Resistance-mediating Mutations Research Articles

Genetic basis of macrolide resistance in porcine Pasteurella multocida isolates from the German national resistance monitoring program GERM-Vet 2008-2021.

To analyse porcine Pasteurella multocida isolates obtained from the national resistance monitoring program GERM-Vet 2008-2021 in Germany for phenotypic and genotypic macrolide resistance. The antimicrobial susceptibility of the isolates was determined by broth microdilution according to CLSI standards. Closed genomes were obtained by WGS via Illumina MiSeq and MinION platforms followed by a hybrid assembly. Of 1114 porcine P. multocida isolates, only four isolates (0.36%), one each from 2008, 2010, 2019 and 2021, exhibited resistance to at least one macrolide tested. The isolate from 2010 was only resistant to erythromycin and WGS analysis neither revealed a macrolide resistance gene nor a macrolide resistance-mediating mutation. The isolates from 2008 and 2019 were resistant to erythromycin, tilmicosin, tildipirosin, tulathromycin and gamithromycin and showed either only the A2058G mutation in all six 23S rRNA operons or the chromosomally located macrolide resistance genes msr(E) and mph(E), respectively. The isolate from 2021 was resistant to erythromycin, tulathromycin, gamithromycin and tylosin and carried a novel integrative and conjugative element of 64 966 bp, designated Tn7730, in its chromosomal DNA. It harboured the macrolide resistance genes mef(C), mph(G) and estT, the lincosamide resistance gene lnu(H), and the tetracycline resistance gene tet(Y), the last two were detected for the first time in P. multocida. Macrolide resistance in German porcine P. multocida can be due to resistance-mediating mutations or resistance genes. The presence of the novel Tn7730 carrying three different macrolide resistance genes is alarming and should be monitored.

Susceptibility of Ugandan Plasmodium falciparum Isolates to the Antimalarial Drug Pipeline.

Malaria, especially Plasmodium falciparum infection, remains an enormous problem, and its treatment and control are seriously challenged by drug resistance. New antimalarial drugs are needed. To characterize the Medicines for Malaria Venture pipeline of antimalarials under development, we assessed the ex vivo drug susceptibilities to 19 compounds targeting or potentially impacted by mutations in P. falciparum ABC transporter I family member 1, acetyl-CoA synthetase, cytochrome b, dihydroorotate dehydrogenase, elongation factor 2, lysyl-tRNA synthetase, phenylalanyl-tRNA synthetase, plasmepsin X, prodrug activation and resistance esterase, and V-type H+ ATPase of 998 fresh P. falciparum clinical isolates collected in eastern Uganda from 2015 to 2022. Drug susceptibilities were assessed by 72-h growth inhibition (half-maximum inhibitory concentration [IC50]) assays using SYBR green. Field isolates were highly susceptible to lead antimalarials, with low- to midnanomolar median IC50s, near values previously reported for laboratory strains, for all tested compounds. However, outliers with decreased susceptibilities were identified. Positive correlations between IC50 results were seen for compounds with shared targets. We sequenced genes encoding presumed targets to characterize sequence diversity, search for polymorphisms previously selected with in vitro drug pressure, and determine genotype-phenotype associations. We identified many polymorphisms in target genes, generally in <10% of isolates, but none were those previously selected in vitro with drug pressure, and none were associated with significantly decreased ex vivo drug susceptibility. Overall, Ugandan P. falciparum isolates were highly susceptible to 19 compounds under development as next-generation antimalarials, consistent with a lack of preexisting or novel resistance-conferring mutations in circulating Ugandan parasites. IMPORTANCE Drug resistance necessitates the development of new antimalarial drugs. It is important to assess the activities of compounds under development against parasites now causing disease in Africa, where most malaria cases occur, and to determine if mutations in these parasites may limit the efficacies of new agents. We found that African isolates were generally highly susceptible to the 19 studied lead antimalarials. Sequencing of the presumed drug targets identified multiple mutations in these genes, but these mutations were generally not associated with decreased antimalarial activity. These results offer confidence that the activities of the tested antimalarial compounds now under development will not be limited by preexisting resistance-mediating mutations in African malaria parasites.

