Implementation of Bedaquiline, Pretomanid, and Linezolid in the United States: Experience Using a Novel All-Oral Treatment Regimen for Treatment of Rifampin-Resistant or Rifampin-Intolerant Tuberculosis Disease.

BACKGROUND: Rifampin-resistant tuberculosis is a leading cause of morbidity worldwide; only one-third of persons start treatment, and outcomes are often inadequate. Several trials demonstrate 90% efficacy using an all-oral, 6-month regimen of bedaquiline, pretomanid, and linezolid (BPaL), but significant toxicity occurred using 1200-mg linezolid. After US Food and Drug Administration approval in 2019, some US clinicians rapidly implemented BPaL using an initial 600-mg linezolid dose adjusted by serum drug concentrations and clinical monitoring. METHODS: Data from US patients treated with BPaL between 14 October 2019 and 30 April 2022 were compiled and analyzed by the BPaL Implementation Group (BIG), including baseline examination and laboratory, electrocardiographic, and clinical monitoring throughout treatment and follow-up. Linezolid dosing and clinical management was provider driven, and most patients had linezolid adjusted by therapeutic drug monitoring. RESULTS: Of 70 patients starting BPaL, 2 changed to rifampin-based therapy, 68 (97.1%) completed BPaL, and 2 of the 68 (2.9%) experienced relapse after completion. Using an initial 600-mg linezolid dose daily adjusted by therapeutic drug monitoring and careful clinical and laboratory monitoring for adverse effects, supportive care, and expert consultation throughout BPaL treatment, 3 patients (4.4%) with hematologic toxicity and 4 (5.9%) with neurotoxicity required a change in linezolid dose or frequency. The median BPaL duration was 6 months. CONCLUSIONS: BPaL has transformed treatment for rifampin-resistant or intolerant tuberculosis. In this cohort, effective treatment required less than half the duration recommended in 2019 US guidelines for drug-resistant tuberculosis. Use of individualized linezolid dosing and monitoring likely enhanced safety and treatment completion. The BIG cohort demonstrates that early implementation of new tuberculosis treatments in the United States is feasible.

Carbapenem-Resistant Klebsiella pneumoniae in Large Public Acute-Care Healthcare System, New York, New York, USA, 2016-2022.

Controlling the spread of carbapenem-resistant Enterobacterales is a global priority. Using National Healthcare Safety Network data, we characterized the changing epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) in a large public health system in New York, New York, USA. During 2016-2020, CRKP cases declined; however, during 2021-June 2022, a notable increase occurred. Of 509 cases, 262 (51%) were considered community-onset, including 149 in patients who were living at home. Of 182 isolates with proven or presumptive (ceftazidime/avibactam susceptible) enzymes, 143 were serine carbapenemases; most confirmed cases were K. pneumoniae carbapenemase. The remaining 39 cases were proven or presumptive metallo-ß-lactamases; all confirmed cases were New Delhi metallo-ß-lactamases. After 2020, a marked increase occurred in the percentage of isolates possessing metallo-ß-lactamases. Most patients with metallo-ß-lactamases originated from long-term care facilities. An aggressive and universal program involving surveillance and isolation will be needed to control the spread of CRKP in the city of New York.

Novel 6-Month Treatment for Drug-Resistant Tuberculosis, United States.

The US Food and Drug Administration approved a 6-month regimen of pretomanid, bedaquiline, and linezolid for extensively drug-resistant or multidrug-intolerant tuberculosis after a trial in South Africa demonstrated 90% effectiveness 6 months posttreatment. We report on a patient who completed the regimen using a lower linezolid dose.

Considerations from the College of American Pathologists for Implementation of an Assay for SARS-CoV-2 Testing after a Change in Regulatory Status.

The U.S. Food & Drug Administration (FDA) regulates the marketing of manufacturers' in vitro diagnostic tests (IVDs), including assays for the detection of SARS-CoV-2. The U.S. government's Clinical Laboratory Improvement Amendments (CLIA) of 1988 regulates the studies that a clinical diagnostic laboratory needs to perform for an IVD before placing it into use. Until recently, the FDA has authorized the marketing of SARS-CoV-2 IVDs exclusively through the Emergency Use Authorization (EUA) pathway. The regulatory landscape continues to evolve, and IVDs will eventually be required to pass through conventional non-EUA FDA review pathways once the emergency declaration is terminated, in order to continue to be marketed as an IVD in the United States. When FDA regulatory status of an IVD changes or is anticipated to change, the laboratory should review manufacturer information and previously performed internal verification studies to determine what, if any, additional studies are needed before implementing the non-EUA version of the IVD in accordance with CLIA regulations. Herein, the College of American Pathologists' Microbiology Committee provides guidance for how to approach regulatory considerations when an IVD is converted from EUA to non-EUA status.

