Impact of active surveillance and decolonization strategies for methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit.

OBJECTIVE: To assess the impact of active surveillance and decolonization strategies on methicillin-resistant Staphylococcus aureus (MRSA) infection rates in a NICU. STUDY DESIGN: MRSA infection rates were compared before (2014-2016) and during (2017-2022) an active surveillance program. Eligible infants were decolonized with chlorohexidine gluconate (CHG) bathing and/or topical mupirocin. Successful decolonization and rates of recolonization were assessed. RESULTS: Fifty-two (0.57%) of 9 100 hospitalized infants had invasive MRSA infections from 2014 to 2022; infection rates declined non-significantly. During the 6-year surveillance program, the risk of infection was 16.9-times [CI95 8.4, 34.1] higher in colonized infants than uncolonized infants. Those colonized with mupirocin-susceptible MRSA were more likely successfully decolonized (aOR 9.7 [CI95 4.2, 22.5]). Of 57 infants successfully decolonized who remained hospitalized, 34 (60%) became recolonized. CONCLUSIONS: MRSA infection rates did not significantly decline in association with an active surveillance and decolonization program. Alternatives to mupirocin and CHG are needed to facilitate decolonization.

Evaluation of a Zoonotic Orthopoxvirus PCR Assay for the Detection of Mpox Virus Infection.

An epidemic caused by an outbreak of mpox (formerly monkeypox) in May 2022 rapidly spread internationally, requiring an urgent response from the clinical diagnostics community. A detailed description of the clinical validation and implementation of a laboratory-developed real-time PCR test for detecting nonvariola Orthopoxvirus-specific DNA based on the newly designed RealStar Zoonotic Orthopoxvirus assay is presented. The validation was performed using an accuracy panel (n = 97) comprising skin lesion swabs in universal transport media and from mpox virus genomic DNA spiked into pooled mpox virus-negative remnant universal transport media of lesion specimens submitted for routine clinical testing in the NewYork-Presbyterian Hospital clinical laboratory system. Accuracy testing demonstrated excellent assay agreement between expected and observed results and comparable diagnostic performance to three different reference tests. Analytical sensitivity with 95% detection probability was 126 copies/mL, and analytical specificity, clinical sensitivity, and clinical specificity were 100%. In summary, the RealStar Zoonotic Orthopoxvirus assay provides a sensitive and reliable method for routine diagnosis of mpox infections.

Assessing respiratory viral exclusion and affinity interactions through co-infection incidence in a pediatric population during the 2022 resurgence of influenza and RSV.

Introduction: In the Northeast US, respiratory viruses such as influenza and respiratory syncytial virus (RSV), which were largely suppressed by COVID-19-related social distancing, made an unprecedented resurgence during 2022, leading to a substantial rise in viral co-infections. However, the relative rates of co-infection with seasonal respiratory viruses over this period have not been assessed. Methods: Here we reviewed multiplex respiratory viral PCR data (BioFire FilmArray™ Respiratory Panel v2.1 [RPP]) from patients with respiratory symptoms presenting to our medical center in New York City to assess co-infection rates of respiratory viruses, which were baselined to total rates of infection for each virus. We examined trends in monthly RPP data from adults and children during November 2021 through December 2022 to capture the full seasonal dynamics of respiratory viruses across periods of low and high prevalence. Results: Of 50,022 RPPs performed for 34,610 patients, 44% were positive for at least one target, and 67% of these were from children. The overwhelming majority of co-infections (93%) were seen among children, for whom 21% of positive RPPs had two or more viruses detected, as compared to just 4% in adults. Relative to children for whom RPPs were ordered, children with co-infections were younger (3.0 vs 4.5 years) and more likely to be seen in the ED or outpatient settings than inpatient and ICU settings. In children, most viral co-infections were found at significantly reduced rates relative to that expected from the incidence of each virus, especially those involving SARS-CoV-2 and influenza. SARS-CoV-2 positive children had an 85%, 65% and 58% reduced rate of co-infection with influenza, RSV, and Rhino/enteroviruses, respectively, after compensating for the incidence of infection with each virus (p< 0.001). Discussion: Our results demonstrate that most respiratory viruses peaked in different months and present in co-infections less than would be expected based on overall rates of infection, suggesting a viral exclusionary effect between most seasonal respiratory viruses, including SARS-CoV-2, influenza and RSV. We also demonstrate the significant burden of respiratory viral co-infections among children. Further work is necessary to understand what predisposes certain patients for viral co-infection despite this exclusionary effect.