Genetic Organization of Acquired Antimicrobial Resistance Genes and Detection of Resistance-Mediating Mutations in a Gallibacterium anatis Isolate from a Calf Suffering from a Respiratory Tract Infection

Gallibacterium (G.) anatis isolates associated with respiratory diseases in calves and harboring acquired antimicrobial resistance genes have been described in Belgium. The aim of this study was to analyze the genetic organization of acquired resistance genes in the G. anatis isolate IMT49310 from a German calf suffering from a respiratory tract infection. The isolate was submitted to antimicrobial susceptibility testing, and a closed genome was obtained by a hybrid assembly of Illumina MiSeq short-reads and MinION long-reads. Isolate IMT49310 showed elevated MIC values for macrolides, aminoglycosides, florfenicol, tetracyclines, and trimethoprim/sulfamethoxazole. The acquired resistance genes catA1, floR, aadA1, aadB, aphA1, strA, tet(M), tet(B), erm(B), and sul2 were identified within three resistance gene regions in the genome, some of which were associated with IS elements, such as ISVsa5-like or IS15DII. Furthermore, nucleotide exchanges within the QRDRs of gyrA and parC, resulting in amino acid exchanges S83F and D87A in GyrA and S80I in ParC, were identified. Even if the role in the pathogenesis of respiratory tract infections in cattle needs to be further investigated, the identification of a G. anatis isolate with reduced susceptibility to regularly used antimicrobial agents in cases of fatal bovine respiratory tract infections is worrisome, and such isolates might also act as a reservoir for antimicrobial resistance genes.

Plasma Sequencing for Patients with GIST-Limitations and Opportunities in an Academic Setting.

Circulating tumor DNA (ctDNA) from circulating free DNA (cfDNA) in GIST is of interest for the detection of heterogeneous resistance mutations and treatment monitoring. However, methodologies for use in a local setting are not standardized and are error-prone and difficult to interpret. We established a workflow to evaluate routine tumor tissue NGS (Illumina-based next generation sequencing) panels and pipelines for ctDNA sequencing in an academic setting. Regular blood collection (Sarstedt) EDTA tubes were sufficient for direct processing whereas specialized tubes (STRECK) were better for transportation. Mutation detection rate was higher in automatically extracted (AE) than manually extracted (ME) samples. Sensitivity and specificity for specific mutation detection was higher using digital droplet (dd)PCR compared to NGS. In a retrospective analysis of NGS and clinical data (133 samples from 38 patients), cfDNA concentration correlated with tumor load and mutation detection. A clinical routine pipeline and a novel research pipeline yielded different results, but known and resistance-mediating mutations were detected by both and correlated with the resistance spectrum of TKIs used. In conclusion, NGS routine panel analysis was not sensitive and specific enough to replace solid biopsies in GIST. However, more precise methods (hybridization capture NGS, ddPCR) may comprise important research tools to investigate resistance. Future clinical trials need to compare methodology and protocols.

Genomic and Evolutionary Analysis of Salmonella enterica Serovar Kentucky Sequence Type 198 Isolated From Livestock In East Africa.