Practical Guidance for Clinical Microbiology Laboratories: Mycobacteria.

Mycobacteria are the causative organisms for diseases such as tuberculosis (TB), leprosy, Buruli ulcer, and pulmonary nontuberculous mycobacterial disease, to name the most important ones. In 2015, globally, almost 10 million people developed TB, and almost half a million patients suffered from its multidrug-resistant form. In 2016, a total of 9,287 new TB cases were reported in the United States. In 2015, there were 174,608 new case of leprosy worldwide. India, Brazil, and Indonesia reported the most leprosy cases. In 2015, the World Health Organization reported 2,037 new cases of Buruli ulcer, with most cases being reported in Africa. Pulmonary nontuberculous mycobacterial disease is an emerging public health challenge. The U.S. National Institutes of Health reported an increase from 20 to 47 cases/100,000 persons (or 8.2% per year) of pulmonary nontuberculous mycobacterial disease among adults aged 65 years or older throughout the United States, with 181,037 national annual cases estimated in 2014. This review describes contemporary methods for the laboratory diagnosis of mycobacterial diseases. Furthermore, the review considers the ever-changing health care delivery system and stresses the laboratory's need to adjust and embrace molecular technologies to provide shorter turnaround times and a higher quality of care for the patients who we serve.

Outbreak of Tattoo-associated Nontuberculous Mycobacterial Skin Infections.

BACKGROUND: On 29 April 2015, the Florida Department of Health in Miami-Dade County (DOH Miami-Dade) was notified by a local dermatologist of 3 patients with suspected nontuberculous mycobacterial (NTM) infection after receiving tattoos at a local tattoo studio. METHODS: DOH Miami-Dade conducted interviews and offered testing, described below, to tattoo studio clients reporting rashes. Culture of clinical isolates and identification were performed at the Florida Bureau of Public Health Laboratories. Characterization of NTM was performed by the Centers for Disease Control and Prevention and the US Food and Drug Administration (FDA), respectively. Whole-genome sequencing (WGS) and single-nucleotide polymorphism (SNP) analyses were used to construct a phylogeny among 21 Mycobacterium isolates at the FDA. RESULTS: Thirty-eight of 226 interviewed clients were identified as outbreak-associated cases. Multivariate logistic regression revealed that individuals who reported gray tattoo ink in their tattoos were 8.2 times as likely to report a rash (95% confidence interval, 3.1-22.1). Multiple NTM species were identified in clinical and environmental specimens. Phylogenetic results from environmental samples and skin biopsies indicated that 2 Mycobacterium fortuitum isolates (graywash ink and a skin biopsy) and 11 Mycobacterium abscessus isolates (5 from the implicated bottle of graywash tattoo ink, 2 from tap water, and 4 from skin biopsies) were indistinguishable. In addition, Mycobacterium chelonae was isolated from 5 unopened bottles of graywash ink provided by 2 other tattoo studios in Miami-Dade County. CONCLUSIONS: WGS and SNP analyses identified the tap water and the bottle of graywash tattoo ink as the sources of the NTM infections.

Initial Public Health Laboratory Response After Hurricane Maria - Puerto Rico, 2017.

Hurricane Maria made landfall in Puerto Rico on September 20, 2017, causing major damage to infrastructure and severely limiting access to potable water, electric power, transportation, and communications. Public services that were affected included operations of the Puerto Rico Department of Health (PRDOH), which provides critical laboratory testing and surveillance for diseases and other health hazards. PRDOH requested assistance from CDC for the restoration of laboratory infrastructure, surveillance capacity, and diagnostic testing for selected priority diseases, including influenza, rabies, leptospirosis, salmonellosis, and tuberculosis. PRDOH, CDC, and the Association of Public Health Laboratories (APHL) collaborated to conduct rapid needs assessments and, with assistance from the CDC Foundation, implement a temporary transport system for shipping samples from Puerto Rico to the continental United States for surveillance and diagnostic and confirmatory testing. This report describes the initial laboratory emergency response and engagement efforts among federal, state, and nongovernmental partners to reestablish public health laboratory services severely affected by Hurricane Maria. The implementation of a sample transport system allowed Puerto Rico to reinitiate priority infectious disease surveillance and laboratory testing for patient and public health interventions, while awaiting the rebuilding and reinstatement of PRDOH laboratory services.

