmed), Embase, Web of Science and Iranian Database. Meta-analysis was performed by Comprehensive Meta-Analysis (Biostat V2.2) software. RESULTS: Of 250 articles published from January 2000 to September 2016, 25 studies that reported incidence of GBS colonization in pregnant women were included in this review. The meta-analyses showed that the prevalence of GBS colonization among Iranian pregnant women was 9.8% (95% confidence interval, 7.9-12). CONCLUSIONS: The results of this study indicate that GBS screening measures and chemoprophylaxis guidelines concerning GBS infections must be established for pregnant women in Iran, and these guidelines must provide guidance for obstetricians, midwives and neonatologists on the prevention of GBS infections.

Prevalence of methicillin-resistant Staphylococcus aureus isolated from burn patients in Iran: A systematic review and meta-analysis.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) is considered a major cause of infection and mortality in burn patients, especially when nosocomially acquired. However, limited information is available on the prevalence of MRSA among burn patients in Iran. METHODS: A systematic search was performed using different electronic databases including Medline (via PubMed), Embase, Web of Science and Iranian Database. A meta-analysis was performed using Comprehensive Meta-Analysis Software (BioStat v.2.2). Use of a fixed- or random-effects model was used based on a heterogeneity test, and publication bias was assessed using Begg rank correlation and Egger weighted regression methods. RESULTS: Of 555 articles published from January 2000 to January 2016, 13 studies were included in this review. The meta-analyses showed that the prevalence of MRSA infection in burn patients was 77.9% (95% confidence interval 70.2-84.0%) among culture-positive cases. CONCLUSIONS: The results of this study indicate that the prevalence of MRSA among burn patients is very high in Iran. Thus, a comprehensive infection control strategy based on hand hygiene, education and training in antibiotic prescribing, environmental cleaning, contact precautions, good antibiotic stewardship and an active surveillance system on the basis of international criteria is urgently needed.

Extraintestinal pathogenic Escherichia coli sequence type 131 H30-R and H30-Rx subclones in retail chicken meat, Italy.

Extraintestinal pathogenic Escherichia coli sequence type 131 (ST131), typically fluoroquinolone-resistant (FQ-R) and/or extended-spectrum ß-lactamase (ESBL)-producing, has emerged globally. Among clinical isolates, ST131, primarily its H30-R and H30-Rx subclones, accounts for most antimicrobial-resistant E. coli and is the dominant E. coli strain worldwide. We assessed its prevalence and characteristics among raw chicken meat samples on sale in Palermo, Italy. A collection of 237 fluoroquinolone resistant and ESBL/AmpC producing E. coli isolates, which had been isolated from processed retail chicken meat in the period May 2013-April 2015, was analyzed. Established polymerase chain reaction methods were used to define ST131 and its H30 subclones, ESBL, AmpC, and plasmid-mediated quinolone resistance (PMQR) determinants. Amplified Fragment Length Polymorphism (AFLP) was performed to assess the relatedness among ST131 isolates. Out of the 237 E. coli isolates, 12 isolates belonged to the phylogenetic group B2. Based on the molecular definition of ExPEC, all isolates were attributed with the status of ExPEC. SNP-PCR results confirmed that nine isolates were ST131. SNP-PCR for H30-R and H30-Rx subclones showed that six and three ExPEC ST131 were positive for H30-R and H30-Rx, respectively. The results of AFLP showed that, except for four isolates grouped into two clusters which proved to be indistinguishable, the isolates under study were genetically heterogeneous. To the best of our knowledge, this is the first report of H30-R and H30-Rx subclones in animal food samples. Our findings appear to support the role of food chain in their transmission to humans.

Development of a multiplex Q-PCR to detect Trichoderma harzianum Rifai strain T22 in plant roots.