Since its emergence in the beginning of the 90’s, multidrug-resistant (MDR) Salmonella enterica subsp. enterica serovar Kentucky has become a significant public health problem, especially in East Africa. This study aimed to investigate the antimicrobial resistance profile and the genotypic relatedness of Salmonella Kentucky isolated from animal sources in Ethiopia and Kenya (n=19). We also investigated population evolutionary dynamics through phylogenetic and pangenome analyses with additional publicly available Salmonella Kentucky ST198 genomes (n=229). All the 19 sequenced Salmonella Kentucky isolates were identified as ST198. Among these isolates, the predominant genotypic antimicrobial resistance profile observed in ten (59.7%) isolates included the aac(3)-Id, aadA7, strA-strB, bla TEM-1B, sul1, and tet(A) genes, which mediated resistance to gentamicin, streptomycin/spectinomycin, streptomycin, ampicillin, sulfamethoxazole and tetracycline, respectively; and gyrA and parC mutations associated to ciprofloxacin resistance. Four isolates harbored plasmid types Incl1 and/or Col8282; two of them carried both plasmids. Salmonella Pathogenicity islands (SPI-1 to SPI-5) were highly conserved in the 19 sequenced Salmonella Kentucky isolates. Moreover, at least one Pathogenicity Island (SPI 1–4, SPI 9 or C63PI) was identified among the 229 public Salmonella Kentucky genomes. The phylogenetic analysis revealed that almost all Salmonella Kentucky ST198 isolates (17/19) stemmed from a single strain that has accumulated ciprofloxacin resistance-mediating mutations. A total of 8,104 different genes were identified in a heterogenic and still open Salmonella Kentucky ST198 pangenome. Considering the virulence factors and antimicrobial resistance genes detected in Salmonella Kentucky, the implications of this pathogen to public health and the epidemiological drivers for its dissemination must be investigated.

Whole genome sequencing-based drug resistance predictions of multidrug-resistant Mycobacterium tuberculosis isolates from Tanzania.

BackgroundRifampicin- or multidrug-resistant (RR/MDR) Mycobacterium tuberculosis complex (MTBC) strains account for considerable morbidity and mortality globally. WGS-based prediction of drug resistance may guide clinical decisions, especially for the design of RR/MDR-TB therapies.MethodsWe compared WGS-based drug resistance-predictive mutations for 42 MTBC isolates from MDR-TB patients in Tanzania with the MICs of 14 antibiotics measured in the Sensititre™ MycoTB assay. An isolate was phenotypically categorized as resistant if it had an MIC above the epidemiological-cut-off (ECOFF) value, or as susceptible if it had an MIC below or equal to the ECOFF.ResultsOverall, genotypically non-wild-type MTBC isolates with high-level resistance mutations (gNWT-R) correlated with isolates with MIC values above the ECOFF. For instance, the median MIC value (mg/L) for rifampicin-gNWT-R strains was >4.0 (IQR 4.0–4.0) compared with 0.5 (IQR 0.38–0.50) in genotypically wild-type (gWT-S, P < 0.001); isoniazid-gNWT-R >4.0 (IQR 2.0–4.0) compared with 0.25 (IQR 0.12–1.00) among gWT-S (P = 0.001); ethionamide-gNWT-R 15.0 (IQR 10.0–20.0) compared with 2.50 (IQR; 2.50–5.00) among gWT-S (P < 0.001). WGS correctly predicted resistance in 95% (36/38) and 100% (38/38) of the rifampicin-resistant isolates with ECOFFs >0.5 and >0.125 mg/L, respectively. No known resistance-conferring mutations were present in genes associated with resistance to fluoroquinolones, aminoglycosides, capreomycin, bedaquiline, delamanid, linezolid, clofazimine, cycloserine, or p-amino salicylic acid.ConclusionsWGS-based drug resistance prediction worked well to rule-in phenotypic drug resistance and the absence of second-line drug resistance-mediating mutations has the potential to guide the design of RR/MDR-TB regimens in the future.

Isocitrate dehydrogenase gene variants in cancer and their clinical significance.

Human isocitrate dehydrogenase (IDH) genes encode for the IDH1, 2 & 3 isoenzymes which catalyse the formation of 2-oxoglutarate from isocitrate and are essential for normal mammalian metabolism. Although mutations in these genes in cancer were long thought to lead to a ‘loss of function’, combined genomic and metabolomic studies led to the discovery that a common IDH 1 mutation, present in low-grade glioma and acute myeloid leukaemia (AML), yields a variant (R132H) with a striking change of function leading to the production of (2R)-hydroxyglutarate (2HG) which consequently accumulates in large quantities both within and outside cells. Elevated 2HG is proposed to promote tumorigenesis, although the precise mechanism by which it does this remains uncertain. Inhibitors of R132H IDH1, and other subsequently identified cancer-linked 2HG producing IDH variants, are approved for clinical use in the treatment of chemotherapy-resistant AML, though resistance enabled by additional substitutions has emerged. In this review, we provide a current overview of cancer linked IDH mutations focussing on their distribution in different cancer types, the effects of substitution mutations on enzyme activity, the mode of action of recently developed inhibitors, and their relationship with emerging resistance-mediating double mutations.