A case of novel, rapidly-growing Mycolicibacter kumamotonensis infection in a patient with severe pulmonary disease treated in New York City.

INTRODUCTION: Mycolicibacter kumamotonensis is a slowly growing, non-chromogenic non-tuberculous mycobacteria (NTM) that was initially distinguished from the M. terrae complex in 2006. Since then it has been rarely reported as the cause of pulmonary and soft-tissue infections in both immunocompromised and immunocompetent patients. CASE PRESENTATION: We present a case of severe pulmonary disease due to Mycolicibacter kumamotonensis in a 57-year-old male who was immunocompetent at time of diagnosis, with a history of interstitial lung disease and a prior diagnosis of tuberculosis (TB). After initial treatment for TB in 2017, his condition stabilized until a recurrence in September 2021, leading to an evaluation for lung transplant in the setting of pulmonary fibrosis and emphysema which led to the identification of Mycolicibacter kumamotonensis. A lung transplant was completed, and the patient was successfully treated with a combination of Ethambutol, Azithromycin, and Rifabutin. CONCLUSIONS: This represents the first case reported of M. kumamotonensis in a patient undergoing lung transplant, and the first case with rapid culture growth during identification of the organism (4 days). This report highlights the need for consideration of M. kumamotonensis as a pathogen in humans, with the potential for rapid growth in liquid media, and the importance of early identification to inform empiric therapy.

Partial ORF1ab Gene Target Failure with Omicron BA.2.12.1.

Mutations in the genome of SARS-CoV-2 can affect the performance of molecular diagnostic assays. In some cases, such as S-gene target failure, the impact can serve as a unique indicator of a particular SARS-CoV-2 variant and provide a method for rapid detection. Here, we describe partial ORF1ab gene target failure (pOGTF) on the cobas SARS-CoV-2 assays, defined by a ≥2-thermocycle delay in detection of the ORF1ab gene compared to that of the E-gene. We demonstrate that pOGTF is 98.6% sensitive and 99.9% specific for SARS-CoV-2 lineage BA.2.12.1, an emerging variant in the United States with spike L452Q and S704L mutations that may affect transmission, infectivity, and/or immune evasion. Increasing rates of pOGTF closely mirrored rates of BA.2.12.1 sequences uploaded to public databases, and, importantly, increasing local rates of pOGTF also mirrored increasing overall test positivity. Use of pOGTF as a proxy for BA.2.12.1 provides faster tracking of the variant than whole-genome sequencing and can benefit laboratories without sequencing capabilities.

Partial ORF1ab Gene Target Failure with Omicron BA.2.12.1.

Mutations in the viral genome of SARS-CoV-2 can impact the performance of molecular diagnostic assays. In some cases, such as S gene target failure, the impact can serve as a unique indicator of a particular SARS-CoV-2 variant and provide a method for rapid detection. Here we describe partial ORF1ab gene target failure (pOGTF) on the cobas ® SARS-CoV-2 assays, defined by a ≥2 thermocycles delay in detection of the ORF1ab gene compared to the E gene. We demonstrate that pOGTF is 97% sensitive and 99% specific for SARS-CoV-2 lineage BA.2.12.1, an emerging variant in the United States with spike L452Q and S704L mutations that may impact transmission, infectivity, and/or immune evasion. Increasing rates of pOGTF closely mirrored rates of BA.2.12.1 sequences uploaded to public databases, and, importantly increasing local rates of pOGTF also mirrored increasing overall test positivity. Use of pOGTF as a proxy for BA.2.12.1 provides faster tracking of the variant than whole-genome sequencing and can benefit laboratories without sequencing capabilities.

