MYCOPLASMA GENITALIUM FEASABILITY AND PITFALLS OF ADJUNCT TESTING FOR MACROLIDE RESISTANCE (a.k.a. no test is 100%)

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1 MYCOPLASMA GENITALIUM FEASABILITY AND PITFALLS OF ADJUNCT TESTING FOR MACROLIDE RESISTANCE (a.k.a. no test is 100%) Collette Bromhead, Peter Lowe, Litty Tan, Arlo Upton The New Zealand Sexual Health Society Annual Conference 7 th 9th th September 2017

2 Disclosures Hologic USA provided funding to Massey University for this study SpeeDx Pty Australia supplied their reagents free of charge for this study I have previously received conference and travel funding, and honoraria from Roche Diagnostics NZ Ltd

3 Usual symptoms of MG infection i.e. it s a problem because it can look like lots of other common STIs

4 But MG has superpowers It has no cell wall so it can bend into funny shapes Instrinsic resistance to penicillins and cephalosporins Attended the School of Antibiotic Resistance with the Neisseria cohort..

5 Mycoplasma s Can hide inside Trichomonads to avoid antibiotics Dessi et al Mycoplasma hominis and Trichomonas vaginalis: a unique case of symbiotic relationship between two obligate human parasites. Frontiers in Bioscience 11,

6 Rationale, aims and ethics Prevalence and antibiogram of MG in NZ is unknown and unmonitored Aimed to approximate the prevalence of MG in a cohort of 2643 Auckland clinical samples collected from Trial commercial PCR assays for MG and Macrolide Resistance SNPs 23SrRNA sequencing of resistant samples to identify epidemiology of individual resistance mutations How good is the kit at calling genotypic resistance? Ethical approval granted by Massey University Human Ethics Committee: SOA 16/18 and HDEC 15/CEN/136/AM01

7 Study Design 2643 Urine, urethral, vaginal and cervical swabs sent for STI testing Auckland region Aptima MG Test performed at Labtests Auckland 135 MG Positives MG Negative Aptima swab and urine samples de-identified, DNA extracted (column) at Massey University DNA tested by SpeeDx for MG,+ 5 x 18SrRNA mutations MG Positive MG Negative MG 23SrRNA amplified by HRM MG 23SrRNA products sequenced at NZGL (Massey) MG 23SrRNA genotype MG Positive MG Negative MG Macrolide Resistant or Sensistive

8 Aptima MG Results A total of 135 (5.1%) urogenital samples were positive Similar rate of infections in males (6.5%) and females (5.3%) High rate of co-infections, particularly with Chlamydia MG infection Co-infection CT Co-infection NG Co-infection TV Co-infection with another STI (%) (23) ** Data courtesy of Dr Arlo Upton, Labtests Auckland

9 SpeeDx MG Results Received 131 Aptima samples from 130 patients who were MG positive Of these 24 were males (18%) and 106 were females (82%) M. genitalium N = % Not Detected 51 39% Detected 80 61% Total 131 Large difference in sensitivity between Aptima and SpeeDx for MG Similar results shown by Le Roy et al J Clin Microbiol 2017

10 SpeeDx Macrolide Resistance Results Macrolide Susceptibility N = % Resistant 33 42% Susceptible 46 58% Total 79

11 Demographics SpeeDx - Results Gender M F Detected Not Detected Mutant 5 28 WT 9 37 % Mutant 35.7% 43.1% % Not Detected 41.7% 38.7% SpeeDx - Results Age yrs yrs yrs yrs >=55 yrs Detected Not Detected Mutant WT % Mutant 46.8% 39.1% 25.0% 25.0% 0.0% % Not Detected 29.9% 50.0% 63.6% 20.0% 0.0%

12 Sequencing vs SpeeDx Results SpeeDx 23S rrna Sequencing No Sequence Detected Not Detected Mutant WT WT Mutant KEY 1. Discrepant detection rates between both tests, in particular 8 samples MG positive by SpeeDx that couldn t be sequenced but there was agreement between the two methods on 64/79 (81%) of the samples 2. SpeeDx miscalled the genotypes of 7 (9%) samples: 1 was miscalled resistant 6 samples were miscalled susceptible Sensitivity for macrolide resistance = 83% Specificity = 97%

13 23S rrna Genotypes Sequenced Genotypes detected 27 x A2059G 8 x A2058G 4 x A2059C Tabrizi et al 2016 showed the limit of detection for the 23S rrna gene targets was: 10 copies for A2058C, A2058T and A2059G 12 copies for A2058G 15 copies for A2059C? Our cohort had a high rate of SNPs SpeeDx is less sensitive for?

14 Feasibility Aptima MG is ideal frontline screening assay and allows CT, NG, TV and MG on one sample Reflex SpeeDx MG ResPlus on Aptima MG positive samples for fast turnaround Unfortunately the Panther nukes its DNA extracts so can t use them on a third-party test without an interim step Hologic has a RUO assay for Macrolide R detection As sensitive as Aptima MG RT-PCR for 23s rrna; identifies WT and MUT in same population

15 Pitfalls of antimicrobial genotyping 1. There will be discordance between Aptima and SpeeDx MG results 2. Laboratory reports need to emphasise the possibility of an incorrect result 3. GENOTYPE PHENOTYPE 4. Clinicians need to be aware of partner discordance in susceptibility results: RETEST

16 Summary There is always a gap between the performance of screening assays vs specific genotyping assays and NO TEST HAS 100% PERFORMANCE The SpeeDx assay is user friendly and easy to implement as a reflex test with 83% sensitivity and 97% specificity for macrolide resistance mutations If there is a test result discrepancy affecting patient management, retest! Do not underestimate the ability of Mycoplasma genitalium to ruin your day

17 Acknowledgements Arlo Upton (LabTests Auckland) Peter Lowe (Hologic) for data analysis Litty Tan (SpeeDx) for being a LightCycler Ninja Karishma Deo (Massey) Liselle Bisessor (LabTests Auckland) FUNDING: Hologic USA SpeeDx Pty Ltd (Australia)

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