Defining Fluoroquinolone Resistance-Mediating Mutations from Non-Resistance Polymorphisms in Mycoplasma hominis Topoisomerases.

Often dismissed as a commensal, Mycoplasma hominis is an increasingly prominent target of research due to its role in septic arthritis and organ transplant failure in immunosuppressed patients, particularly lung transplantation. As a mollicute, its highly reductive genome and structure render it refractile to most forms of treatment and growing levels of resistance to the few sources of treatment left, such as fluoroquinolones. We examined antimicrobial susceptibility (AST) to fluoroquinolones on 72 isolates and observed resistance in three (4.1%), with corresponding mutations in the quinolone resistance-determining region (QRDR) of S83L or E87G in gyrA and S81I or E85V in parC. However, there were high levels of polymorphism identified between all isolates outside of the QRDR, indicating caution for a genomics-led approach for resistance screening, particularly as we observed a further two quinolone-susceptible isolates solely containing gyrA mutation S83L. However, both isolates spontaneously developed a second spontaneous E85K parC mutation and resistance following prolonged incubation in 4 mg/L levofloxacin for an extra 24–48 h. Continued AST surveillance and investigation is required to understand how gyrA QRDR mutations predispose M. hominis to rapid spontaneous mutation and fluoroquinolone resistance, absent from other susceptible isolates. The unusually high prevalence of polymorphisms in M. hominis also warrants increased genomics’ surveillance.

High Prevalence of Macrolide and Fluoroquinolone Resistance-Mediating Mutations in Mycoplasma genitalium-Positive Urine Specimens From Saskatchewan.

Mycoplasma genitalium is an emerging, sexually transmitted infection, which is more prevalent than Chlamydia trachomatis in some regions. An increase in antibiotic resistance, that is, azithromycin and moxifloxacin, recommended for treating M. genitalium infections has been noted. This is the first detailed report on the prevalence of M. genitalium and its antimicrobial resistance in Saskatchewan, Canada. Aptima urine specimens (n = 1977), collected for the diagnosis of C. trachomatis/Neisseria gonorrhoeae, were tested for M. genitalium using the Aptima M. genitalium assay (MG-TMA). Antimicrobial resistance was ascertained using polymerase chain reaction and DNA sequencing of 23S rRNA (azithromycin) and parC (moxifloxacin) from Aptima M. genitalium assay-positive specimens; mutations predictive of resistance were noted. The prevalence of M. genitalium was 9.6% (189/1977). Predicted resistance to azithromycin (substitutions at positions 2058/2059 in 23S rRNA) was observed in 63.6% (70/110) of the specimens tested, whereas resistance to moxifloxacin (S83I in ParC) was observed in 10.6% (9/85) of the specimens. Mutations in both 23S rRNA and ParC were observed in 2.12% (4/189) of the specimens. Women aged 20 to 24 years had the highest prevalence (18.3%, P < 0.001), and in females, M. genitalium was significantly associated with C. trachomatis or N. gonorrhoeae/C. trachomatis (P < 0.001) coinfection. The prevalence of M. genitalium (9.6%) in the province of Saskatchewan was higher than that of the other 2 bacterial sexually transmitted infections (N. gonorrhoeae (3.09%) and C. trachomatis (6.85%). The prevalence of M. genitalium (9.6%) and associated resistance to azithromycin (63.6%) in Saskatchewan high, suggesting that empiric azithromycin therapy may not be adequate for treating M. genitalium infections.