Multicenter Evaluation of the Unyvero Platform for Testing Bronchoalveolar Lavage Fluid.

Bronchoalveolar lavage (BAL) culture is a standard, though time-consuming, approach for identifying microorganisms in patients with severe lower respiratory tract (LRT) infections. The sensitivity of BAL culture is relatively low, and prior antimicrobial therapy decreases the sensitivity further, leading to overuse of empirical antibiotics. The Unyvero LRT BAL Application (Curetis GmbH, Germany) is a multiplex molecular panel that detects 19 bacteria, 10 antibiotic resistance markers, and a fungus, Pneumocystis jirovecii, in BAL fluid in ∼4.5 h. Its performance was evaluated using 1,016 prospectively collected and 392 archived specimens from 11 clinical trial sites in the United States. Overall positive and negative percent agreements with culture results for identification of bacteria that grow in routine cultures were 93.4% and 98.3%, respectively, with additional potential pathogens identified by Unyvero in 21.7% of prospectively collected specimens. For detection of P. jirovecii, the positive percent agreement with standard testing was 87.5%. Antibiotic resistance marker results were compared to standard antibiotic susceptibility test results to determine positive predictive values (PPVs). PPVs ranged from 80 to 100%, based on the microorganism and specific resistance marker(s). The Unyvero LRT BAL Application provides accurate detection of common agents of bacterial pneumonia and of P. jirovecii The sensitivity and rapidity of this panel suggest significant clinical value for choosing appropriate antibiotics and for antibiotic stewardship.

Globicatella sanguinis Osteomyelitis and Bacteremia: Review of an Emerging Human Pathogen with an Expanding Spectrum of Disease.

BACKGROUND: Globicatella sanguinis is an uncommon pathogen that may be misdiagnosed as viridans group streptococci. We review the literature of Globicatella and report 2 clinical cases in which catalase-negative Gram-positive cocci resembling viridans group streptococci with elevated minimum inhibitory concentrations (MICs) to ceftriaxone were inconsistently identified phenotypically, with further molecular characterization and ultimate identification of G sanguinis. METHODS: Two clinical strains (from 2 obese women; 1 with a prosthetic hip infection and the other with bacteremia) were analyzed with standard identification methods, followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, 16S recombinant ribonucleic acid (rRNA), and sodA polymerase chain reaction (PCR). The existing medical literature on Globicatella also was reviewed. RESULTS: Standard phenotypic methods failed to consistently identify the isolates. 16S PCR yielded sequences that confirmed Globicatella species. sodA sequencing provided species-level identification of G sanguinis. The review of literature reveals G sanguinis as an increasingly reported cause of infections of the urine, meninges, and blood. To our knowledge, this is the first reported case of an orthopedic infection caused by Globicatella sanguinis. A review of the 37 known cases of G sanguinis infection revealed that 83% of patients are female, and 89% are at the extremes of age (<5 or >65 years). CONCLUSIONS: Globicatella sanguinis, an uncommon pathogen with elevated minimum inhibitory concentrations to third-generation cephalosporins, is difficult to identify by phenotypic methods and typically causes infections in females at the extremes of age. It may colonize skin or mucosal surfaces. Advanced molecular techniques utilizing 16S rRNA with sodA PCR accurately identify G sanguinis.

Multicenter evaluation of Candida QuickFISH BC for identification of Candida species directly from blood culture bottles.

