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ABSTRACTThe technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosisisolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncAcoding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-type pncAgene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n= 88), and concordance values between the Bactec 460, the Wayne test, or pncAgene Sanger sequencing and NGS results were 82% (n= 88), 83% (n= 88), and 89% (n= 88), respectively. NGS confirmed the majority of pncAmutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates, pncAmutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS of pncAimproved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies.

ABSTRACTWidespread infections with community-associated (CA) methicillin-resistant Staphylococcus aureus(MRSA) have occurred in the United States with the dissemination of the USA300 strain beginning in 2000. We examined 105 isolates obtained from children treated at the University of Chicago from 1994 to 1997 (75 methicillin-susceptible S. aureus[MSSA] and 30 MRSA isolates) in order to investigate for possible evidence of USA300 during this period. Infections were defined epidemiologically based on medical record review. The isolates underwent multilocus sequence typing (MLST), as well as assays for the Panton-Valentine leukocidin (PVL) genes, the protein A gene (spa), and arcAand opp3, proxy markers for the arginine catabolic mobile element (ACME), characteristic of USA300 MRSA. MRSA isolates also underwent staphylococcal cassette chromosome mec(SCCmec) typing and pulsed-field gel electrophoresis (PFGE) subtyping. MSSA isolates belonged to 17 sequence type (ST) groups. The 12 epidemiologically defined CA-MRSA infection isolates were either ST1 (n= 4) or ST8 (n= 8). They belonged to 3 different PFGE types: USA100 (n= 1), USA400 (n= 5), and USA500 (n= 6). Among the CA-MRSA infection isolates, 8 (67%) were PVL+. None of the MRSA or MSSA isolates contained arcAor opp3. Only one MRSA isolate was USA300 by PFGE. This was a health care-associated (HA) MRSA isolate, negative for PVL, that carried SCCmectype II. USA300 with its characteristic features was not identified in the collection from the years 1994 to 1997.

ABSTRACTStaphylococcus aureusis a commensal species that can also be a formidable pathogen. In the United States, an epidemic of community-acquired methicillin-resistant Staphylococcus aureus(MRSA) infections has been occurring for the last 15 years. In the context of a study in which we identified patients with skin and soft tissue infections (SSTIs) and randomized them to receive one of two antimicrobial treatment regimens, we assessed S. aureuscolonization in the nares, throat, and perianal skin on the day of enrollment and 40 days after therapy. We compared the prevalence of colonization between the SSTI patients and an uninfected control population. A total of 144 subjects and 130 controls, predominantly African American, participated in this study, and 116 returned for a 40-day follow-up visit. Of the SSTI patients, 76% were colonized with S. aureusat enrollment, as were 65% of the controls. Patients were more likely than the controls to be colonized with USA300 MRSA (62/144 [43.1%] versus 11/130 [8.5%], respectively; P< 0.001). The nares were not the most common site of colonization. The colonization prevalence diminished somewhat after antibiotic treatment but remained high. The isolates that colonized the controls were more likely than those in the patients to be methicillin-susceptible S. aureus(MSSA) (74/84 [88.1%] versus 56/106 [52.8%], respectively; P< 0.001). In conclusion, the prevalence of S. aureuscolonization among SSTI patients was high and often involved USA300 MRSA. The prevalence diminished somewhat with antimicrobial therapy but remained high at the 40-day follow-up visit. Control subjects were also colonized at a high prevalence but most often with a genetic background not associated with a clinical infection in this study. S. aureusis a commensal species and a pathogen. Plans for decolonization or eradication should take this distinction into account.

ABSTRACTIn 928 Dallas County Jail detainees, nasal carriage of Staphylococcus aureuswas found in 32.8% (26.5% methicillin-susceptible Staphylococcus aureus[MSSA] and 6.3% methicillin-resistant S. aureus[MRSA]), and hand carriage was found in 24.9% (20.7% MSSA and 4.1% MRSA). Among MRSA nasal carriers, 41% had hand MRSA carriage; 29% with hand MRSA carriage had no nasal S. aureuscarriage. The prevalence of carriage was not associated with duration of the jail stay up to 180 days.