Performance of the G4 Xpert® MTB/RIF assay for the detection of Mycobacterium tuberculosis and rifampin resistance: a retrospective case-control study of analytical and clinical samples from high- and low-tuberculosis prevalence settings.

BACKGROUND: The Xpert® MTB/RIF (Xpert) assay is a rapid PCR-based assay for the detection of Mycobacterium tuberculosis complex DNA (MTBc) and mutations associated with rifampin resistance (RIF). An updated version introduced in 2011, the G4 Xpert, included modifications to probe B and updated analytic software. METHODS: An analytical study was performed to assess Xpert detection of mutations associated with rifampin resistance in rifampin-susceptible and -resistant isolates. A clinical study was performed in which specimens from US and non-US persons suspected of tuberculosis (TB) were tested to determine Xpert performance characteristics. All specimens underwent smear microscopy, mycobacterial culture, conventional drug-susceptibility testing and Xpert testing; DNA from isolates with discordant rifampin resistance results was sequenced. RESULTS: Among 191 laboratory-prepared isolates in the analytical study, Xpert sensitivity for detection of rifampin resistance associated mutations was 97.7% and specificity was 90.8%, which increased to 99.0% after DNA sequencing analysis of the discordant samples. Of the 1,096 subjects in the four clinical studies, 49% were from the US. Overall, Xpert detected MTBc in 439 of 468 culture-positive specimens for a sensitivity of 93.8% (95% confidence interval [CI]: 91.2%-95.7%) and did not detect MTBc in 620 of 628 culture-negative specimens for a specificity of 98.7% (95% CI: 97.5%-99.4%). Sensitivity was 99.7% among smear-positive cases, and 76.1% among smear-negative cases. Non-determinate MTBc detection and false-positive RIF resistance results were low (1.2 and 0.9%, respectively). CONCLUSIONS: The updated Xpert assay retained the high sensitivity and specificity of the previous assay versions and demonstrated low rates of non-determinate and RIF resistance false positive results.

Bioterrorism: a laboratory who does it?

In October 2001, the first disseminated biological warfare attack was perpetrated on American soil. Initially, a few clinical microbiology laboratories were testing specimens from acutely ill patients and also being asked to test nasal swabs from the potentially exposed. Soon after, a significant number of clinical microbiology and public health laboratories received similar requests to test the worried well or evaluate potentially contaminated mail or environmental materials, sometimes from their own break rooms. The role of the clinical and public health microbiology laboratory in response to a select agent event or act of bioterrorism is reviewed.

Direct detection of drug-resistant Mycobacterium tuberculosis using targeted next generation sequencing.

Mycobacterium tuberculosis complex (MTBC) infections are treated with combinations of antibiotics; however, these regimens are not as efficacious against multidrug and extensively drug resistant MTBC. Phenotypic (growth-based) drug susceptibility testing on slow growing bacteria like MTBC requires many weeks to months to complete, whereas sequencing-based approaches can predict drug resistance (DR) with reduced turnaround time. We sought to develop a multiplexed, targeted next generation sequencing (tNGS) assay that can predict DR and can be performed directly on clinical respiratory specimens. A multiplex PCR was designed to amplify a group of thirteen full-length genes and promoter regions with mutations known to be involved in resistance to first- and second-line MTBC drugs. Long-read amplicon libraries were sequenced with Oxford Nanopore Technologies platforms and high-confidence resistance mutations were identified in real-time using an in-house developed bioinformatics pipeline. Sensitivity, specificity, reproducibility, and accuracy of the tNGS assay was assessed as part of a clinical validation study. In total, tNGS was performed on 72 primary specimens and 55 MTBC-positive cultures and results were compared to clinical whole genome sequencing (WGS) performed on paired patient cultures. Complete or partial susceptibility profiles were generated from 82% of smear positive primary specimens and the resistance mutations identified by tNGS were 100% concordant with WGS. In addition to performing tNGS on primary clinical samples, this assay can be used to sequence MTBC cultures mixed with other mycobacterial species that would not yield WGS results. The assay can be effectively implemented in a clinical/diagnostic laboratory with a two to three day turnaround time and, even if batched weekly, tNGS results are available on average 15 days earlier than culture-derived WGS results. This study demonstrates that tNGS can reliably predict MTBC drug resistance directly from clinical specimens or cultures and provide critical information in a timely manner for the appropriate treatment of patients with DR tuberculosis.

Mycobacterium tuberculosis complex whole-genome sequencing in New York State: Implementation of a reduced phenotypic drug susceptibility testing algorithm.