Decreased Susceptibility to Dihydrofolate Reductase Inhibitors Associated With Genetic Polymorphisms in Ugandan Plasmodium falciparum Isolates.

The Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors pyrimethamine and cycloguanil (the active metabolite of proguanil) have important roles in malaria chemoprevention, but drug resistance challenges their efficacies. A new compound, P218, was designed to overcome resistance, but drug-susceptibility data for P falciparum field isolates are limited. We studied ex vivo PfDHFR inhibitor susceptibilities of 559 isolates from Tororo and Busia districts, Uganda, from 2016 to 2020, sequenced 383 isolates, and assessed associations between genotypes and drug-susceptibility phenotypes. Median half-maximal inhibitory concentrations (IC50s) were 42 100 nM for pyrimethamine, 1200 nM for cycloguanil, 13000 nM for proguanil, and 0.6 nM for P218. Among sequenced isolates, 3 PfDHFR mutations, 51I (100%), 59R (93.7%), and 108N (100%), were very common, as previously seen in Uganda, and another mutation, 164L (12.8%), had moderate prevalence. Increasing numbers of mutations were associated with decreasing susceptibility to pyrimethamine, cycloguanil, and P218, but not proguanil, which does not act directly against PfDHFR. Differences in P218 susceptibilities were modest, with median IC50s of 1.4 nM for parasites with mixed genotype at position 164 and 5.7 nM for pure quadruple mutant (51I/59R/108N/164L) parasites. Resistance-mediating PfDHFR mutations were common in Ugandan isolates, but P218 retained excellent activity against mutant parasites.

Evaluation of Clinical, Gram Stain, and Microbiological Cure Outcomes in Men Receiving Azithromycin for Acute Nongonococcal Urethritis: Discordant Cures Are Associated With Mycoplasma genitalium Infection.

In men with nongonococcal urethritis (NGU), clinicians and patients rely on clinical cure to guide the need for additional testing/treatment and when to resume sex, respectively; however, discordant clinical and microbiological cure outcomes do occur. How accurately clinical cure reflects microbiological cure in specific sexually transmitted infections (STIs) is unclear. Men with NGU were tested for Neisseria gonorrhoeae, Chlamydia trachomatis (CT), Mycoplasma genitalium (MG), Trichomonas vaginalis, urethrotropic Neisseria meningitidis ST-11 clade strains, and Ureaplasma urealyticum (UU). Men received azithromycin 1 g and returned for a 1-month test-of-cure visit. In MG infections, we evaluated for the presence of macrolide resistance-mediating mutations (MRMs) and investigated alternate hypotheses for microbiological treatment failure using in situ shotgun metagenomic sequencing, phylogenetic analysis, multilocus sequence typing analyses, and quantitative PCR. Of 280 men with NGU, 121 were included in this analysis. In the monoinfection group, 52 had CT, 16 had MG, 7 had UU, 10 had mixed infection, and 36 men had idiopathic NGU. Clinical cure rates were 85% for CT, 100% for UU, 50% for MG, and 67% for idiopathic NGU. Clinical cure accurately predicted microbiological cure for all STIs, except MG. Discordant results were significantly associated with MG-NGU and predominantly reflected microbiological failure in men with clinical cure. Mycoplasma genitalium MRMs, but not MG load or strain, were strongly associated with microbiological failure. In azithromycin-treated NGU, clinical cure predicts microbiological cure for all STIs, except MG. Nongonococcal urethritis management should include MG testing and confirmation of microbiological cure in azithromycin-treated MG-NGU when MRM testing is unavailable.