ABSTRACTSequence type 398 (ST398) Staphylococcus aureus, frequently carried by livestock, has caused severe human infections and often carries transmissible antibiotic resistance genes. Among methicillin-susceptible S. aureusisolates colonizing Dallas County Jail detainees, 13.2% were ST398, spatype t571, and were genetically similar to human colonization isolates from New York, Chicago, and the Dominican Republic.

ABSTRACTMethicillin-resistant Staphylococcus aureus(MRSA) has become a common cause of skin infections and invasive infections in community dwellers in the United States since the late 1990s. Isolates characterized as USA300 by pulsed-field gel electrophoresis (PFGE) are the predominant strain type in these infections. USA100 and USA500 strains commonly cause health care-associated infections. We compared PFGE with a number of other methods of genotyping in a sample of 149 clinical MRSA isolates from the University of Chicago Medical Center. The 5 USA500 isolates yielded 3 spatypes and 2 multilocus sequence types (MLSTs). Among the 24 USA100 isolates, 21 (88%) were of spatype t002, 19 (79%) were of ST5, 2 carried arcAand opp3, and 1 was Panton-Valentine leukocidin positive (PVL+). Among the 102 USA300 isolates, 96 (94%) were of ST8 and 94 (92%) were of spatype t008. The combination of traits that provided the best sensitivity (98%), specificity (97%), positive predictive value (PPV) (99%), and negative predictive value (NPV) (95%) for identifying USA300 isolates were the presence of the arcAgene and the presence of the PVL genes (area under the curve, 0.980; 95% confidence interval [CI], 0.955 to 1.0). PFGE did not delineate a homogeneous group of MRSA genetic backgrounds, as documented for other typing methods, particularly for USA500 and USA100 pulsotypes. Documenting the presence of arcAand PVL genes by PCR was an efficient and accurate means of identifying USA300 in a collection of MRSA isolates in which USA300 is common. None of the tested genotyping methods provided an accurate means of identifying the next most common PFGE-based backgrounds, USA100 and USA500.

ABSTRACTA novel protocol for full-length Mycobacterium tuberculosisgene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine (PGM). Five genes—rpoB(rifampin), katG(isoniazid), pncA(pyrazinamide), gyrA(ofloxacin/fluoroquinolone), and rrs(aminoglycosides)—were amplified and sequenced, and results were compared to those obtained by genotypic Hain line probe assay (LPA) and phenotypic Bactec MGIT 960 analysis using 26 geographically diverse South African clinical isolates collected between July and November 2011. Ion Torrent sequencing exhibited 100% (26/26) concordance to phenotypic resistance obtained by MGIT 960 culture and genotypic rpoBand katGresults by LPA. In several rifampin-resistant isolates, Ion Torrent sequencing revealed uncommon substitutions (H526R and D516G) that did not have a defined mutation by LPA. Importantly, previously uncharacterized mutations in rpoB(V194I), rrs(G878A), and pncA(Q122Stop) genes were observed. Ion Torrent sequencing may facilitate tracking and monitoring geographically diverse multidrug-resistant and extensively drug-resistant strains and could potentially be integrated into selected regional and reference settings throughout Africa, India, and China.

