No widespread dissemination of Chlamydia trachomatis diagnostic-escape variants and the impact of Neisseria gonorrhoeae positivity on the Aptima Combo 2 assay

ObjectivesA Finnish Chlamydia trachomatis (CT) new variant was detected in 2019 that escaped detection in the Hologic Aptima Combo 2 (AC2) assay due to a C1515T mutation in the CT 23S rRNA target region. Reflex testing of CT-negative/CT-equivocal specimens as well as those positive for Neisseria gonorrhoeae (NG) with the Hologic Aptima CT (ACT) assay was recommended to identify any CT variants.MethodsFrom June to October 2019, specimens with discrepant AC2/ACT CT results were submitted to Public Health England and screened for detectable CT DNA using an inhouse real-time (RT)-PCR. When enough DNA was present, partial CT 23S rRNA gene sequencing was performed. Analysis of available relative light units and interpretative data was performed.ResultsA total of 317 discordant AC2/ACT specimens were collected from 315 patients. Three hundred were tested on the RT-PCR; 53.3% (n=160) were negative and 46.7% (n=140) were positive. Due to low DNA load in most specimens, sequencing was successful for only 36 specimens. The CT 23S rRNA wild-type sequence was present in 32 specimens, and two variants with C1514T or G1523A mutation were detected in four specimens from three patients. Of the discordant specimens with NG interpretation, 36.6% of NG-negative/CT-negative AC2 specimens had detectable CT DNA on the inhouse RT-PCR vs 53.3% of NG-positive/CT-negative specimens.ConclusionsNo widespread dissemination of AC2 diagnostic-escape CT variants has occurred in England. We however identified the impact of NG positivity on the discordant AC2/ACT specimens; a proportion appeared due to NG positivity and the associated NG signal, rather than any diagnostic-escape variants or low DNA load. Several patients with gonorrhoea may therefore receive false-negative AC2 CT results. Single diagnostic targets and multiplex diagnostic assays have their limitations such as providing selection pressure for escape mutants and potentially reduced sensitivity, respectively. These limitations must be considered when establishing diagnostic pathways.

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The Finnish New Variant of Chlamydia trachomatis with a Single Nucleotide Polymorphism in the 23S rRNA Target Escapes Detection by the Aptima Combo 2 Test

Microorganisms ◽

10.3390/microorganisms7080227 ◽

2019 ◽

Vol 7 (8) ◽

pp. 227 ◽

Cited By ~ 8

Author(s):

Kati Hokynar ◽

Kaisu Rantakokko-Jalava ◽

Antti Hakanen ◽

Marika Havana ◽

Laura Mannonen ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Chlamydia Trachomatis ◽

False Negative ◽

23S Rrna ◽

Rrna Gene ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Continuous Increase ◽

New Variant ◽

23S Rrna Gene

In 2019, more than 200 cases of Chlamydia trachomatis negative/equivocal by the Aptima Combo 2 assay (AC2, target: 23S rRNA) with slightly elevated relative light units (RLUs), but positive by the Aptima Chlamydia trachomatis assay (ACT, target: 16S rRNA) have been detected in Finland To identify the cause of the AC2 CT false-negative specimens, we sequenced parts of the CT 23S rRNA gene in 40 specimens that were AC2 negative/equivocal but ACT positive. A single nucleotide polymorphism (SNP; C1515T in the C. trachomatis 23S rRNA gene) was revealed in 39 AC2/ACT discordant specimens. No decrease in the number of mandatorily notified C. trachomatis cases was observed nationally in Finland in 2010–2019. When RLUs obtained for AC2 negative specimens were retrospectively evaluated in 2011–2019, a continuous increase in the proportion of samples with RLUs 10–19 was observed since 2014, and a slight increase in the proportion of samples with RLUs 20–84 in 2017–2019, indicating that the Finnish new variant of C. trachomatis might have been spreading nationally for several years. This emphasizes that careful surveillance of epidemiology, positivity rate and test performance are mandatory to detect any changes affecting detection of infections.