Candida species are common causes of bloodstream infections (BSI), with high mortality. Four species cause >90% of Candida BSI: C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis. Differentiation of Candida spp. is important because of differences in virulence and antimicrobial susceptibility. Candida QuickFISH BC, a multicolor, qualitative nucleic acid hybridization assay for the identification of C. albicans (green fluorescence), C. glabrata (red fluorescence), and C. parapsilosis (yellow fluorescence), was tested on Bactec and BacT/Alert blood culture bottles which signaled positive on automated blood culture devices and were positive for yeast by Gram stain at seven study sites. The results were compared to conventional identification. A total of 419 yeast-positive blood culture bottles were studied, consisting of 258 clinical samples (89 C. glabrata, 79 C. albicans, 23 C. parapsilosis, 18 C. tropicalis, and 49 other species) and 161 contrived samples inoculated with clinical isolates (40 C. glabrata, 46 C. albicans, 36 C. parapsilosis, 19 C. tropicalis, and 20 other species). A total of 415 samples contained a single fungal species, with C. glabrata (n = 129; 30.8%) being the most common isolate, followed by C. albicans (n = 125; 29.8%), C. parapsilosis (n = 59; 14.1%), C. tropicalis (n = 37; 8.8%), and C. krusei (n = 17; 4.1%). The overall agreement (with range for the three major Candida species) between the two methods was 99.3% (98.3 to 100%), with a sensitivity of 99.7% (98.3 to 100%) and a specificity of 98.0% (99.4 to 100%). This study showed that Candida QuickFISH BC is a rapid and accurate method for identifying C. albicans, C. glabrata, and C. parapsilosis, the three most common Candida species causing BSI, directly from blood culture bottles.

Rapid detection of Enterococcus spp. direct from blood culture bottles using Enterococcus QuickFISH method: a multicenter investigation.

The performance of a diagnostic method for detection and identification of Enterococcus spp. directly from positive blood culture was evaluated in a clinical study. The method, Enterococcus QuickFISH BC, is a second-generation peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) test, which uses a simplified, faster assay procedure. The test uses fluorescently labeled PNA probes targeting 16S rRNA to differentiate Enterococcus faecalis from other Enterococcus spp. by the color of the cellular fluorescence. Three hundred fifty-six routine blood culture samples were tested; only 2 discordant results were recorded. The sensitivities for detection of Enterococcus faecalis and non-faecalis Enterococcus were 100% (106/106) and 97.0% (65/67), respectively, and the combined specificity of the assay was 100%. The combined positive and negative predictive values of the assay were 100% (171/171) and 98.9% (185/187), respectively.

Multicenter evaluation of the Staphylococcus QuickFISH method for simultaneous identification of Staphylococcus aureus and coagulase-negative staphylococci directly from blood culture bottles in less than 30 minutes.

A novel rapid peptide nucleic acid fluorescence in situ hybridization (FISH) method, Staphylococcus QuickFISH, for the direct detection of Staphylococcus species from positive blood culture bottles was evaluated in a multicenter clinical study. The method utilizes a microscope slide with predeposited positive- and negative-control organisms and a self-reporting 15-min hybridization step, which eliminates the need for a wash step. Five clinical laboratories tested 722 positive blood culture bottles containing gram-positive cocci in clusters. The sensitivities for detection of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) were 99.5% (217/218) and 98.8% (487/493), respectively, and the combined specificity of the assay was 89.5% (17/19). The combined positive and negative predictive values of the assay were 99.7% (696/698) and 70.8% (17/24), respectively. Studies were also performed on spiked cultures to establish the specificity and performance sensitivity of the method. Staphylococcus QuickFISH has a turnaround time (TAT) of <30 min and a hands-on time (HOT) of <5 min. The ease and speed of the method have the potential to improve the accuracy of therapeutic intervention by providing S. aureus/CoNS identification simultaneously with Gram stain results.

Rapid detection of Klebsiella pneumoniae carbapenemase genes in enterobacteriaceae directly from blood culture bottles by real-time PCR.