ABSTRACTMupirocin is a topical antimicrobial used to eradicate methicillin-resistant Staphylococcus aureus(MRSA) colonization, usually in the absence of susceptibility testing. We hypothesized that high-level (HL) mupirocin resistance was associated with multidrug resistance (MDR). To this end, unique patient isolates identified at our institution during 2008 were stratified into those resistant to =3 non-ß-lactam antimicrobial classes (MDR) and non-MDR MRSA. HL mupirocin resistance was screened by mupAPCR on all MDR isolates (n= 191) and a 20% random sample (n= 130) of non-MDR isolates; E-testing confirmed HL resistance. We found that among MDR isolates, 13 (6.8%) carried mupA, whereas none of the non-MDR isolates did (P= 0.001). Thus, although the overall prevalence of HL mupirocin resistance is low among MRSA isolates at our institution, an association exists between mupAcarriage and MDR. Using genotyping and antimicrobial susceptibility profiling, we identified nine HL mupirocin-resistant clones. Whereas the majority of mupA-negative MDR isolates had a health care-associated MRSA (HA-MRSA) genotype (multilocus sequence type 5 [ST5] or SCCmectype II), the majority of mupA-positive MDR isolates had a community-associated MRSA (CA-MRSA) genotype (ST8 or SCCmectype IV). However, CA- and HA-MRSA genotypes were more evenly distributed among mupA-positive isolates compared to mupA-negative MDR isolates. Thus, in Chicago, mupAis circulating among both CA- and HA-MRSA backgrounds.

ABSTRACTThe BD GeneOhm methicillin-resistant Staphylococcus aureus(MRSA) assay is a molecular screening test for detection of MRSA in nasal colonization. This assay coamplifies the extremity of staphylococcal chromosome cassette mec(SCCmec) and adjacent chromosomal DNA at the SCCmecinsertion site. Increasing reports of novel SCCmectypes and the diverse genetic backgrounds of MRSA strains prompted us to test the accuracy of the BD GeneOhm MRSA kit with 914 MRSA isolates with a variety of SCCmectypes harbored in 21 genetic backgrounds, as determined by the multilocus sequence type (ST). The BD GeneOhm MRSA assay was performed on colony lysates; purified genomic DNA (0.2 pg/µl and 0.2 ng/µl) was tested to confirm negative results from lysates. Of 914 MRSA isolates tested, 911 tested positive (detection rate, 99.7%). The SCCmectypes carried by assay-positive isolates were I, II, III, IV, V, V(5C2&5), VI, and VIII and SCCmeccomposite islands with mecclass A and ccrcomplexes 2 and 4. One of the assay-negative isolates had a community-associated genotype: ST8, SCCmectype IV. However, this was an outlier among the 99.8% (434/435) ST8, SCCmectype IV-containing isolates that tested positive. The two other assay-negative isolates had a health care-associated genotype (ST5); both carried a distinct, uncommon, composite SCCmectype. In summary, the BD GeneOhm MRSA assay had a high rate of detection of MRSA isolates harboring common and uncommon SCCmectypes from the United States and Taiwan.

ABSTRACTIn the 1990s, community-associated methicillin-resistant Staphylococcus aureus(CA-MRSA) strains emerged as pathogens outside of the health care environment. Epidemic foci of CA-MRSA infections were reported in jails and prisons, but risk factors for MRSA infection there are not known. All skin and soft tissue infections (SSTIs) cultured in the Cook County Jail in March 2004 to August 2005 were reviewed. Demographic and clinical risk factors were compared among patients with methicillin-susceptible S. aureus(MSSA) SSTIs and those with MRSA SSTIs. Antibiotic susceptibilities were recorded, and we performed multilocus sequence typing on a sample of MRSA isolates. There were 378 SSTIs from different patients requiring culture, of which 240 (63.5%) were of MRSA and 43 (11.4%) were of MSSA; 84.8% of S. aureusisolates were MRSA. MRSA- and MSSA-infected patients were similar with regard to age, gender, ethnicity, previous exposure to the jail, and comorbidities. In the 12 months prior to the index culture, MRSA patients were more likely to have received a ß-lactam antibiotic (25% versus 9%; P= 0.02). Among 26 MRSA strains, 24 (92%) had the sequence type 8 (ST8) genotype. Within 6 months, 14% (95% confidence interval, 8.7% to 22.3%) of MRSA SSTI patients in the jail had a recurrent SSTI compared with 8.8% (95% confidence interval, 2.1% to 32.6%) of MSSA SSTI patients (P= 0.004). MRSA is the predominant cause of SSTIs requiring culture in the jail. Few risk factors differentiated MRSA from MSSA SSTIs, and detainee patients with MRSA SSTIs are at high risk for recurrent SSTIs.

