The emergence and spread of antibiotic resistance among pathogenic bacteria has been a rising problem for public health in recent decades. It is becoming increasingly recognized that not only ...antibiotic resistance genes (ARGs) encountered in clinical pathogens are of relevance, but rather, all pathogenic, commensal as well as environmental bacteria-and also mobile genetic elements and bacteriophages-form a reservoir of ARGs (the resistome) from which pathogenic bacteria can acquire resistance via horizontal gene transfer (HGT). HGT has caused antibiotic resistance to spread from commensal and environmental species to pathogenic ones, as has been shown for some clinically important ARGs. Of the three canonical mechanisms of HGT, conjugation is thought to have the greatest influence on the dissemination of ARGs. While transformation and transduction are deemed less important, recent discoveries suggest their role may be larger than previously thought. Understanding the extent of the resistome and how its mobilization to pathogenic bacteria takes place is essential for efforts to control the dissemination of these genes. Here, we will discuss the concept of the resistome, provide examples of HGT of clinically relevant ARGs and present an overview of the current knowledge of the contributions the various HGT mechanisms make to the spread of antibiotic resistance.
According to the current guidelines for laboratory diagnosis of sexually transmitted infections (STIs), nucleic acid amplification tests (NAATs) are the preferred diagnostic method for Chlamydia ...trachomatis (CT) infections. However, NAATs amplify the available target DNA without discriminating between DNA originating from viable or non-viable CT. Assessing CT viability will provide more insights in the clinical and public health relevance of a CT positive test result. The aim of this study was to technically validate and implement viability-PCR (V-PCR) to asses CT viability.
Technical validation of V-PCR was performed by the assessment of predefined viability ratios of CT. Samples were subjected to V-PCR which consisted of propidium monoazide (PMA) treatment prior to DNA extraction followed by quantitative PCR (qPCR) targeting the ompA gene for the detection of CT DNA. Finally, V-PCR was applied to vaginal swabs of 50 CT positive patients, as indicated by routine NAAT, collected at our outpatient STD clinics before antimicrobial treatment.
Technical validation of V-PCR showed that PMA treatment of heat-inactivated CT culture resulted in an almost complete loss of qPCR signal. PMA treated samples of the fresh viable CT culture showed no marked reduction of PCR signal, indicating that all DNA from viable CT could be detected. Applying V-PCR to clinical samples showed that in 36% of samples (18/50) less than 1% of CT DNA originated from viable bacteria.
V-PCR showed to be a fast and easy method to assess CT viability in clinical samples, without the need of traditional challenging cell culture methods. Furthermore, V-PCR results of clinical samples have indicated that a substantial amount of the amplified CT DNA originated from non-viable cells. Although results might be influenced by cell death during transport, this study suggests that there is a potential overestimation of quantitative CT positivity by currently used NAATs.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
There has been a growing body of evidence that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant (B.1.617.2) shows enhanced transmissibility and increased viral ...loads compared to other variants. A recent study has even suggested that respiratory samples from people infected with the Delta variant can harbor up to 1000 times higher viral loads compared to samples with variants that are more closely related to the original Wuhan strain, although the sample size of this study (n = 125) was very limited. Here, we have compared the viral load in 16,185 samples that were obtained in periods during which non-VOC, the Alpha (B.1.1.7) or Delta variant (B.1.617.2) were dominant as evidenced by genomic surveillance. We found that the Delta variant contained about fourfold higher viral loads across all age groups compared to the non-VOC or Alpha variants, which is significantly lower than reported earlier. Interestingly, the increased viral load for the Delta variant seemed to be age-dependent, regardless of sex, as the viral load was about 14-fold higher for Delta compared to the non-VOC or Alpha variant in age group 0–20 years and fourfold higher in age group 21–40 years, while there was no difference in viral load between variants in age groups 41–60 and 61+ years, most likely as a consequence of a higher degree of vaccination in the older age groups.
The bacterial load of Chlamydia trachomatis (CT) is assumed to play a role in transmission and sequelae. We assessed urogenital CT cycle quantification (Cq) values, as an indicator for CT load, of ...men and women diagnosed by general practitioners (GPs), hospital physicians and the STI clinic.
Urogenital CT-positive samples (n = 2,055 vaginal swabs, n = 77 cervical swabs, n = 1,519 urine samples and n = 19 urethral swabs) diagnosed by GPs, hospital physicians and the STI clinic from the Maastricht Medical Microbiology Laboratory were included (2012-2016). The outcome measure 'urogenital Cq values' was used as an inversely proportional measure for CT load. Among all patients, multivariate linear regression analyses were used to assess primary determinants for mean urogenital Cq values, stratified by sex. Additional clinical determinants were assessed among STI clinic patients.
In men, mean urogenital Cq values were similar between GPs, hospital physicians and the STI clinic (32.7 and 33.5 vs. 32.7; p>0.05). Women visiting the GP had lower urogenital Cq values than women visiting the STI clinic (30.2 vs. 30.9; p = <0.001). Women visiting the hospital had higher urogenital Cq values than women visiting the STI clinic (32.4 vs. 30.9; p = <0.001). Among STI clinic women, urogenital Cq values were lower in women with concurrent anorectal CT and in rectally untested women compared to anorectal CT-negative women (30.7 and 30.6 vs. 33.9; p = <0.001).