ISOLATION AND CHARACTERIZATION OF MULTIDRUG RESISTANT SALMONELLA SPECIES FROM ABATTOIR WASTEWATER IN ABIA STATE, NIGERIA

Over the years, zoonotic bacteria of the genus Salmonella, have acquired antimicrobial resistance properties, with a wide variety of resistance genes and resistance-mediating mutations being identified. This study isolated and characterized multiple drug resistant Salmonella species isolated from abattoir wastewaters in Abia State, Nigeria. Seven hundred wastewater samples from three abattoirs: Aba (300), Ubakala (250) and Lokpa (150) samples were analyzed from 2016 to 2019. Standard microbiological procedures were followed in isolation and identification of the Salmonella spp isolates. The antibiotic susceptibility test was done using the Kirby Bauer disk diffusion method. The results showed moderate but significant prevalence of Salmonella spp in the three abattoir locations; 135(45.0%) from Aba, 108 (43.2%) from Ubakala and 74 (49.3%) from Lokpa. The antibiotic susceptibility pattern of the Salmonella spp isolates show that the organisms were highly susceptible to Ofloxacin (84.4% in Aba, 89.8% in Ubakala and 82.4% in Lokpa) and highly resistant to Ampicillin (97.0% in Aba, 91.7% in Ubakala and 98.6% in Lokpa). Most isolates recorded Multiple Antibiotic Resistance (MAR) index greater than (&gt;0.2). This result emphasizes the urgent need for regulation in the use of antibiotics in man and animals and their subsequent disposal into the environment.

Workflow and Throughput of Commercial Assays to Detect Mycoplasma genitalium and Macrolide Resistance-Mediating Mutations.

Aptima Mycoplasma genitalium (MG) required the shortest and STD6 the longest time to detect MG in clinical samples. ResistancePlus MG detected MG and macrolide resistance-mediating mutations simultaneously. Times were influenced by specimen numbers. M. genitalium positives from the other 2 assays required increased time for macrolide resistance-mediating mutation sequencing.

Molecular detection of Mycoplasma genitalium in endocervical swabs and associated rates of macrolide and fluoroquinolone resistance in Hong Kong.

There is a global trend of increasing macrolide and fluoroquinolone resistance in Mycoplasma genitalium (MG), such that international guidelines recommend molecular detection of resistance if a patient has MG-positive test results. Tests for MG are not routinely performed in Hong Kong. This study examined the detection of MG in endocervical swabs and the associated macrolide and fluoroquinolone resistance rates. Endocervical swabs received from two sexual health clinics in Hong Kong for routine assessments of Chlamydia trachomatis and Neisseria gonorrhoeae were also subjected to detection of MG. All MG-positive samples were tested for resistance-mediating mutations in 23S rRNA, parC, and gyrA genes. Laboratory records and past results for each patient were analysed. In total, endocervical swabs from 285 patients were included in this study. Mycoplasma genitalium was detected in swabs from 21 patients (7.4%) by real-time polymerase chain reaction with a commercial kit. Among MG-positive samples which were successfully analysed further, macrolide resistance-mediating mutations in 23S rRNA were found in 42.1% (8/19); fluoroquinolone resistance-related mutations in parC and gyrA were found in 65% (13/20) and 0% (0/20), respectively. All macrolide-resistant MG strains were also fluoroquinolone-resistant (42.1%, 8/19). No assessed factors were associated with the detection of MG or resistance-related mutations. In Hong Kong, MG was detected in endocervical swabs from 7.4% of patients in sexual health clinics, with high rates of macrolide and fluoroquinolone resistance. These findings warrant careful review of testing, clinical correlation, and treatment strategies for MG in the context of increasing antibiotic resistance.

High Prevalence of Vaginal and Rectal Mycoplasma genitalium Macrolide Resistance Among Female Sexually Transmitted Disease Clinic Patients in Seattle, Washington.