ABSTRACTMethicillin-resistant Staphylococcus aureus(MRSA) isolates carry the methicillin resistance gene (mecA) on a horizontally transferred genetic element called the staphylococcal chromosome cassette mec(SCCmec). Community-acquired MRSA (CAMRSA) isolates usually carry SCCmectype IV. We previously reported that 76% of 17 CAMRSA isolates (multilocus sequence type 59) obtained from pediatric patients with skin and soft tissue infections (SSTI) from Taipei did not carry SCCmectypes I to IV. We used DNA sequence analysis to determine that the element harbored by these nontypeable isolates is a novel subtype of SCCmecV called SCCmecVT.It contains a ccrCrecombinase gene variant (ccrC2) and meccomplex C2. One SSTI isolate contained molecular features of SCCmecIV but also contained ccrC2(a feature of SCCmecVT), suggesting that it may harbor a composite SCCmecelement. The genes lukS-PVand lukF-PVencoding the Panton-Valentine leukocidin (PVL) were present in all CAMRSA SSTI isolates whether they contained SCCmectype IV or VT. SCCmecVTwas also present in 5 of 34 (14.7%) CAMRSA colonization isolates collected from healthy children from Taipei who lacked MRSA risk factors. Four (80%) of the these isolates contained lukS-PVand lukF-PV, as did 1 of 27 (3.7%) SCCmecIV-containing colonization isolates. A total of 63% (10 of 16) of the SSTI isolates and 61.7% (21 of 34) of the colonization isolates tested were resistant to at least four classes of non-ß-lactam antimicrobials. SCCmecVTis a novel SCCmecvariant that is found in multiply resistant CAMRSA strains with sequence type 59 in Taipei in association with the PVL leukotoxin genes.

ABSTRACTViral culture isolation has been widely accepted as the “gold standard” for laboratory confirmation of viral infection; however, it requires ultralow temperature specimen storage. Storage of specimens in ethanol at room temperature could expand our ability to conduct active surveillance and retrospective screenings of viruses with rapid and inexpensive real-time PCR tests, including isolates from remote regions where freezing specimens for culture is not feasible. Molecular methods allow for rapid identification of viral pathogens without the need to maintain viability. We hypothesized that ethanol, while inactivating viruses, can preserve DNA and RNA for PCR-based methods. To evaluate the use of ethanol-stored specimens for augmenting surveillance for detection of influenza viruses A and B and adenoviruses (AdV), paired nasal swab specimens were collected from 384 recruits with febrile respiratory illness at Fort Jackson, S.C., in a 2-year study. One swab was stored at ambient temperature in 100% ethanol for up to 6 months, and the other swab was stored at -70°C in viral medium. For viral detection, frozen specimens were cultured for a variety of respiratory viruses, and ethanol-fixed specimens were tested with TaqMan (TM) probe and LightCycler SYBR green (SG) melting curve assays with at least two different PCR targets for each virus. The sensitivities of the TM and SG assays on specimens stored in ethanol for 1 month were 75% and 58% for influenza A, 89% and 67% for influenza B, and 93 to 98% and 57% for AdV, respectively. Lower specificities of the real-time assays corresponded to the increased detection of PCR-positive but culture-negative specimens. Influenza virus RNA was detected as well or better after 6 months of storage in ethanol.