The fungal species Trichoderma harzianum is widely used as a biological agent in crop protection. To verify the continued presence of this fungus on plant roots manually inoculated with T. harzianum strain T22, a Q-PCR was designed using specific probes for this particular strain. To develop these molecular diagnostic tools, genome mining was first carried out to retrieve putative new regions by which different strains of T. harzianum could be distinguished. Subsequently, Sanger sequencing of the L-aminoacid oxidase gene (aox1) in T. harzianum was applied to determine the mutations differing between various strains isolated from the Trichoderma collection of Koppert Biological Systems. Based on the sequence information obtained, a set of hydrolysis probes was subsequently developed which discriminated T. harzianum T22 strains varying in only a single nucleotide. Probes designed for two strains uniquely recognized the respective strains in Q-PCR with a detection limit of 12,5ng DNA. Titration assays in which T. harzianum DNA from distinct strains was varied further underscored the specificity of the probes. Lastly, fungal DNA extracted from roots of greenhouse cultured tomato plants was analyzed using the probe-based assay. DNA from T. harzianum strain T22 could readily be identified on roots of greenhouse reared tomato plants inoculated with varying concentrations up to one week after treatment with a detection limit of 3e6 colony forming units of T. harzianum T22. We conclude that the Q-PCR method is a reliable and robust method for assessing the presence and quantity of T. harzianum strain T22 in manually inoculated plant material. Our method provides scope for the development of DNA based strain specific identification of additional strains of Trichoderma and other fungal biological control agents.

Variable number of tandem repeat profiles and antimicrobial resistance patterns of Staphylococcus haemolyticus strains isolated from blood cultures in children.

Staphylococcus haemolyticus is a healthcare-associated pathogen and can cause a variety of lifethreatening infections. Additionally, multi-drug resistance (MDR), in particular methicillin-resistant S. haemolyticus (MRSH) isolates, have emerged. Dissemination of such strains can be of great concern in the hospital environment. A total number of 20S. haemolyticus isolates from blood cultures obtained from children were included in this study. A high prevalence of MDR-MRSH isolates with high MIC values to vancomycin was found and 35% of the isolates were intermediate resistant to vancomycin. Multilocus variable number of tandem repeats analysis (MLVF) revealed 5 MLVF types among 20 isolates of S. haemolyticus. Twelve isolates shared the same MLVF type and were isolated from different wards in a pediatric hospital in Iran. This is a serious alarm for infection control; i.e. in the absence of adequate infection diagnostics and infection control guidelines, these resistant strains can spread to other sectors of a hospital and possibly among the community.

Resistance to Antibiotics and Antifungal Medicinal Products: Can Complementary and Alternative Medicine Help Solve the Problem in Common Infection Diseases? The Introduction of a Dutch Research Consortium.

The increase of antibiotic resistance worldwide, rising numbers of deaths and costs associated with this, and the fact that hardly any new antimicrobial drugs have been developed during the last decade have increased the interest in Complementary and Alternative Medicine (CAM) therapeutic interventions, if proven safe and effective. Observational studies on clinical CAM practices demonstrate positive effects of treatment of infections with CAM therapies (clinical effects, patient satisfaction) in combination with small percentages of antibiotics prescription. However, Cochrane reviews and other studies demonstrate that in most instances the quality of clinical trials on CAM treatment of infections is currently too low to provide sufficient evidence. Therefore a Dutch consortium on (in vitro and clinical) scientific research on CAM and antibiotic resistance has been formed. The aim and objective of the consortium is to establish an enduring partnership and to develop expertise to further develop and investigate safe and effective CAM treatments for infectious diseases of humans (and animals). A first ongoing project on the development of safe and effective biobased CAM antimycotics in women with (recurrent) vaginal candidiasis infection is introduced.

Prevention of catheter-related bacteremia with a daily ethanol lock in patients with tunnelled catheters: a randomized, placebo-controlled trial.

