Investigating the cause of increased tetracycline-resistant Neisseria gonorrhoeae in England, 2016-20.

BACKGROUND: Antimicrobial resistance in Neisseria gonorrhoeae is a global public health concern. Tetracycline resistance (TetR) increased from 39.4% to 75.2% between 2016 and 2021 in N. gonorrhoeae isolates collected through national surveillance in England, despite the absence of use of tetracyclines for the treatment of gonorrhoea. OBJECTIVES: We investigated whether there was correlation between bacterial sexually transmitted infection (STI) tests performed and treatment with antimicrobials, with increased TetR in N. gonorrhoeae. METHODS: We examined correlations between bacterial STI tests, antimicrobial treatment and TetR in N. gonorrhoeae, using national surveillance data from three large sexual health services (SHS) in London during 2016-20. Doxycycline prescribing data and antibiograms of a non-STI pathogen from distinct patient groups (sexual health, obstetric and paediatric), at a large London hospital, were analysed to identify if doxycycline use in SHS was associated with resistance in a non-STI organism. RESULTS: A substantial increase in TetR was observed, particularly in isolates from gay, bisexual and other MSM (GBMSM). Strong positive correlations were observed exclusively in GBMSM between N. gonorrhoeae TetR and both bacterial STI tests (r = 0.97, P = 0.01) and antimicrobial treatment (r = 0.87, P = 0.05). Doxycycline prescribing increased dramatically during the study period in SHS. Prevalence of TetR in Staphylococcus aureus was higher in isolates sourced from SHS attendees than those from other settings. CONCLUSIONS: Frequent screening of GBMSM at higher risk of STIs, such as those on pre-exposure prophylaxis (PrEP) leading to/and increased use of doxycycline for the treatment of diagnosed infections, may account for the increase in TetR in N. gonorrhoeae.

High-resolution genotyping of Lymphogranuloma Venereum (LGV) strains of Chlamydia trachomatis in London using multi-locus VNTR analysis-ompA genotyping (MLVA-ompA).

BACKGROUND: Lymphogranuloma venereum (LGV) is caused by Chlamydia trachomatis strains with ompA genotypes L1 to L3. An LGV epidemic associated with the L2b genotype has emerged in the past few decades amongst men who have sex with men (MSM). C. trachomatis genotypes can be discriminated by outer membrane protein A gene (ompA) sequencing, however this method has limited resolution. This study employed a high-resolution genotyping method, namely, multi-locus tandem repeat (VNTR) analysis with ompA sequencing (MLVA-ompA), to assess the distribution of LGV MLVA-ompA genotypes amongst individuals attending genitourinary medicine (GUM) clinics in London. METHODS: Clinical specimens were collected from individuals attending eight London-based GUM clinics. Specimens that tested positive for C. trachomatis by commercial nucleic acid amplification test (NAAT) were confirmed as LGV by pmpH real-time PCR. LGV-positive DNA extracts were subsequently genotyped using MLVA-ompA. RESULTS: Two hundred and thirty DNA extracts were confirmed as LGV, and 162 (70%) yielded complete MLVA-ompA genotypes. Six LGV MLVA-ompA genotypes were identified: 1.9.2b-L2, 1.9.3b-L2b, 1.9.2b-L2b, 1.9.2b-L2b/D, 1.4a.2b-L2b, and 5.9.2b-L1. The following LGV ompA genotypes were identified (in descending order of abundance): L2, L2b, L2b/D, and L1. Eight ompA sequences with the hybrid L2b/D profile were detected. The hybrid sequence was identical to the ompA of a recombinant L2b/D strain detected in Portugal in 2017. CONCLUSIONS: The L2 ompA genotype was found to predominate in the London study population. The study detected an unusual hybrid L2b/D ompA profile that was previously reported in Portugal. We recommend further monitoring and surveillance of LGV strains within the UK population.

Sample Preparation Free Mass Spectrometry Using Laser-Assisted Rapid Evaporative Ionization Mass Spectrometry: Applications to Microbiology, Metabolic Biofluid Phenotyping, and Food Authenticity.

Mass spectrometry has established itself as a powerful tool in the chemical, biological, medical, environmental, and agricultural fields. However, experimental approaches and potential application areas have been limited by a traditional reliance on sample preparation, extraction, and chromatographic separation. Ambient ionization mass spectrometry methods have addressed this challenge but are still somewhat restricted in requirements for sample manipulation to make it suitable for analysis. These limitations are particularly restrictive in view of the move toward high-throughput and automated analytical workflows. To address this, we present what we consider to be the first automated sample-preparation-free mass spectrometry platform utilizing a carbon dioxide (CO2) laser for sample thermal desorption linked to the rapid evaporative ionization mass spectrometry (LA-REIMS) methodology. We show that the pulsatile operation of the CO2 laser is the primary factor in achieving high signal-to-noise ratios. We further show that the LA-REIMS automated platform is suited to the analysis of three diverse biological materials within different application areas. First, clinical microbiology isolates were classified to species level with an accuracy of 97.2%, the highest accuracy reported in current literature. Second, fecal samples from a type 2 diabetes mellitus cohort were analyzed with LA-REIMS, which allowed tentative identification of biomarkers which are potentially associated with disease pathogenesis and a disease classification accuracy of 94%. Finally, we showed the ability of the LA-REIMS system to detect instances of adulteration of cooking oil and determine the geographical area of production of three protected olive oil products with 100% classification accuracy.