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae are endemic in New York City hospitals and have been associated with serious infections globally. A real-time polymerase chain reaction (RT-PCR) assay was developed to detect carbapenem resistance attributable to KPC from blood culture bottles positive for gram-negative bacilli. Culture confirmation of carbapenemase production included automated imipenem and meropenem susceptibility testing and ertapenem susceptibility testing by disk-diffusion. A total of 323 Enterobacteriaceae isolates were tested, of which 8.7% (n = 28) demonstrated carbapenem-resistance by automated and manual susceptibility testing methods or by RT-PCR. The sensitivity, specificity, and positive and negative predictive values of the RT-PCR assay when compared with the automated method were 92.9%, 99.3%, 92.9%, and 99.3%, respectively, and 96.4%, 99.7%, 96.4%, and 99.7%, respectively, when compared with the ertapenem disk-diffusion method. RT-PCR is a rapid and reliable means of detecting carbapenem resistance due to KPC-plasmids in Enterobacteriaceae directly from blood culture bottles.

Identification of a novel Acinetobacter baumannii clone in a US hospital outbreak by multilocus polymerase chain reaction/electrospray-ionization mass spectrometry.

We investigated the relatedness of multidrug-resistant Acinetobacter baumannii isolates from a burn intensive care unit (BICU) outbreak, control isolates, and isolates from a previous 2007 outbreak by 3 molecular typing methods (repetitive sequence-based polymerase chain reaction [rep-PCR], broad-range PCR and electrospray ionization mass spectrometry [PCR/ESI-MS], and pulsed-field gel electrophoresis [PFGE]). Partial rpoB gene sequencing confirmed all tested isolates as A. baumannii. Molecular typing analysis showed that 17 of 19 outbreak isolates were indistinguishable or closely related to each other. Three of 4 non-BICU outbreak control isolates and 5 of 6 isolates from the previous outbreak closely matched the BICU outbreak genotype. The outbreak strain represented a novel strain type, ST96, on PCR/ESI-MS with a new combination of alleles not previously seen in the United States. The ST96 strain also represented a novel rpoB Seqtype. Results of PCR/ESI-MS and PFGE genotyping were most closely correlated, while rep-PCR and PCR/ESI-MS systems generated rapid results.

Identification of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa in blood cultures: a multicenter performance evaluation of a three-color peptide nucleic acid fluorescence in situ hybridization assay.

A multicenter evaluation was undertaken to evaluate the performance of a new three-color peptide nucleic acid fluorescence in situ hybridization assay that identifies isolates directly from blood cultures positive for Gram-negative bacilli (GNB). The assay correctly identified 100% (186/186) of the Escherichia coli isolates, 99.1% (109/110) of the Klebsiella pneumoniae isolates, and 95.8% (46/48) of the Pseudomonas aeruginosa isolates in this study. Negative assay results were correctly obtained for 162 of 165 other GNB (specificity, 98.2%).

Comparison of polymyxin B, tigecycline, cefepime, and meropenem MICs for KPC-producing Klebsiella pneumoniae by broth microdilution, Vitek 2, and Etest.

We report MIC agreement and error rates between broth microdilution (BMD), Vitek 2, and Etest against 48 clinical KPC-producing Klebsiella pneumoniae isolates for polymyxin B, tigecycline, cefepime, and meropenem. Both commercial testing methods were useful for tigecycline testing; Etest provided a conservative estimate of polymyxin B susceptibility. We suggest that laboratories consider the supplemental use of reference BMD or Etest for cefepime and meropenem for susceptibility testing of KPC-producing K. pneumoniae, as Vitek 2 did not provide reliable results.

Quality monitoring of microbial contamination of cryopreserved parathyroid tissue.

Cryopreservation of parathyroid tissue (PT) provides patients undergoing parathyroidectomy with an option for delayed autologous heterotopic parathyroid transplantation. A standard protocol for quality monitoring of PT has not been established. This article describes a method for detecting the presence of bacterial contamination in PT tissue intended for autologous transplantation. PT was received in the tissue bank, processed under aseptic conditions, and placed into cryopreservation medium. Sterility testing was performed at 2 time points prior to cryopreservation. From January 2005 to October 2008, 47 PT samples were cryopreserved. The following bacteria were isolated from 11 PT specimens: Staphylococcus epidermidis, Staphylococcus capitis subspecies ureolyticus, Staphylococcus lugdunensis, Bacillus pumilus, and corynebacteria (diphtheroids). 23% of PTs were contaminated at the time of collection, predominantly with indigenous bacteria. Quality monitoring using this protocol is a useful tool to identify tissues contaminated with bacteria.