ABSTRACTInfections of adenovirus type 4 (Ad4) and Ad7 were discovered among previously vaccinated individuals through febrile respiratory illness surveillance at military recruit camps. Genetic analysis was performed on these isolates and a sample of adenovirus isolates from unvaccinated patients. Antigenic regions of the adenovirus hexon gene from 21 vaccinated and 31 unvaccinated patients were sequenced and compared to homologous regions of Ad4 and Ad7 vaccine strains and of other representative hexon sequences archived in GenBank. The phylogenetic distribution of sequences from vaccinated individuals closely resembled those from unvaccinated individuals. The most common Ad7 strain was the Ad7d2 hexon genotype, and the most common Ad4 strain was a genotype nearly identical to the recently discovered Z-G 95-873 Ad4 variant. Near exclusive isolation of Ad4 since 1999 indicates that the Ad4 variant is currently responsible for the vast majority of adenovirus morbidity in military recruit camps. Different ratios of nonsynonymous to synonymous nucleotide substitution rates in known antigenic regions compared to nonantigenic regions indicated positive selection for diversity in the antigenic regions and purifying selection in the nonantigenic regions.

ABSTRACTA real-time reverse transcriptase PCR (RRT-PCR) assay based on the avian influenza virus matrix gene was developed for the rapid detection of type A influenza virus. Additionally, H5 and H7 hemagglutinin subtype-specific probe sets were developed based on North American avian influenza virus sequences. The RRT-PCR assay utilizes a one-step RT-PCR protocol and fluorogenic hydrolysis type probes. The matrix gene RRT-PCR assay has a detection limit of 10 fg or approximately 1,000 copies of target RNA and can detect 0.1 50% egg infective dose of virus. The H5- and H7-specific probe sets each have a detection limit of 100 fg of target RNA or approximately 103to 104gene copies. The sensitivity and specificity of the real-time PCR assay were directly compared with those of the current standard for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs and hemagglutinin subtyping by hemagglutination inhibition (HI) assay. The comparison was performed with 1,550 tracheal and cloacal swabs from various avian species and environmental swabs obtained from live-bird markets in New York and New Jersey. Influenza virus-specific RRT-PCR results correlated with VI results for 89% of the samples. The remaining samples were positive with only one detection method. Overall the sensitivity and specificity of the H7- and H5-specific RRT-PCR were similar to those of VI and HI.

A total of 114 strains of Haemophilus influenza were characterized with respect to beta-lactamase production and ampicillin MIC. Of this total, 41 strains produced a TEM-type beta-lactamase, and ampicillin MICs for these strains were greater than or equal to 2.0 microgram/ml. It was found that 54 strains lacked TEM-type beta-lactamase activity, and ampicillin MICs for them were less than or equal to 0.5 microgram/ml. The remaining 19 strains were beta-lactamase negative, but ampicillin MICs were greater than or equal to 2.0 micrograms/ml. Disk diffusion susceptibility tests were performed with two media, i.e., Mueller-Hinton agar containing 1.0% hemoglobin and 1.0% IsoVitaleX supplement (CHOC-MHA) and enriched chocolate agar (CHOC), by using disks containing 10 and 2 micrograms of ampicillin. If strains of H. influenzae for which ampicillin MICs were greater than or equal to 2.0 micrograms/ml were considered resistant, while strains for which MICs were less than or equal to 0.5 microgram/ml were considered susceptible, the following zone diameter interpretive criteria were identified as indicating ampicillin susceptibility: CHOC-MHA (10-micrograms disks), greater than or equal to 20 mm; CHOC-MHA (2-micrograms disks), greater than or equal to 17 mm; CHOC (10-micrograms disks), greater than or equal to 25 mm; and CHOC (2-micrograms disks), greater than or equal to 20 mm. In all cases, zones of inhibition less than those listed above would be interpreted as indicating resistance. Each of these four combinations was found to be essentially equivalent in identifying susceptible and resistant strains of H. influenzae, irrespective of beta-lactamase production.