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Finnish new variant of Chlamydia trachomatis escaping detection in the Aptima Combo 2 assay also present in Örebro County, Sweden, May 2019

Eurosurveillance ◽

10.2807/1560-7917.es.2019.24.26.1900370 ◽

2019 ◽

Vol 24 (26) ◽

Cited By ~ 10

Author(s):

Magnus Unemo ◽

Marit Hansen ◽

Ronza Hadad ◽

Ylva Lindroth ◽

Hans Fredlund ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

23S Rrna ◽

Rrna Gene ◽

New Variant ◽

23S Rrna Gene ◽

International Awareness

We identified the first two cases of the Finnish new variant of Chlamydia trachomatis (F-nvCT) beyond Finland in two clinical urogenital specimens in Örebro County, Sweden. These Aptima Combo 2 assay-negative specimens were Aptima Chlamydia trachomatis (CT) assay positive and had the characteristic C1515T mutation in the 23S rRNA gene. From 22 March to 31 May 2019, 1.3% (2/158) of the CT-positive cases in Örebro County were missed because of the F-nvCT. International awareness, investigations and actions are essential.

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Molecular pathways to high-level azithromycin resistance in Neisseria gonorrhoeae

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkab084 ◽

2021 ◽

Cited By ~ 1

Author(s):

J G E Laumen ◽

S S Manoharan-Basil ◽

E Verhoeven ◽

S Abdellati ◽

I De Baetselier ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Ribosomal Proteins ◽

Efflux Pump ◽

23S Rrna ◽

Rrna Gene ◽

23S Rrna Gene ◽

Evolution Of Resistance ◽

High Level ◽

Azithromycin Resistance ◽

Level Resistance

Abstract Background The prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide. Objectives To characterize the genetic pathways leading to high-level azithromycin resistance. Methods A customized morbidostat was used to subject two N. gonorrhoeae reference strains (WHO-F and WHO-X) to dynamically sustained azithromycin pressure. We tracked stepwise evolution of resistance by whole genome sequencing. Results Within 26 days, all cultures evolved high-level azithromycin resistance. Typically, the first step towards resistance was found in transitory mutations in genes rplD, rplV and rpmH (encoding the ribosomal proteins L4, L22 and L34 respectively), followed by mutations in the MtrCDE-encoded efflux pump and the 23S rRNA gene. Low- to high-level resistance was associated with mutations in the ribosomal proteins and MtrCDE efflux pump. However, high-level resistance was consistently associated with mutations in the 23S ribosomal RNA, mainly the well-known A2059G and C2611T mutations, but also at position A2058G. Conclusions This study enabled us to track previously reported mutations and identify novel mutations in ribosomal proteins (L4, L22 and L34) that may play a role in the genesis of azithromycin resistance in N. gonorrhoeae.

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Molecular characterization of a ceftriaxone-resistant Neisseria gonorrhoeae strain found in Switzerland: a case report

Annals of Clinical Microbiology and Antimicrobials ◽

10.1186/s12941-021-00456-5 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Konrad Egli ◽

Anna Roditscheff ◽

Ursula Flückiger ◽

Martin Risch ◽

Lorenz Risch ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

23S Rrna ◽

Rrna Gene ◽

Limited Information ◽

Specific Pcr ◽

Appropriate Treatment ◽

23S Rrna Gene ◽

Allele Type ◽

Ciprofloxacin Resistance

Abstract Background The resistance of Neisseria gonorrhoeae to ceftriaxone is unusual in Switzerland. The underlying genotype responsible for resistance is suspected to be novel. Generally, resistance in Neisseria gonorrhoeae (Ng) involves a comprehensive set of genes with many different mutations leading to resistance to different β-lactams and fluoroquinolones. Case presentation A patient had a positive result from specific PCR for Ng. We routinely culture all clinical specimens with a positive NG-PCR. In this particular case, we isolated a strain with resistance to ceftriaxone in Switzerland. A total of seven different genes (penA, ponA, porinB, mtr, gyrA, parC, 23S rRNA gene) in this strain were partially sequenced for comparison with phenotypic susceptibility testing. Interestingly, two different mutations in the porinB gene were observed, and data on this gene are limited. Information on the identified allele type of the penA gene is very limited as well. Three different mutations of parC and gyrA that correlate with ciprofloxacin resistance were found. The combination of ceftriaxone and ciprofloxacin resistance makes an appropriate treatment difficult to obtain due to multidrug resistance. Conclusion The combined results for all genes show the appearance of new mutations in central Europe either due to worldwide spread or the emergence of new genetic combinations of mutations.