Whole-genome sequencing (WGS) can predict drug resistance and antimicrobial susceptibility in Mycobacterium tuberculosis complex (MTBC) and has shown promise in partially replacing culture-based phenotypic drug susceptibility testing (pDST). We performed a two-year side by side study comparing the prediction of drug resistance and antimicrobial susceptibility by WGS molecular DST (mDST) to pDST to determine resistance at the critical concentration by Mycobacterial Growth Indicator Tube (MGIT) and agar proportion testing. Negative predictive values of WGS results were consistently high for the first-line drugs: rifampin (99.9%), isoniazid (99.0%), pyrazinamide (98.5%), and ethambutol (99.8%); the rates of resistance to these drugs, among strains in our population, are 2.9%, 10.4%, 46.3%, and 2.3%, respectively. WGS results were available an average 8 days earlier than first-line MGIT pDST. Based on these findings, we implemented a new testing algorithm with an updated WGS workflow in which strains predicted pan-susceptible were no longer tested by pDST. This algorithm was applied to 1177 isolates between October 2018 and September 2020, eliminating pDST for 66.6% of samples and reducing pDST for an additional 22.0%. This algorithm change resulted in faster turnaround times and decreased cost while maintaining comprehensive antimicrobial susceptibility profiles of all culture-positive MTBC cases in New York.

Phenotypic and molecular characterization of Solobacterium moorei isolates from patients with wound infection.

Though seldom reported, Solobacterium moorei, which was first described in 2000, has been identified in specimens from patients with root canals, periradicular lesions, periodontal disease, dentoalveolar abscesses, bacteremia, septic thrombophlebitis, and halitosis. In the present study, we describe 9 cases of mixed wound infection, from a pool of 400 surgical wound infections that we have studied, in which S. moorei was isolated or found in a clone library. All isolates of S. moorei were identified by 16S rRNA gene sequence analysis, and then six were examined for their physiological and biochemical characteristics and for antimicrobial susceptibility. The results of the present study indicate that Solobacterium moorei may be a significant component in some mixed surgical wound infections and that surgical management and antimicrobial therapy may be indicated when these bacteria are identified in significant situations.

Characterization of Slackia exigua isolated from human wound infections, including abscesses of intestinal origin.

Eleven clinical strains isolated from infected wound specimens were subjected to polyphasic taxonomic analysis. Sequence analysis of the 16S rRNA gene showed that all 11 strains were phylogenetically related to Slackia exigua. Additionally, conventional and biochemical tests of 6 of the 11 strains were performed as supplementary methods to obtain phenotypic identification by comparison with the phenotypes of the relevant type strains. S. exigua has been considered an oral bacterial species in the family Coriobacteriaceae. This organism is fastidious and grows poorly, so it may easily be overlooked. The 16S rRNA gene sequences and the biochemical characteristics of four of the S. exigua strains isolated for this study from various infections indicative of an intestinal source were almost identical to those of the validated S. exigua type strain from an oral source and two of the S. exigua strains from oral sources evaluated in this study. Thus, we show for the first time that S. exigua species can be isolated from extraoral infections as well as from oral infections. The profiles of susceptibility to selected antimicrobials of this species were also investigated for the first time.

Comparative Performance of the Reverse Algorithm Using Architect Syphilis TP Versus the Traditional Algorithm Using Rapid Plasma Reagin in Florida's Public Health Testing Population.

In Florida, where syphilis is a reportable disease, the number of primary and secondary (P&S) syphilis cases has increased from 3,266 in 2008-2010 to 5,340 in 2013-2015, a 63% increase. The objective of this study was to compare the performance and sensitivity of the syphilis reverse algorithm with the traditional algorithm for detecting P&S (infectious) syphilis cases. Clinical specimens from individuals who self-referred for syphilis testing at public health clinics were processed using the traditional algorithm (non-treponemal rapid plasma reagin (RPR) test followed by a confirmatory treponemal (EIA) test) and then further tested with the Architect Syphilis TP (ASTP) immunoassay (Abbott Diagnostics, Chicago, IL, USA) or by RPR confirmation, if needed (reverse algorithm). Of 1,079 specimens, 59 were positive for syphilis. The sensitivity of the reverse algorithm was 98.3% (58/59) and of the traditional algorithm was 72.9% (43/59). Based on clinical evidence, of the 16 traditional algorithm-negative but reverse algorithm-positive cases, 68.8% (11/16) were classified as missed P&S infections (treatment naïve) and 31.2% (5/16) were classified as missed past syphilis (latent or infections with documented linkage to care). The reverse algorithm enables the detection of additional P&S syphilis cases missed by our current traditional algorithm.

