P3-S6.08 Detection of the 23S rRNA point mutations (A2058G and A2059G) associated with azithromycin resistance in treponema pallidum using a TaqMan-based real-time Triplex-PCR assay

ABSTRACT We describe a real-time PCR assay for the discrimination of azithromycin-resistant and -susceptible strains of Treponema pallidum. This assay is rapid and allows for as many as 30 clinical specimens to be analyzed simultaneously without the need for DNA sequencing.

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Treponema pallidum in female sex workers from the Brazilian Marajó Archipelago: prevalence, risk factors, drug-resistant mutations and coinfections

Transactions of the Royal Society of Tropical Medicine and Hygiene ◽

10.1093/trstmh/traa127 ◽

2020 ◽

Author(s):

Evelen C Coelho ◽

Samara B Souza ◽

Camila Carla S Costa ◽

Luana M Costa ◽

Luiz Marcelo L Pinheiro ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Sex Workers ◽

Female Sex Workers ◽

Treponema Pallidum ◽

Point Mutations ◽

Unprotected Sex ◽

23S Rrna ◽

Factors Associated ◽

Female Sex

Abstract Background Female sex workers (FSWs) are an especially vulnerable group for syphilis and other sexually transmitted infection (STIs). This study determined the prevalence of syphilis in FSWs and factors associated with this disease in the Marajó Archipelago (northern Brazil), as well as the frequency of point mutations (A2058G and A2059G) in the 23S rRNA gene of Treponema pallidum and coinfections with hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis D virus (HDV). Methods FSWs were diagnosed using a rapid qualitative test and the isolates were evaluated for the presence of point mutations by real-time PCR. Blood samples with T. pallidum were tested for the presence of HBV, HCV and HDV by ELISA and confirmed by real-time PCR. The factors associated with syphilis were identified using Poisson regression models. Results Overall, 41.1% FSWs tested positive for syphilis and 23.5% were infected with strains having A2058G/A2059G point mutations. HBV (23.0%) and HCV (8.1%) were detected among FSWs with syphilis. Six factors were associated with syphilis: low levels of education, reduced income, drug use, unprotected sex, a lengthy career in prostitution and a lack of regular medical check-ups. Conclusions These findings indicate an urgent need for implementation of effective strategies to diagnose, prevent and treat syphilis, as well as other STIs, in this Brazilian region.

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P1.09 Diagnosingmycoplasma genitalium: comparison of four nucleic acid amplification tests and use of the speedx real-time pcr assay for azithromycin resistance

10.1136/sextrans-2017-053264.117 ◽

2017 ◽

Author(s):

Charlotte Gaydos ◽

Justin Hardick ◽

Susan Tuddenham ◽

Maria Trent

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Nucleic Acid Amplification ◽

Pcr Assay ◽

Nucleic Acid Amplification Tests ◽

Azithromycin Resistance

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Development and Application of Cas13a-based Diagnostic Assay for Neisseria Gonorrhoeae Detection and Identification of Azithromycin Resistance

10.1101/2021.05.20.445076 ◽

2021 ◽

Author(s):

Hao Luo ◽

Wentao Chen ◽

Zhi-Da Mai ◽

Xiaomian Lin ◽

Jianjiang Yang ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Clinical Decision Making ◽

Point Mutations ◽

Test Group ◽

Dual Therapy ◽

23S Rrna ◽

Resistance Testing ◽

Rrna Gene ◽

Resistance Mutations ◽

Azithromycin Resistance

Gonorrhea caused by Neisseria gonorrhoeae has spread world-wide. Antimicrobial-resistant strains have emerged to an alarming level to most antibiotics, including to the ceftriaxone-azithromycin combination, currently recommended as first-line dual therapy. Rapid testing for antimicrobial resistance will contribute to clinical decision-making for rational drug use and will slow this trend. Herein, we developed a Cas13a-based assay for N. gonorrhoeae detection (porA target) and azithromycin resistance identification (A2059G and C2611T point mutations). We evaluated the sensitivity and specificity of this method, and 10 copies per reaction can be achieved in porA detection and C2611T identification, with no cross-reactions. Comparison of the Cas13a-based assay (porA target) with Roche Cobas 4800 assay (n=23 urine samples) revealed 100% concordance. Isolated N. gonorrhoeae strains were used to validate the identification of A2059G and C2611T resistance mutations. All tested strains (8 A2059G strains, 8 C2611T strains, and 8 wild-type strains) were successfully distinguished by our assay and verified by testing MIC for azithromycin and sequencing the 23S rRNA gene. We adopted lateral flow for the SHERLOCK assay readout, which showed a visible difference between test group and NC group results. To further evaluate the capability of our assay, we tested 27 urethral swabs from patients with urethritis for N. gonorrhoeae detection and azithromycin-resistance identification. Of these, 62.96% (17/27) strains were detected with no mutant strains and confirmed by sequencing. In conclusion, the novel Cas13a-based assay for rapid and accurate N. gonorrhoeae detection combined with azithromycin drug resistance testing is a promising assay for application in clinical practice.