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Performance of Unobserved Self-Collected Nasal Swabs for Detection of SARS-CoV-2 by RT-PCR Utilizing a Remote Specimen Collection Strategy

Open Forum Infectious Diseases ◽

10.1093/ofid/ofab039 ◽

2021 ◽

Author(s):

Ron M Kagan ◽

Amy A Rogers ◽

Gwynngelle A Borillo ◽

Nigel J Clarke ◽

Elizabeth M Marlowe

Keyword(s):

Return To Work ◽

Screening Program ◽

False Negative ◽

N Gene ◽

Rt Pcr ◽

Pooled Testing ◽

Specimen Collection ◽

Asymptomatic Patients ◽

Nasal Swabs ◽

The Impact

Abstract Background The use of a remote specimen collection strategy employing a kit designed for unobserved self-collection for SARS-CoV-2 RT-PCR can decrease the use of PPE and exposure risk. To assess the impact of unobserved specimen self-collection on test performance, we examined results from a SARS-CoV-2 qualitative RT-PCR test for self-collected specimens from participants in a return-to-work screening program and assessed the impact of a pooled testing strategy in this cohort. Methods Self-collected anterior nasal swabs from employee return to work programs were tested using the Quest Diagnostics SARS-CoV-2 RT-PCR EUA. The Ct values for the N1 and N3 N-gene targets and a human RNase P (RP) gene control target were tabulated. For comparison, we utilized Ct values from a cohort of HCP-collected specimens from patients with and without COVID-19 symptoms. Results Among 47,923 participants, 1.8% were positive. RP failed to amplify for 13/115,435 (0.011%) specimens. The median (IQR) Cts were 32.7 (25.0-35.7) for N1 and 31.3 (23.8-34.2) for N3. Median Ct values in the self-collected cohort were significantly higher than those of symptomatic, but not asymptomatic patients. Based on Ct values, pooled testing with 4 specimens would have yielded inconclusive results in 67/1,268 (5.2%) specimens but only a single false-negative result. Conclusions Unobserved self-collection of nasal swabs provides adequate sampling for SARS-CoV-2 RT-PCR testing. These findings alleviate concerns of increased false negatives in this context. Specimen pooling could be used for this population as the likelihood of false negative results is very low due when using a sensitive, dual-target methodology.

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Long-Term Exposure of Agricultural Soil to Veterinary Antibiotics Changes the Population Structure of Symbiotic Nitrogen-Fixing Rhizobacteria Occupying Nodules of Soybeans (Glycine max)

Applied and Environmental Microbiology ◽

10.1128/aem.00109-18 ◽

2018 ◽

Vol 84 (9) ◽

Cited By ~ 4

Author(s):

Cécile Revellin ◽

Alain Hartmann ◽

Sébastien Solanas ◽

Edward Topp

Keyword(s):