BACKGROUND: Catheter-related bloodstream infection (CRBSI) results in significant attributable morbidity and mortality. In this randomized, double-blind, placebo-controlled trial, we studied the efficacy and safety of a daily ethanol lock for the prevention of CRBSI in patients with a tunnelled central venous catheter (CVC). METHODOLOGY: From 2005 through 2008, each lumen of the CVC of adult hematology patients was locked for 15 minutes per day with either 70%-ethanol or placebo, where after the lock solution was flushed through. As a primary endpoint, the incidence rates of endoluminal CRBSI were compared. PRINCIPAL FINDINGS: The intent-to-treat analysis was based on 376 patients, accounting for 448 CVCs and 27,745 catheter days. For ethanol locks, the incidence of endoluminal CRBSI per 1000 CVC-days was 0.70 (95%-CI, 0.4-1.3), compared to 1.19 (95% confidence interval, 0.7-1.9) for placebo (incidence rate-ratio, 0.59; 95% confidence interval, 0.27-1.30; P = .19). For endoluminal CRBSI according to the strictest definition (positive hub culture and identical bacterial strain in blood), a 3.6-fold, non-significant, reduction was observed for patients receiving ethanol (2 of 226 versus 7 of 222; P = .103). No life-threatening adverse events were observed. More patients receiving ethanol discontinued lock-therapy (11 of 226 versus 1 of 222; P = .006) or continued with decreased lock-frequency (10 of 226 versus 0 of 222; P = .002), due to non-severe adverse events. CONCLUSIONS: In this study, the reduction in the incidence of endoluminal CRBSI using preventive ethanol locks was non-significant, although the low incidence of endoluminal CRBSI precludes definite conclusions. Therefore, the lack of statistical significance may partially reflect a lack of power. Significantly more patients treated with ethanol locks discontinued their prophylactic treatment due to adverse effects, which were non-severe but reasonably ethanol related. Additional studies should be performed in populations with higher incidence of (endoluminal) CRBSI. Alternative sources of bacteremia, like exoluminal CRBSI or microbial translocation during chemotherapy-induced mucositis may have been more important in our patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT00122642.

Comparison of the DiversiLab system, Pulsed-Field Gel Electrophoresis and Multi-Locus Sequence Typing for the characterization of epidemic reference MRSA strains.

We have analyzed a representative selection of the HARMONY meticillin-resistant Staphylococcus aureus strain collection originating from 11 European countries (Cookson, B.D. et al., 2007, J. Clin. Microbiol. 45: 1830-1837) with the DiversiLab System, Pulsed-Field Gel Electrophoresis (PFGE) and Multi-Locus Sequence Typing (MLST). Simpson's diversity indices were 0.905, 0.877 and 0.860 for PFGE, MLST and DiversiLab, respectively. All methods displayed concordant classification of the MRSA strains, although with divergent resolution and reproducibility.

Co-evolutionary aspects of human colonisation and infection by Staphylococcus aureus.

Although Staphylococcus aureus is a bacterial species of medical significance, only approximately 30% of all humans carry staphylococcal cells persistently but asymptomatically in their nasopharynx and/or other body sites. This goes largely unnoticed by the host, which shows that in the natural situation the human ecosystem is hospitable or at least receptive to the bacteria and that by a process of co-evolution this has lead to a state of mutual acceptance or tolerance. However, upon disturbance of this balanced, neutral state, localized or disseminated invasive infection can occur. Unfortunately, the events leading to infection are still largely unknown and especially the causal events leading to the transition from colonization to infection are ill-defined in vivo. Whether certain genotypes of S. aureus are more prone to colonise and/or infect humans is still quite heavily debated. The genetic population structure of S. aureus has been largely solved by using a number of different DNA polymorphism-interrogating laboratory methods. However, even this major effort has not (yet) revealed major clues with respect to colonisation and infection potency of the clonal lineages that were thus identified, except for the fact that certain lineages are highly epidemic. The overall picture is that in principle all S. aureus strains can become invasive given the proper circumstances. What these, primarily host-defined circumstances are is still enigmatic. However, a large variety of staphylococcal virulence and colonization factors have been identified as well as a number of host' colonisation and infection susceptibility traits. How these are specifically involved in colonisation and infection has been experimentally substantiated in only a limited number of cases. The present review paper will explore the relevance of these and other, for instance environmental factors that define the colonisation or infection state in humans. When the nature of these states would be known in more detail, this knowledge could be used to design novel and empirical, knowledge-driven means of preventing colonisation from proceeding into S. aureus infection.

Optical fingerprinting in bacterial epidemiology: Raman spectroscopy as a real-time typing method.

Hospital-acquired infections (HAI) increase morbidity and mortality and constitute a high financial burden on health care systems. An effective weapon against HAI is early detection of potential outbreaks and sources of contamination. Such monitoring requires microbial typing with sufficient reproducibility and discriminatory power. Here, a microbial-typing method is presented, based on Raman spectroscopy. This technique provides strain-specific optical fingerprints in a few minutes instead of several hours to days, as is the case with genotyping methods. Although the method is generally applicable, we used 118 Staphylococcus aureus isolates to illustrate that the discriminatory power matches that of established genotyping techniques (numerical index of diversity [D]=0.989) and that concordance with the gold standard (pulsed-field gel electrophoresis) is high (95%). The Raman clustering of isolates was reproducible to the strain level for five independent cultures, despite the various culture times from 18 h to 24 h. Furthermore, this technique was able to classify stored (-80 degrees C) and recent isolates of a methicillin-resistant Staphylococcus aureus-colonized individual during surveillance studies and did so days earlier than established genotyping techniques did. Its high throughput and ease of use make it suitable for use in routine diagnostic laboratory settings. This will set the stage for continuous, automated, real-time epidemiological monitoring of bacterial infections in a hospital, which can then be followed by timely corrective action by infection prevention teams.