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A Real-Time PCR Assay for Rapid Identification of Inducible and Acquired Clarithromycin Resistance in Mycobacterium abscessus

10.21203/rs.3.rs-38897/v5 ◽

2020 ◽

Author(s):

Meenu Kaushal Sharma ◽

Yanni La ◽

Debra Janella ◽

Hafid Soualhine

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Acquired Resistance ◽

Rapid Identification ◽

Mycobacterium Abscessus ◽

23S Rrna ◽

Pcr Assay ◽

Constitutive Resistance ◽

Clarithromycin Resistance ◽

Severe Infections

Abstract Background: Mycobacterium abscessus is a rapidly growing mycobacteria involved in severe infections of the lung, skin, or soft tissue. Macrolides such as clarithromycin are the recommended first line drugs for treatment of M. abscessus infections. However, M. abscessus has dual mechanisms of resistance to macrolides, making treatment by macrolides difficult. A functional erm(41) gene confers for inducible resistance while acquired mutations on the 23S rRNA rrl gene confer for constitutive resistance.Methods: We have developed a real-time PCR assay to detect both inducible and acquired resistance to clarithromycin, and compared the results to traditional erm(41) and rrl sequencing and phenotypic susceptibility testing using Sensititre™ plates. Results: Of the total 126 M. abscessus isolates tested, truncated erm(41) was found in 23/126 (18.3%) of the samples, 27/126 (21.4%) had a T28C mutation in erm(41), and 2/126 (1.6%) had an acquired A2058C mutation in rrl. The phenotypic results correlated with the expected sequencing results in 121/126 samples (96%). Phenotypic testing compared to real-time PCR resolved 2 of these discrepancies by showing the existence of both erm(41) alleles in the isolates that sequencing missed. One culture was found to be mixed with two M. abscessus subsp. as per hsp65 sequencing and 2 isolates had discordance between molecular and phenotypic results. It was presumed that 3 isolates showed discrepancy between sequencing and real-time PCR, but one culture was mixed and other 2 detected both alleles by real-time PCR leading to 100% concordance when compared to sequencing.Conclusion: In conclusion, real-time PCR is more accurate for detection of both acquired and induced clarithromycin resistance, specifically when mixed genic profiles are present in a sample.

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A real-time PCR assay for rapid identification of inducible and acquired clarithromycin resistance in Mycobacterium abscessus

BMC Infectious Diseases ◽

10.1186/s12879-020-05686-0 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Meenu Kaushal Sharma ◽

Yanni La ◽

Debra Janella ◽

Hafid Soualhine

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Acquired Resistance ◽

Rapid Identification ◽

Mycobacterium Abscessus ◽

23S Rrna ◽

Pcr Assay ◽

Constitutive Resistance ◽

Clarithromycin Resistance ◽

Severe Infections

Abstract Background Mycobacterium abscessus is a rapidly growing mycobacteria involved in severe infections of the lung, skin, or soft tissue. Macrolides such as clarithromycin are the recommended first line drugs for treatment of M. abscessus infections. However, M. abscessus has dual mechanisms of resistance to macrolides, making treatment by macrolides difficult. A functional erm(41) gene confers for inducible resistance while acquired mutations on the 23S rRNA rrl gene confer for constitutive resistance. Methods We have developed a real-time PCR assay to detect both inducible and acquired resistance to clarithromycin, and compared the results to traditional erm(41) and rrl sequencing and phenotypic susceptibility testing using Sensititre™ plates. Results Of the total 126 M. abscessus isolates tested, truncated erm(41) was found in 23/126 (18.3%) of the samples, 27/126 (21.4%) had a T28C mutation in erm(41), and 2/126 (1.6%) had an acquired A2058C mutation in rrl. The phenotypic results correlated with the expected sequencing results in 121/126 samples (96%). Phenotypic testing compared to real-time PCR resolved 2 of these discrepancies by showing the existence of both erm(41) alleles in the isolates that sequencing missed. One culture was found to be mixed with two M. abscessus subsp. as per hsp65 sequencing and 2 isolates had discordance between molecular and phenotypic results. It was presumed that 3 isolates showed discrepancy between sequencing and real-time PCR, but one culture was mixed and other 2 detected both alleles by real-time PCR leading to 100% concordance when compared to sequencing. Conclusion In conclusion, real-time PCR is more accurate for detection of both acquired and induced clarithromycin resistance, specifically when mixed genic profiles are present in a sample.

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