Agricultural Soil ◽

23S Rrna ◽

Rrna Gene ◽

Nodule Occupancy ◽

Nitrogen Fixing ◽

Swine Production ◽

Soybean Roots ◽

The Impact ◽

Field Plots

ABSTRACTAntibiotics are entrained in agricultural soil through the application of manures from medicated animals. In the present study, a series of small field plots was established in 1999 that receive annual spring applications of a mixture of tylosin, sulfamethazine, and chlortetracycline at concentrations ranging from 0.1 to 10 mg · kg−1soil. These antibiotics are commonly used in commercial swine production. The field plots were cropped continuously for soybeans, and in 2012, after 14 annual antibiotic applications, the nodules from soybean roots were sampled and the occupying bradyrhizobia were characterized. Nodules and isolates were serotyped, and isolates were distinguished using 16S rRNA gene and 16S to 23S rRNA gene intergenic spacer region sequencing, multilocus sequence typing, and RSα fingerprinting. Treatment with the antibiotic mixture skewed the population of bradyrhizobia dominating the nodule occupancy, with a significantly larger proportion ofBradyrhizobium liaoningenseorganisms even at the lowest dose of 0.1 mg · kg−1soil. Likewise, all doses of antibiotics altered the distribution of RSα fingerprint types. Bradyrhizobia were phenotypically evaluated for their sensitivity to the antibiotics, and there was no association betweenin situtreatment and a decreased sensitivity to the drugs. Overall, long-term exposure to the antibiotic mixture altered the composition of bradyrhizobial populations occupying nitrogen-fixing nodules, apparently through an indirect effect not associated with the sensitivity to the drugs. Further work evaluating agronomic impacts is warranted.IMPORTANCEAntibiotics are entrained in agricultural soil through the application of animal or human waste or by irrigation with reused wastewater. Soybeans obtain nitrogen through symbiotic nitrogen fixation. Here, we evaluated the impact of 14 annual exposures to antibiotics commonly used in swine production on the distribution of bradyrhizobia occupying nitrogen-fixing nodules on soybean roots in a long-term field experiment. By means of various sequencing and genomic fingerprinting techniques, the repeated exposure to a mixture of tylosin, sulfamethazine, and chlortetracycline each at a nominal soil concentration of 0.1 mg · kg−1soil was found to modify the diversity and identity of bradyrhizobia occupying the nodules. Nodule occupancy was not associated with the level of sensitivity to the antibiotics, indicating that the observed effects were not due to the direct toxicity of the antibiotics on bradyrhizobia. Altogether, these results indicate the potential for long-term impacts of antibiotics on this agronomically important symbiosis.

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Emergence and dissemination of three mild outbreaks of Neisseria gonorrhoeae with high-level resistance to azithromycin in Barcelona, 2016–18

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa536 ◽

2020 ◽

Author(s):

P Salmerón ◽

A Moreno-Mingorance ◽

J Trejo ◽

R Amado ◽

B Viñado ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Neisseria Gonorrhoeae ◽

23S Rrna ◽

Phylogenomic Analysis ◽

Rrna Gene ◽

Snp Analysis ◽

Enhanced Resolution ◽

23S Rrna Gene ◽

High Level ◽

Level Resistance

Abstract Background Neisseria gonorrhoeae (NG) isolates with high-level azithromycin resistance (HL-AziR) have emerged worldwide in recent decades, threatening the sustainability of current dual-antimicrobial therapy. Objectives This study aimed to characterize the first 16 NG isolates with HL-AziR in Barcelona between 2016 and 2018. Methods WGS was used to identify the mechanisms of antimicrobial resistance, to establish the MLST ST, NG multiantigen sequence typing (NG-MAST) ST and NG sequence typing for antimicrobial resistance (NG-STAR) ST and to identify the clonal relatedness of the isolates with other closely related NG previously described in other countries based on a whole-genome SNP analysis approach. The sociodemographic characteristics of the patients included in the study were collected by comprehensive review of their medical records. Results Twelve out of 16 HL-AziR isolates belonged to the MLST ST7823/NG-MAST ST5309 genotype and 4 to MLST ST9363/NG-MAST ST3935. All presented the A2059G mutation in all four alleles of the 23S rRNA gene. MLST ST7823/NG-MAST ST5309 isolates were only identified in men who have sex with women and MLST ST9363/NG-MAST ST3935 were found in MSM. Phylogenomic analysis revealed the presence of three transmission clusters of three different NG strains independently associated with sexual behaviour. Conclusions Our findings support the first appearance of three mild outbreaks of NG with HL-AziR in Spain. These results highlight the continuous capacity of NG to develop antimicrobial resistance and spread among sexual networks. The enhanced resolution of WGS provides valuable information for outbreak investigation, complementing the implementation of public health measures focused on the prevention and dissemination of MDR NG.