Rectal Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) are increasingly recognized as common infections among women. Little is known about the prevalence of rectal Mycoplasma genitalium (MG), rectal MG/CT/GC coinfection, or MG antimicrobial resistance patterns among women. In 2017 to 2018, we recruited women at high risk for CT from Seattle's municipal sexually transmitted disease clinic. Participants self-collected vaginal and rectal specimens for CT/GC nucleic acid amplification testing. We retrospectively tested samples for vaginal and rectal MG using nucleic acid amplification testing and tested MG-positive specimens for macrolide resistance-mediating mutations (MRM) and ParC quinolone resistance-associated mutations (QRAMs). Of 50 enrolled women, 13 (26%) tested positive for MG, including 10 (20%) with vaginal MG and 11 (22%) with rectal MG; 8 (62%) had concurrent vaginal/rectal MG. Five (38%) were coinfected with CT, none with GC. Only 2 of 11 women with rectal MG reported anal sex in the prior year. Of MG-positive specimens, 100% of rectal and 89% of vaginal specimens had an MRM. There were no vaginal or rectal MG-positive specimens with ParC QRAMs previously associated with quinolone failure. Five MG-infected women received azithromycin for vaginal CT, 4 of whom had a MG MRM detected in their vaginal and/or rectal specimens. We observed a high prevalence of macrolide-resistant vaginal and rectal MG among a population of women at high risk for CT. This study highlights how the use of antimicrobials designed to treat an identified infection-in this case, CT-could influence treatment outcomes and antimicrobial susceptibility in other unidentified infections.

Clinical evaluation of the ResistancePlus MG FleXible test on the GeneXpert Infinity-48s instrument: a near-patient assay for simultaneous detection of Mycoplasma genitalium and macrolide resistance

Antibiotic resistance in Mycoplasma genitalium (MG) is rising globally, especially to macrolides. In response to this challenge, assays reporting both the detection of MG and macrolide resistance-mediating mutations (MRMM) allow therapy to be tailored to the individual. The study evaluated the performance of the ResistancePlus® MG FleXible assay for the detection of MG and MRMM. Overall, the test performed well for the detection of MG compared to the AllplexTM STI Essential assay, used as a reference, with a kappa value of 0.926 (95% CI, 0.863–0.990). The kit also performed well for the detection of MRMM when compared with Sanger sequencing of the 23S rRNA gene, with a kappa value of 0.901 (95% CI, 0.807–0.996). The rate of MRMM in MG among the study population was 41.8%. In conclusion, the ResistancePlus® MG FleXible is a rapid, simple, and accurate cartridge-based assay for simultaneous detection of MG and MRMM in clinical settings.

Mycoplasma genitalium macrolide resistance update: Rate among a 2016–2017 cohort of patients in Barcelona, Spain

IntroductionMycoplasma genitalium is a major cause of urethritis and other genital syndromes. Antibiotic resistance, especially to macrolides, is increasing at an alarming rate worldwide. The aim of this study was to estimate the rate of macrolide resistance in M. genitalium among a 2016–2017 cohort of patients in Barcelona, Spain; and to compare this estimate with previous data from 2013 to 2014 in this region. MethodsThe study was conducted retrospectively with M. genitalium-positive samples collected between December 2016 and February 2017 at the Hospital Vall d’Hebron Microbiology Department. Genotypic markers of macrolide resistance were primarily detected using the ResistancePlus® MG molecular assay (SpeeDx). Mutations were then confirmed by sequencing. ResultsMacrolide resistance-mediating mutations were detected in 30/83 infections (36.1% [95% CI, 25.9%–47.4%]). This resistance was more frequent among men who have sex with men (55.0% [95% CI, 38.5%–70.7%]) compared to heterosexual men (27.3% [95% CI, 10.7%–50.2%]) and women (9.5% [95% CI, 1.3%–30.4%]), p<0.001. Additionally, macrolide resistance did not significantly increase in this cohort when compared with previous investigations. ConclusionDespite the current notable rate of macrolide resistance in M. genitalium, resistance did not significantly increase between 2013–2014 and 2016–2017 in our region. Nevertheless, strict local surveillance and the implementation of rapid diagnostic tests that combine the detection of the bacterium and resistance-mediating mutations may facilitate the optimization of antibiotic administration and reduce the transmission of resistance in M. genitalium.

Evaluation of the ResistancePlus MG FleXible Assay for Detection of Wild-Type and 23S rRNA-Mutated Mycoplasma genitalium Strains.