Trends in methicillin-resistant Staphylococcus aureus anovaginal colonization in pregnant women in 2005 versus 2009.

In 2005, the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) anovaginal colonization in pregnant women at our center (Columbia University Medical Center) was 0.5%, and MRSA-colonized women were less likely to carry group B streptococcus (GBS). In this study, our objectives were to identify changing trends in the prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) anovaginal colonization in pregnant women, to assess the association between MRSA and GBS colonization, and to characterize the MRSA strains. From February to July 2009, Lim broths from GBS surveillance samples were cultured for S. aureus. MRSA strains were identified by resistance to cefoxitin and characterized by MicroScan, staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), spa typing, and Panton-Valentine leukocidin PCR. A total of 2,921 specimens from different patients were analyzed. The prevalences of MSSA, MRSA, and GBS colonization were 11.8%, 0.6% and 23.3%, respectively. GBS colonization was associated with S. aureus colonization (odds ratio [OR], 1.9; 95% confidence interval [95% CI], 1.5 to 2.4). The frequencies of GBS colonization were similar in MRSA-positive (34.2%) versus MRSA-negative patients (21.8%) (P = 0.4). All MRSA isolates from 2009 and 13/14 isolates from 2005 were SCCmec type IV or V, consistent with community-associated MRSA; 12/18 (2009) and 0/14 (2005) isolates were the USA300 clone. Levofloxacin resistance increased from 14.3% (2005) to 55.6% (2009) (P = 0.028). In conclusion, the prevalence of MRSA anovaginal colonization in pregnant women in New York City, NY, remained stable from 2005 to 2009, and USA300 emerged as the predominant clone with a significant increase in levofloxacin-resistant isolates.

Changes in the molecular epidemiological characteristics of methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit.

OBJECTIVE: To determine whether the molecular epidemiological characteristics of methicillin-resistant Staphylococcus aureus (MRSA) had changed in a level III neonatal intensive care unit (NICU). DESIGN: Retrospective review of medical records. SETTING: Level III NICU of a university-affiliated children's hospital in New York, New York. PATIENTS: Case patients were neonates hospitalized in the NICU who were colonized or infected with MRSA. METHODS: Rates of colonization and infection with MRSA during the period from 2000 through 2008 were assessed. Staphylococcal chromosomal cassette (SCC) mecA analysis and genotyping for S. aureus encoding protein A (spa) were performed on representative MRSA isolates from each clonal pulsed-field gel electrophoresis pattern. RESULTS: Endemic MRSA infection and colonization occurred throughout the study period, which was punctuated by 4 epidemiologic investigations during outbreak periods. During the study period, 93 neonates were infected and 167 were colonized with MRSA. Surveillance cultures were performed for 1,336 neonates during outbreak investigations, and 115 (8.6%) neonates had MRSA-positive culture results. During 2001-2004, healthcare-associated MRSA clones, carrying SCC mec type II, predominated. From 2005 on, most MRSA clones were community-associated MRSA with SCC mec type IV, and in 2007, USA300 emerged as the principal clone. CONCLUSIONS: Molecular analysis demonstrated a shift from healthcare-associated MRSA (2001-2004) to community-associated MRSA (2005-2008).

Multicenter evaluation of a new shortened peptide nucleic acid fluorescence in situ hybridization procedure for species identification of select Gram-negative bacilli from blood cultures.

A shortened protocol for two peptide nucleic acid fluorescence in situ hybridization (PNA FISH) assays for the detection of Gram-negative bacilli from positive blood cultures was evaluated in a multicenter trial. There was 100% concordance between the two protocols for each assay (368 of 368 and 370 of 370 results) and 99.7% (367 of 368 and 369 of 370 results) agreement with routine laboratory techniques.