Two hemolytic phenotypes of Vibrio damsela, isolated from the tissue of a patient with a fatal wound infection, were characterized. The patient had underlying disease, and the wound was associated with an injury inflicted during the handling of a catfish. The phenotypes were morphologically and biochemically similar except for their lecithinase, lipase, and hemolytic activities. When grown on rabbit blood agar, one phenotype (LZ) produced a large zone of hemolysis (10 mm) around the colony, whereas the other type (SZ) produced only a small zone (1 to 2 mm). On sheep blood agar, the differences in hemolytic activity were more subtle. By a modified CAMP test in which V. damsela was streaked perpendicularly to Staphylococcus aureus, it was determined that a factor elaborated by the LZ phenotype (but not the SZ phenotype) protected sheep erythrocytes from the hemolysis normally caused by S. aureus toxins. Cell-free filtrates of broth cultures of each phenotype had the same effects on erythrocytes as did the organisms themselves.

ABSTRACTIn June 2001, an outbreak of acute gastroenteritis among 109 attendees of a church picnic in Kerr County, Texas, was reported. A 5'-nuclease PCR assay was used to screen for Salmonellain nine food items from the buffet line. Barbeque chicken B tested positive for Salmonella, and no amplification was detected in the remaining food items. These PCR findings were consistent with culture results and were confirmed by direct nucleotide sequencing. Salmonella entericaserotype Panama was cultured from both food and patient stool samples.

We cared for a 4-year-old male with nosocomially acquired epiglottitis caused by Streptococcus pneumoniae. He had been receiving ceftazidime therapy when this infection was recognized. The S. pneumoniae isolate was of serotype 15B and was resistant to beta-lactam antibiotics, cephalosporins (including those with extended spectra), and trimethoprim-sulfamethoxazole. Clinicians and clinical microbiologists must be aware that cephalosporin susceptibility may no longer be assumed for penicillin-resistant S. pneumoniae isolates and that susceptibility testing for the extended-spectrum cephalosporins should be performed whenever this species is isolated from a normally sterile body fluid.

The activity of chloramphenicol against 100 different strains of Haemophilus influenzae was assessed by a macrotube broth dilution technique and by a standardized disk diffusion method using both enriched chocolate agar (CHOC) and Mueller-Hinton agar containing 1.0% hemoglobin and 1.0% IsoVitaleX (BBL Microbiology Systems, Cockeysville, Md.) supplement (CHOC-MHA). Filter disks containing 30 micrograms of chloramphenicol were used with the disk diffusion procedure. The following zone diameter interpretive criteria were defined: CHOC-MHA, less than or equal to 25 mm = resistant [corrected] and greater than or equal to 26 mm = susceptible [corrected]; CHOC, less than or equal to 28 mm = resistant [corrected] and greater than or equal to 29 mm = susceptible [corrected]. all of the H. influenzae strains examined were also characterized by using two rapid assays for chloramphenicol acetyltransferase (CAT) activity: a 1-h tube method (t-CAT) and a 30-min procedure which used commercially available reagent-impregnated disks (d-CAT). The t-CAT procedure was found to be significantly more accurate than the d-CAT procedure as a means for demonstrating production of CAT.

ABSTRACTThe Ukraine ranks among the top 20 countries with the highest number of multidrug-resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosiscases in the world. However, little is known of the genetic diversity, i.e., resistance signatures, in clinical isolates from this region. We analyzed seven of most prevalent MDR/XDR antibiotic resistance-conferring genes from clinical isolates (n= 75) collected from geographically diverse Ukrainian oblasts and the southern Crimean peninsula. Genomic analysis revealed that 6 (8%) were sensitive, 3 (4%) were resistant to at least one antibiotic but were not MDR, 40 (53%) were MDR, and 26 (35%) were XDR. The majority of isolates (81%) were of the Beijing-like lineage. This is the first study to use next-generation sequencing (NGS) of clinical isolates from the Ukraine to characterize mutations in genes conferring M. tuberculosisdrug resistance. Several isolates harbored drug resistance signatures that have not been observed in other countries with high-burden tuberculosis. Most notably, the absence of inhAgene promoter mutations, a diversity of mutation types in the rpoBresistance-determining region, and detection of heteroresistance provide a broader understanding of MDR/XDR from this area of the world.