Antibiotic resistance in Mycoplasma genitalium is rising globally, and resistance-guided diagnostics can facilitate targeted and timely treatment. The ResistancePlus MG FleXible (RPMG Flex) assay for the detection of M. genitalium and macrolide resistance-mediating mutations (MRMM) was evaluated for analytical sensitivity, specificity, reproducibility, and inhibition in the presence of interfering substances by simulating M. genitalium-negative pooled urine and swab matrices with M. genitalium cultures. Furthermore, the clinical sensitivity of the assay was evaluated and compared with a reference real-time PCR assay. The analytical sensitivity of the RPMG Flex assay was 157 genomes/ml for wild-type (WT) and 387 genomes/ml for MRMM strains in both matrices. For clinical specimens, the RPMG assay had an overall sensitivity of 96.1% (95% urine: 10/10 WT, 9/10 MRMM; 96.5% swab: 25/26 WT, 26/29 MRMM) compared to 85.7% for the MgPa/MagNAPure24 assay (95% urine: 19/20; 87% swab: 48/57). Clinical specificity was 100% for urine and 98.5% for swab specimens, respectively. No inhibition due to the presence of any of the tested interfering substances was observed. The RPMG Flex assay was more sensitive than the reference MgPa assay, in particular, for swab specimens. The implementation of this assay may increase ease of use and considerably decrease hands-on time for sample preparation compared to a standard block-based assay. The RPMG Flex assay for the GeneXpert Dx system provides a much-needed platform for the simultaneous detection of MG and MRMM and may thereby facilitate resistance-guided therapy for M. genitalium infections.

Macrolide-Resistant Mycoplasma genitalium in Southeastern Region of the Netherlands, 2014-2017.

Mycoplasma genitalium infections of the urogenital tract are usually treated with azithromycin; however, for the past several years, rates of azithromycin treatment failure have increased. To document the occurrence and frequency of macrolide resistance-mediating mutations (MRMMs) in M. genitalium infections, we collected 894 M. genitalium-positive samples during April 2014-December 2017 and retrospectively tested them for MRMMs. We designated 67 samples collected within 6 weeks after a positive result as test-of-cure samples; of these, 60 were MRMM positive. Among the remaining 827 samples, the rate of MRMM positivity rose from 22.7% in 2014 and 22.3% in 2015 to 44.4% in 2016 but decreased to 39.7% in 2017. Because of these high rates of MRMMs in M. genitalium infections, we recommend that clinicians perform tests of cure after treatment and that researchers further explore the clinical consequences of this infection.

Pediatric ALL relapses after allo-SCT show high individuality, clonal dynamics, selective pressure, and druggable targets

AbstractSurvival of patients with pediatric acute lymphoblastic leukemia (ALL) after allogeneic hematopoietic stem cell transplantation (allo-SCT) is mainly compromised by leukemia relapse, carrying dismal prognosis. As novel individualized therapeutic approaches are urgently needed, we performed whole-exome sequencing of leukemic blasts of 10 children with post–allo-SCT relapses with the aim of thoroughly characterizing the mutational landscape and identifying druggable mutations. We found that post–allo-SCT ALL relapses display highly diverse and mostly patient-individual genetic lesions. Moreover, mutational cluster analysis showed substantial clonal dynamics during leukemia progression from initial diagnosis to relapse after allo-SCT. Only very few alterations stayed constant over time. This dynamic clonality was exemplified by the detection of thiopurine resistance-mediating mutations in the nucleotidase NT5C2 in 3 patients' first relapses, which disappeared in the post–allo-SCT relapses on relief of selective pressure of maintenance chemotherapy. Moreover, we identified TP53 mutations in 4 of 10 patients after allo-SCT, reflecting acquired chemoresistance associated with selective pressure of prior antineoplastic treatment. Finally, in 9 of 10 children's post–allo-SCT relapse, we found alterations in genes for which targeted therapies with novel agents are readily available. We could show efficient targeting of leukemic blasts by APR-246 in 2 patients carrying TP53 mutations. Our findings shed light on the genetic basis of post–allo-SCT relapse and may pave the way for unraveling novel therapeutic strategies in this challenging situation.