Utilisation of Ambient Laser Desorption Ionisation Mass Spectrometry (ALDI-MS) Improves Lipid-Based Microbial Species Level Identification.

The accurate and timely identification of the causative organism of infection is important in ensuring the optimum treatment regimen is prescribed for a patient. Rapid evaporative ionisation mass spectrometry (REIMS), using electrical diathermy for the thermal disruption of a sample, has been shown to provide fast and accurate identification of microorganisms directly from culture. However, this method requires contact to be made between the REIMS probe and microbial biomass; resulting in the necessity to clean or replace the probes between analyses. Here, optimisation and utilisation of ambient laser desorption ionisation (ALDI) for improved speciation accuracy and analytical throughput is shown. Optimisation was completed on 15 isolates of Escherichia coli, showing 5 W in pulsatile mode produced the highest signal-to-noise ratio. These parameters were used in the analysis of 150 clinical isolates from ten microbial species, resulting in a speciation accuracy of 99.4% - higher than all previously reported REIMS modalities. Comparison of spectral data showed high levels of similarity between previously published electrical diathermy REIMS data. ALDI does not require contact to be made with the sample during analysis, meaning analytical throughput can be substantially improved, and further, increases the range of sample types which can be analysed in potential direct-from-sample pathogen detection.

An evaluation study of the Becton-Dickinson ProbeTec Qx (BDQx) Trichomonas vaginalis trichomoniasis molecular diagnostic test in two large, urban STD services.

OBJECTIVES: The BASHH guidelines recommend molecular tests to aid diagnosis of Trichomonas vaginalis (TV) infection; however many clinics continue to use relatively insensitive techniques (pH, wet-prep microscopy (WPM) and culture). Our objectives were to establish a laboratory pathway for TV testing with the Becton-Dickinson Qx (BDQx) molecular assay, to determine TV prevalence and to identify variables associated with TV detection. METHODS: A prospective study of 901 women attending two urban sexual health services for STI testing was conducted. Women were offered TV BDQx testing in addition to standard of care. Data collected were demographics, symptoms, results of near-patient tests and BDQx results for TV, Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC). Women with any positive TV result were treated and followed up for test of cure (TOC). RESULTS: 901 women had a TV BDQx test. 472 (53%) were white, 143 (16%) black and 499 (55%) were symptomatic. Infections detected by BDQx were: 11 TV (1.2%), three GC (0.3%) and 44 CT (4.9%). Of the 11 BDQx-detected TV infections, 8 (73%) were in patients of black ethnicity. Of these, four of seven cases (57%) were WPM-positive. All patients received treatment and nine of nine (100%) were BDQx-negative at TOC. In univariate analysis, only black ethnicity was associated with likelihood of a positive TV BDQx result (relative risk (RR) 10.2 (95% CI 2.15 to 48.4)). CONCLUSIONS: The use of the BDQ enhanced detection of TV in asymptomatic and symptomatic populations. Cost-effective implementation of the test will rely on further work to reliably detect demographic and clinical variables that predict positivity.

Effect of Electrode Geometry on the Classification Performance of Rapid Evaporative Ionization Mass Spectrometric (REIMS) Bacterial Identification.

The recently developed automated, high-throughput monopolar REIMS platform is suited for the identification of clinically important microorganisms. Although already comparable to the previously reported bipolar forceps method, optimization of the geometry of monopolar electrodes, at the heart of the system, holds the most scope for further improvements to be made. For this, sharp tip and round shaped electrodes were optimized to maximize species-level classification accuracy. Following optimization of the distance between the sample contact point and tube inlet with the sharp tip electrodes, the overall cross-validation accuracy improved from 77% to 93% in negative and from 33% to 63% in positive ion detection modes, compared with the original 4 mm distance electrode. As an alternative geometry, round tube shaped electrodes were developed. Geometry optimization of these included hole size, number, and position, which were also required to prevent plate pick-up due to vacuum formation. Additional features, namely a metal "X"-shaped insert and a pin in the middle were included to increase the contact surface with a microbial biomass to maximize aerosol production. Following optimization, cross-validation scores showed improvement in classification accuracy from 77% to 93% in negative and from 33% to 91% in positive ion detection modes. Supervised models were also built, and after the leave 20% out cross-validation, the overall classification accuracy was 98.5% in negative and 99% in positive ion detection modes. This suggests that the new generation of monopolar REIMS electrodes could provide substantially improved species level identification accuracies in both polarity detection modes. Graphical abstract.

Translational utility of a hierarchical classification strategy in biomolecular data analytics.