Detection of methicillin-resistant Staphylococcus aureus in a low-prevalence setting by polymerase chain reaction with a selective enrichment broth.

The objective of this study was to evaluate the test characteristics of a modified BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) assay on individual and pooled samples in a setting of low MRSA prevalence. The results of the polymerase chain reaction (PCR) assay were compared with culture results from a selective phenol red mannitol broth subcultured after 48 h. Sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) were calculated. For individual testing, 581 samples from 201 persons were collected; 18 (3.2%) were MRSA culture positive. Five hundred ten broths from 174 persons were combined in 106 pools after overnight incubation; 8 pools (7.5%) contained 1 or more MRSA culture-positive specimens. There were no inhibited PCR tests. The combined sensitivity of individual and pooled specimens was 92% (95% confidence interval [CI], 73-99%), the specificity was 98% (95% CI, 96-99%), and the PPV and NPV were 63% and 99.7%, respectively. Our modified procedure gives satisfactory results, and the pooling of broths may reduce costs.

Methicillin-resistant and -susceptible Staphylococcus aureus sequence type 398 in pigs and humans.

Methicillin-resistant Staphylococcus aureus sequence type 398 (ST398 MRSA) was identified in Dutch pigs and pig farmers. ST398 methicillin-susceptible S. aureus circulates among humans at low frequency (0.2%) but was isolated in 3 human cases of bacteremia (2.1%; p = 0.026). Although its natural host is probably porcine, ST398 MRSA likely causes infections in humans.

Host-microbe interplay in persistent Staphylococcus aureus nasal carriage in HIV patients.

It has been shown that persistent Staphylococcus aureus nasal carriage results in increased bacterial dispersal and a higher risk of infection compared to non-or-intermittent S. aureus carriage. Although many studies investigated S. aureus nasal carriage in HIV patients, none compared persistent carriage to non-persistent carriage nor were studies performed in the HAART era. We investigated the host-microbe interplay of persistent S. aureus nasal carriage in HIV-infected patients by studying host determinants of persistent carriage as well as the genetic structure of S. aureus strains isolated. We compared this genetic structure with the previously determined population structure of S. aureus isolates obtained from healthy individuals. Between February 2004 and June 2005 all HIV patients visiting the outpatient department of Erasmus MC (Rotterdam, The Netherlands) were asked to participate in this study. Participants were interviewed and screened for persistent S. aureus carriage using two semi-quantitative nasal swab cultures. For 443 patients two cultures were available, 131 (29.6%) were persistent carriers, which is significantly higher as compared to healthy individuals from the same geographic region (17.6%; P<0.0001). Male sex (odds ratio [OR], 2.22; 95% confidence interval [CI], 1.32-3.73), current smoking (OR, 0.58; 95% CI, 0.38-0.90), Pneumocystis jiroveci pneumonia (PCP) prophylaxis (OR, 0.39; 95% CI, 0.16-0.97) and antiretroviral therapy (OR, 0.61; 95% CI, 0.38-0.98) were independent determinants of persistent carriage. Only two strains were mecA positive (1.2%) and no PVL positive strains were detected. The population structure of S. aureus strains isolated from HIV patients appeared to be strongly overlapping with that of S. aureus isolates from healthy individuals.

Quality control of direct molecular diagnostics for methicillin-resistant Staphylococcus aureus.

Ten samples containing various amounts of methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible S. aureus, methicillin-resistant Staphylococcus epidermidis (MRSE), and combinations thereof were distributed to 51 laboratories for molecular diagnostics testing. Samples containing 10(2) to 10(3) MRSA cells were frequently reported to be negative. MRSE samples were scored as negative by all commercial tests but by only two out of three in-house tests.