ABSTRACTModern advances in genomics provide an opportunity to reinterpret historical bacterial culture collections. In this study, genotypic antibiotic resistance profiles of Mycobacterium tuberculosisisolates from a historical 20-year-old multidrug-resistant tuberculosis (MDR-TB) culture collection in South Africa are described. DNA samples extracted from the phenotypically MDR-TB isolates (n= 240) were assayed by Hain line probe assay (LPA) for the confirmation of MDR-TB and by Illumina Miseq whole-genome sequencing (WGS) for the characterization of mutations in eight genes (rpoB, katG, inhA, rpsL, pncA, embB, gyrA, and rrs) that are known to code for resistance to commonly used anti-TB agents. LPA identified 71.3% of the TB isolates as MDR-TB, 18.3% as rifampin (RIF) monoresistant, 2% as isoniazid (INH) monoresistant, and 8.3% as susceptible to both RIF and INH (RIF+INH). In a subset of 42 randomly selected isolates designated as RIF+INH resistant by Löwenstein-Jensen (LJ) culture in 1993, LPA and WGS results confirmed MDR-TB. In all five INH-monoresistant isolates by LPA and in all but one (the wild type) of the 34 successfully sequenced RIF-monoresistant isolates, WGS revealed matching mutations. Only 26% of isolates designated as susceptible by LPA, however, were found to be wild type by WGS. Novel mutations were found in the rpoB(Thr480Ala, Gln253Arg, Val249Met, Val251Tyr, Val251Phe), katG(Trp477STOP, Gln88STOP, Trp198STOP, Trp412STOP), embB(Thr11Xaa, Gln59Pro), and pncA(Thr100Ile, Thr159Ala, Ala134Arg, Val163Ala, Thr153Ile, DelGpos7, Phe106Ser) genes. Three MDR-TB isolates showed mutations in both the gyrAand rrsgenes, suggesting that extensively drug-resistant tuberculosis existed in South Africa well before its formal recognition in 2006.

ABSTRACTHealth care-associated methicillin-resistant Staphylococcus aureus(MRSA) infections are a burden on the health care system. Clinical laboratories play a key role in reducing this burden, as the timely identification of MRSA colonization or infection facilitates infection control practices that are effective at limiting invasive MRSA infections. The Xpert MRSA NxG assay recently received FDA clearance for the direct detection of MRSA from nasal swabs. This multicenter study evaluated the clinical performance characteristics of the Xpert MRSA NxG assay with prospectively collected rayon nasal swabs (n= 1,103) and flocked swab (ESwab) nasal specimens (n= 846). Culture-based identification methods and antimicrobial susceptibility testing were used as the reference standards for comparison. According to the reference method, the positivity rates for MRSA in the population evaluated were 11.1% (122/1,103) for rayon swabs and 11.6% (98/846) for flocked swabs. The overall sensitivity and specificity of the rayon swabs were 91.0% (95% confidence interval [CI], 84.6 to 94.9%) and 96.9% (95% CI, 95.7 to 97.8%), respectively, across eight testing sites. The flocked swab specimens were 92.9% sensitive (95% CI, 86.0 to 96.5%) and 97.6% specific (95% CI, 96.2 to 98.5%) for MRSA detection across six testing sites. The sensitivity and specificity of the combined flocked and rayon swab data were 91.8% (95% CI, 87.4 to 94.8%) and 97.2% (95% CI, 96.3 to 97.9%), respectively. The positive predictive value (PPV) for rayon swabs was 78.7%, versus 83.5% for ESwabs. The negative predictive values (NPVs) for rayon swabs and ESwab specimens were 98.9% and 99.1%, respectively. In conclusion, the Xpert MRSA NxG assay is a sensitive and specific assay for the direct detection of MRSA from nasal swab specimens.