APHL 2022 POSTER ABSTRACTS
Infectious Diseases
Can Antimicrobial-resistant Mycobacterium tuberculosis be Diagnosed Directly from Respiratory Specimens using Targeted Next Generation Sequencing ?
S Murphy , C Smith , T Halse , MC Rowlinson , V Escuyer , P LaPierre and K Musser , Wadsworth Center , New York State Department of Health
Mycobacterium tuberculosis ( MTB ) infections are treated with combination antimicrobial therapy ; however , inappropriate therapy with antimicrobials that are not efficacious against multi- and extensively drug-resistant MTB can result in treatment failure . Phenotypic antimicrobial susceptibility testing on slow growing bacteria like MTB requires many weeks to months to complete , whereas sequencing-based approaches can predict antimicrobial resistance ( AR ) with reduced turnaround time and can guide antimicrobial therapy in a timelier manner . The Wadsworth Center currently uses two sequencing-based approaches to predict AR in all NYS TB cases : whole genome sequencing ( WGS ) of MTB-positive cultures and direct pyrosequencing of select loci . We sought to develop a multiplexed , targeted next generation sequencing ( tNGS ) assay that can predict AR directly from respiratory specimens in two days for under $ 100 . PCR pools were designed to amplify thirteen full-length genes ( katG , rpoB , embB , gyrA / B , ethA , eis , rrs , rpsL ) or loci ( mabA-inhA , oxyR-ahpC , pncA , embCA ) with mutations known to confer AR to first line antibiotics ( isoniazid , rifampin , pyrazinamide , ethambutol ) or alternatives ( fluoroquinolones , ethionamide , streptomycin , kanamycin , amikacin ). PCR conditions were optimized and resulted in two PCR pools later combined for NGS . For assay validation , DNA was extracted ( EZ1 , Qiagen ) from MTB-positive or spiked ( H37Rv type strain ) respiratory specimens . Following PCR , amplicon libraries were prepared and sequenced using Oxford Nanopore MinION and analyzed with an in-house developed bioinformatics pipeline . The tNGS assay amplified targets from concentrations as low as 0.1 colony forming unit ( CFU ) per reaction and was determined to be specific for the MTB complex . Intra- and inter-assay reproducibility testing reliably detected pan-susceptible and multidrug-resistant MTB from clinical specimens ( n = 3 ). A blinded and randomized panel of 35 smear-positive retrospective specimens were used to assess tNGS accuracy ; most tests yielded sufficient coverage of all 13 targets ( 71 %) or 11 to 12 targets ( 11 %). All sequenced samples showed 100 % concordance with previous WGS test results . The tNGS samples that did not yield enough material for sequencing ( 17 %) were often from low positive smears ( 1 +) and all had higher Ct values by real-time PCR ( ExtRD9 target , Ct > 34 ). These data indicate that tNGS is highly sensitive and specific and can predict AR in MTB , similar to WGS assays , in the majority of smear-positive primary specimens .
Presenter : Shannon Murphy , Wadsworth Center , New York State Department of Health , shgmurphy @ gmail . com
Antifungal Susceptibility Profiles of Common and Rare Clinical Candida Species Collected 2017-2021 , New York
M Vaidya , V Chaturvedi and S Chaturvedi , Wadsworth Center , New York State Department of Health
Background : Pathogenic yeasts cause serious healthcareassociated infections ( HAIs ). Candida species are the fourth most common cause of HAIs in US hospitals . Currently , three different classes of antifungal agents are used to treat Candida infections . Antifungal susceptibility profiles and breakpoints for azoles and echinocandins are available for the common Candida species . However , the susceptibility breakpoints are not available for rare Candida species . Similarly , the geographical variations in the antifungal susceptibility profiles of common and rare pathogenic yeasts are less understood . Passive laboratory surveillance is an important tool to identify the prevalence of common and rare yeasts and their resistance patterns . In this study , we report antifungal susceptibility profiles of approximately 540 common and 330 rare clinical Candida isolates submitted by New York City and New York State hospitals for 2017-2021 .
Methods : Identification of all clinical isolates was performed by standard mycology procedures and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry ( MALDI-TOF- MS ). Rare yeasts were also confirmed with Sanger Sequencing of ribosomal RNA . Antifungal susceptibility testing was done using CLSI microbroth dilution method and E-tests .
Results : We analyzed 6,900 clinical Candida isolates between 2017-2021 . Of these , about 330 isolates were rare Candida species and 1,200 isolates were common Candida species . Antifungal susceptibility testing was performed on all 330 rare Candida isolates comprising 16 species and 540 of 1,200 isolates from seven common Candida species as per requests from the submitting facilities . Nearly one-fifth of rare Candida isolates comprised Candida fermentati with modest resistance to azoles and rare resistance to anidulafungin . Candida haemulonii and Candida duobushaemulonii constituted 10 % of rare yeast species with 100 % resistance to amphotericin B . Candida metapsilosis , Candida nivarensis and Candida orthopsilosis made up one-fourth of rare yeasts without any resistant isolates . Among 10 Candida blankii , eight isolates were azole-resistant and three isolates were echinocandin-resistant . Three of four Candida famata isolates were resistant to amphotericin B . Among the common Candida species , 49 % of the common yeasts were identified as Candida albicans and nearly 9 % were resistant to at least one of the azoles . Candida parapsilosis comprised 22 % of the common yeast isolates with 5 % resistance to azoles .
Conclusion : The antifungal susceptibility patterns of common and rare clinical Candida species in New York are consistent with published studies from a global collection of yeasts . Azole resistance was notable in common and rare Candida species . Amphotericin B and echinocandin resistance were rare and limited to a few rare Candida species .
Presenter : Mayuri Vaidya , Wadsworth Center , New York State Department of Health , mayuri . vaidya @ health . ny . gov