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Genomic characterization of emerging bacterial uropathogen Neisseria meningitidis misidentified as Neisseria gonorrhoeae by nucleic acid amplification testing

Journal of Clinical Microbiology ◽

10.1128/jcm.01699-20 ◽

2020 ◽

Author(s):

Kimberley V. Sukhum ◽

Sophonie Jean ◽

Meghan Wallace ◽

Neil Anderson ◽

Carey-Ann D. Burnham ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Neisseria Meningitidis ◽

Pathogenic Bacteria ◽

Time Frame ◽

Nucleic Acid Amplification ◽

Rrna Gene ◽

Urogenital System ◽

Urogenital Infection ◽

Routine Identification ◽

The Impact

Neisseria meningitidis (Nm) and Neisseria gonorrhoeae (Ng) are pathogenic bacteria that can cause human infections. While Nm infections are associated with bacterial meningitis and bacteremia, a strain of Nm, isolated from the urogenital system, has recently been associated with urethritis. As this strain is becoming prominent as an emerging pathogen, it is essential to assess identification tools for Nm and Ng urogenital isolates. Consecutive Nm isolates recovered from urogenital cultures of symptomatic patients with presumptive diagnoses of gonorrhea and a random selection of Ng isolates recovered from the same population within the same time frame were characterized with routine identification systems, antimicrobial susceptibility testing, and whole genome sequencing. MALDI-ToF MS, multilocus sequence typing, 16S rRNA gene sequence, and average nucleotide identity methods accurately identified 95% (18/19) of Nm and Ng isolates. 30% (3/10) of Nm isolates were misidentified as Ng with Aptima Combo 2 CT/NG but no misidentifications were found with the Xpert CT/NG NAAT. Phylogenetic core genome and SNP-based grouping analyses showed that urogenital Nm isolates were highly related, and phylogenetically distinct from Ng and respiratory Nm isolates but similar to urogenital Nm isolates from patients with urethritis in the US. Urogenital Nm isolates were predominantly azithromycin resistant while Ng isolates were azithromycin susceptible. These data indicate that urogenital isolates of Nm can cause false-positive detections with Ng diagnostic assays. Misidentification of urogenital Nm isolates may confound public health-related activities for gonorrhea and future studies are needed to understand the impact on clinical outcome of Nm urogenital infection.

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Verification of clinical samples, positive in AMPLICOR Neisseria gonorrhoeae polymerase chain reaction, by 16S rRNA and gyrA compared with culture

International Journal of STD & AIDS ◽

10.1258/0956462054094024 ◽

2005 ◽

Vol 16 (6) ◽

pp. 415-419 ◽

Cited By ~ 4

Author(s):

Åsa Airell ◽

Emma Lindbäck ◽

Ferda Ataker ◽

Kirsti Jalakas Pörnull ◽

Bengt Wretlind

Keyword(s):

Polymerase Chain Reaction ◽

16S Rrna ◽

Neisseria Gonorrhoeae ◽

False Negative ◽

Clinical Samples ◽

Rrna Gene ◽

Chain Reaction ◽

Gyra Gene ◽

Two Samples ◽

Polymerase Chain

We compared 956 samples for AMPLICOR Neisseria gonorrhoeae polymerase chain reaction (PCR) (Roche) with species verification using the 16S rRNA gene to verification using gyrA gene. Control was the culture method. The gyrA verification uses pyrosequencing of the quinolone resistance-determining region of gyrA. Of 52 samples with optical density ≥0.2 in PCR, 27 were negative in culture, two samples from pharynx were false negative in culture and four samples from pharynx were false positives in verification with 16S rRNA. Twenty-five samples showed growth of gonococci, 18 of the corresponding PCR samples were verified by both methods; three urine samples were positive only in gyrA ; and one pharynx specimen was positive only in 16S rRNA. Three samples were lost. We conclude that AMPLICOR N. gonorrhoeae PCR with verification in gyrA gene can be considered as a diagnostic tool in populations with low prevalence of gonorrhoea and that pharynx specimens should not be analysed by PCR.

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