Hierarchical classification (HC) stratifies and classifies data from broad classes into more specific classes. Unlike commonly used data classification strategies, this enables the probabilistic prediction of unknown classes at different levels, minimizing the burden of incomplete databases. Despite these advantages, its translational application in biomedical sciences has been limited. We describe and demonstrate the implementation of a HC approach for "omics-driven" classification of 15 bacterial species at various taxonomic levels achieving 90-100% accuracy, and 9 cancer types into morphological types and 35 subtypes with 99% and 76% accuracy, respectively. Unknown bacterial species were probabilistically assigned with 100% accuracy to their respective genus or family using mass spectra (n = 284). Cancer types were predicted by mRNA data (n = 1960) for most subtypes with 95-100% accuracy. This has high relevance in clinical practice where complete datasets are difficult to compile with the continuous evolution of diseases and emergence of new strains, yet prediction of unknown classes, such as bacterial species, at upper hierarchy levels may be sufficient to initiate antimicrobial therapy. The algorithms presented here can be directly translated into clinical-use with any quantitative data, and have broad application potential, from unlabeled sample identification, to hierarchical feature selection, and discovery of new taxonomic variants.

Rapid Evaporative Ionisation Mass Spectrometry (REIMS) Provides Accurate Direct from Culture Species Identification within the Genus Candida.

Members of the genus Candida, such as C. albicans and C. parapsilosis, are important human pathogens. Other members of this genus, previously believed to carry minimal disease risk, are increasingly recognised as important human pathogens, particularly because of variations in susceptibilities to widely used anti-fungal agents. Thus, rapid and accurate identification of clinical Candida isolates is fundamental in ensuring timely and effective treatments are delivered. Rapid Evaporative Ionisation Mass Spectrometry (REIMS) has previously been shown to provide a high-throughput platform for the rapid and accurate identification of bacterial and fungal isolates. In comparison to commercially available matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF), REIMS based methods require no preparative steps nor time-consuming cell extractions. Here, we report on the ability of REIMS-based analysis to rapidly and accurately identify 153 clinical Candida isolates to species level. Both handheld bipolar REIMS and high-throughput REIMS platforms showed high levels of species classification accuracy, with 96% and 100% of isolates classified correctly to species level respectively. In addition, significantly different (FDR corrected P value < 0.05) lipids within the 600 to 1000 m/z mass range were identified, which could act as species-specific biomarkers in complex microbial communities.

Automated High-Throughput Identification and Characterization of Clinically Important Bacteria and Fungi using Rapid Evaporative Ionization Mass Spectrometry.

Rapid evaporative ionization mass spectrometry (REIMS) has been shown to quickly and accurately speciate microorganisms based upon their species-specific lipid profile. Previous work by members of this group showed that the use of a hand-held bipolar probe allowed REIMS to analyze microbial cultures directly from culture plates without any prior preparation. However, this method of analysis would likely be unsuitable for a high-throughput clinical microbiology laboratory. Here, we report the creation of a customized platform that enables automated, high-throughput REIMS analysis that requires minimal user input and operation and is suitable for use in clinical microbiology laboratories. The ability of this high-throughput platform to speciate clinically important microorganisms was tested through the analysis of 375 different clinical isolates collected from distinct patient samples from 25 microbial species. After optimization of our data analysis approach, we achieved substantially similar results between the two REIMS approaches. For hand-held bipolar probe REIMS, a speciation accuracy of 96.3% was achieved, whereas for high-throughput REIMS, an accuracy of 93.9% was achieved. Thus, high-throughput REIMS offers an alternative mass spectrometry based method for the rapid and accurate identification of clinically important microorganisms in clinical laboratories without any preanalysis preparative steps.

Characterization and identification of clinically relevant microorganisms using rapid evaporative ionization mass spectrometry.

Rapid evaporative ionization mass spectrometry (REIMS) was investigated for its suitability as a general identification system for bacteria and fungi. Strains of 28 clinically relevant bacterial species were analyzed in negative ion mode, and corresponding data was subjected to unsupervised and supervised multivariate statistical analyses. The created supervised model yielded correct cross-validation results of 95.9%, 97.8%, and 100% on species, genus, and Gram-stain level, respectively. These results were not affected by the resolution of the mass spectral data. Blind identification tests were performed for strains cultured on different culture media and analyzed using different instrumental platforms which led to 97.8-100% correct identification. Seven different Escherichia coli strains were subjected to different culture conditions and were distinguishable with 88% accuracy. In addition, the technique proved suitable to distinguish five pathogenic Candida species with 98.8% accuracy without any further modification to the experimental workflow. These results prove that REIMS is sufficiently specific to serve as a culture condition-independent tool for the identification and characterization of microorganisms.

Analysis of intact bacteria using rapid evaporative ionisation mass spectrometry.

An identification system for microorganisms based on recently developed rapid evaporative ionisation mass spectrometry (REIMS) is presented. Nine bacterial species cultured on various growth media were correctly identified to family-, genus-, and species-level based on their different mass spectral fingerprints using a cross-validated maximum margin criterion model.

Lymphogranuloma venereum in the UK in 2003.

Lymphogranuloma venereum in the United Kingdom was detected in 2004, following the introduction of enhanced surveillance. This report describes a previously undiagnosed case, detected retrospectively in a sample obtained in 2003 from a male patient with high-risk behaviour, indicating that this previously rare disease in Europe, was present in the UK in 2003.