Direct transmission of within-host Mycobacterium tuberculosis diversity to secondary cases can lead to variable between-host heterogeneity without de novo mutation: A genomic investigation.

BACKGROUND: Whole genome sequencing (WGS) has enabled the development of new approaches to track Mycobacterium tuberculosis (Mtb) transmission between tuberculosis (TB) cases but its utility may be challenged by the discovery that Mtb diversifies within hosts. Nevertheless, there is limited data on the presence and degree of within-host evolution. METHODS: We profiled a well-documented Mtb transmission cluster with three pulmonary TB cases to investigate within-host evolution and describe its impact on recent transmission estimates. We used deep sequencing to track minority allele frequencies (<50·0% abundance) during transmission and standard treatment. FINDINGS: Pre-treatment (n = 3) and serial samples collected over 2 months of antibiotic treatment (n = 16) from all three cases were analysed. Consistent with the epidemiological data, zero fixed SNP separated all genomes. However, we identified six subclones between the three cases with an allele frequency ranging from 35·0% to 100·0% across sampling intervals. Five subclones were identified within the index case pre-treatment and shared with one secondary case, while only the dominant clone was observed in the other secondary case. By tracking the frequency of these heterogeneous alleles over the two-month therapy, we observed distinct signatures of drift and negative selection, but limited evidence for de novo mutations, even under drug pressure. INTERPRETATION: We document within-host Mtb diversity in an index case, which led to transmission of minority alleles to a secondary case. Incorporating data on heterogeneous alleles may refine our understanding of Mtb transmission dynamics. However, more evidence is needed on the role of transmission bottleneck on observed heterogeneity between cases.

Penicillin G-resistant viridans group streptococcal endocarditis and interpretation of the American Heart Association's Guidelines for the Treatment of Infective Endocarditis.

We report a case of endocarditis due to a penicillin-"resistant" Streptococcus parasanguinis, discuss interpretations of the American Heart Association's guidelines for the treatment of viridans group streptococcal infection, and comment on therapy for infective endocarditis due to penicillin-resistant viridans group streptococci.

Accuracy and reproducibility of the Etest to detect drug-resistant Neisseria gonorrhoeae to contemporary treatment.

PURPOSE: Neisseria gonorrhoeae is a sexually transmitted bacterial pathogen that continues to evolve to become resistant to known antibiotics. In preparing for potential emergence, the Centers for Disease Control and Prevention recommends that clinical laboratories maintain or develop protocols to assess antibiotic susceptibly for this organism. This study examines the intra-laboratory variability of using the Etest method to provide consistent MIC values for N. gonorrhoeae and also compared the results of the Etest to known agar dilution MIC values. METHODOLOGY: Clinical N. gonorrhoeae isolates, 100 paired duplicates, were tested by eight laboratories for antibiotic susceptibility to ceftriaxone, cefixime and azithromycin using Etest strips.Results/Key findings. Overall, >80 % of the paired Etest MIC values were within one log2 dilution of the replicate. When compared to the agar dilution reference method, the cefixime Etest MIC values were consistently underreported by one dilution (seven laboratories) or two dilutions (one laboratory). The azithromycin Etest MIC values agreed 90.7 % with the agar dilution MIC values while the agreement with ceftriaxone was 90.9 %. CONCLUSION: Overall, the Etest method yielded reproducible MIC values within each laboratory with the azithromycin and ceftriaxone MIC results consistent to the reference agar dilution method while the cefixime result tended to provide a lower MIC value.

Associations between filarial and gastrointestinal nematodes.

The possibility that positive or negative associations occur between the filarial nematode Onchocerca volvulus and the gastrointestinal nematodes Ascaris lumbricoides and Trichuris trichiura was investigated in 205 children attending three schools in central Cameroon. Despite the closeness of their locations, marked differences between the schools were detected with respect to the number of species carried, the prevalence of O. volvulus and T. trichiura and the interaction between the prevalence of these two species. The number of species carried and these same prevalences varied significantly across the narrow age range of the study group. In addition, we observed an interaction between the prevalences of O. volvulus and A. lumbricoides that was dependent upon school but independent of host age and sex. Quantitative analyses revealed that the abundances of O. volvulus, A. lumbricoides and T. trichiura were affected by both school and age whereas host sex was significant only for O. volvulus. Finally, we observed significant positive interactions between the intensities of A. lumbricoides and T. trichiura and between O. volvulus and T. trichiura, that were school, age and sex independent. Our data suggest that associations do occur between filarial and gastrointestinal nematodes and that certain individuals are prone to multiple and high-level infections.