Men visiting different STI care providers had similar urogenital Cq values, which could be an indicator for similar CT loads. The lower Cq values of women visiting the GP compared to women visiting the STI clinic could be an indicator for higher CT loads and likely higher transmission potential. Notably, urogenital Cq values of STI clinic women were much lower (>3 Cq) when STI clinic women also had anorectal CT. This finding could indicate higher urogenital CT loads and likely higher chances of transmission and sequelae.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Objectives We evaluated COVID-19 symptoms, case fatality rate (CFR), and viral load among all Long-Term Care Facility (LTCF) residents and staff in South Limburg, the Netherlands (February 2020-June ...2020, wildtype SARS-CoV-2 Wuhan strain). Methods Patient information was gathered via regular channels used to notify the public health services. Ct-values were obtained from the Maastricht University Medical Centre laboratory. Logistic regression analyses were performed to assess associations between COVID-19, symptoms, CFR, and viral load. Results Of 1,457 staff and 1,540 residents, 35.1% and 45.2% tested positive for COVID-19. Symptoms associated with COVID-19 for female staff were fever, cough, muscle ache and loss of taste and smell. Associated symptoms for men were cough, and loss of taste and smell. Associated symptoms for residents were subfebrility, fatigue, and fever for male residents only. LTCF residents had a higher mean viral load compared to staff. Male residents had a higher CFR (35.8%) compared to women (22.5%). Female residents with Ct-values 31 or less had increased odds of mortality. Conclusions Subfebrility and fatigue seem to be associated with COVID-19 in LTCF residents. Therefore, physicians should also consider testing residents who (only) show aspecific symptoms whenever available resources prohibit testing of all residents. Viral load was higher in residents compared to staff, and higher in male residents compared to female residents. All COVID-19 positive male residents, as well as female residents with a medium to high viral load (Ct-values 31 or lower) should be monitored closely, as these groups have an overall increased risk of mortality.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A new two-step filtration protocol followed by a real-time PCR assay based on SYBR green I detection was developed to directly quantitate salmonellae in two types of biological samples: i.e., chicken ...rinse and spent irrigation water. Four prefiltration filters, one type of final filter, and six protocols for recovery of salmonellae from the final filter were evaluated to identify an effective filtration protocol. This method was then combined with a real-time PCR assay based on detection of the invA gene. The best results were obtained by subsequent filtration of 100 ml of chicken rinse or 100 ml of spent irrigation water through filters with pore diameters of >40 μm to remove large particles and of 0.22 μm to recover the Salmonella cells. After this, the Salmonella cells were removed from the filter by vortexing in 1 ml of physiological saline, and this sample was then subjected to real-time quantitative PCR. The whole procedure could be completed within 3 h from sampling to quantitation, and cell numbers as low as 7.5 x 10² CFU per 100-ml sample could be quantified. Below this limit, qualitative detection of concentrations as low as 2.2 CFU/100 ml sample was possible on occasion. This study has contributed to the development of a simple, rapid, and reliable method for quantitation of salmonellae in food without the need for sample enrichment or DNA extraction.
Prospective studies are key study designs when attempting to unravel health mechanisms that are widely applicable. Understanding the internal validity of a prospective study is essential to judge a ...study's quality. Moreover, insights in possible sampling bias and the external validity of a prospective study are useful to judge the applicability of a study's findings. We evaluated participation, retention, and associated factors of women in a multicenter prospective cohort (FemCure) to understand the study's validity.Chlamydia trachomatis (CT) infected adult women, negative for HIV, syphilis, and Neisseria gonorrhoeae were eligible to be preselected and included at three sexually transmitted infection (STI) clinics in the Netherlands (2016-2017). The planned follow-up for participants was 3 months, with two weekly rectal and vaginal CT self-sampling and online questionnaires administered at home and at the clinic. We calculated the proportions of preselected, included, and retained (completed follow-up) women. Associations with non-preselection, noninclusion, and non-retention (called attrition) were assessed (logistic and Cox regression).Among the 4,916 women, 1,763 (35.9%) were preselected, of whom 560 (31.8%) were included. The study population had diverse baseline characteristics: study site, migration background, high education, and no STI history were associated with non-preselection and noninclusion. Retention was 76.3% (n = 427). Attrition was 10.71/100 person/month (95% confidence interval 9.97, 12.69) and was associated with young age and low education. In an outpatient clinical setting, it proved feasible to include and retain women in an intensive prospective cohort. External validity was limited as the study population was not representative (sampling bias), but this did not affect the internal validity. Selective attrition, however (potential selection bias), should be accounted for when interpreting the study results.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Chlamydia trachomatis (CT) is the most common bacterial sexually transmitted infection (STI) worldwide. CT is mainly asymptomatic. Test-and-treat strategies are widely implemented to prevent ...transmission and complications. Strategies are not without controversy in asymptomatic women and men who have sex with men (MSM). Concerns are emerging to test and treat asymptomatic persons for urogenital CT ('Controversy 1') and pharyngeal or rectal CT ('Controversy 2'), whereby testing symptomatic persons is not under debate. Opposed views in CT treatment involve using azithromycin versus doxycycline ('Controversy 3'). The objective of this review is to provide coverage of these public health and clinical controversies by reviewing the current scientific evidence.
A literature search was performed using PubMed for relevant publications between 2018 and September 2021, and iterative retrieval of additional relevant publications.
Controversy 1. In women, the majority of asymptomatic CT are at the urogenital site, and detections mostly include viable CT. CT easily transmits to a partner and potentially also between the vaginal and rectal areas; the clinical impact of urogenital CT is established, although risks for adverse outcomes are uncertain. Wide-scale testing in asymptomatic women has not resulted in reduced prevalence. In MSM, evidence for the clinical impact of asymptomatic urogenital CT is lacking. Controversy 2. Rectal CT is common in women diagnosed with urogenital CT, but the clinical impact of asymptomatic rectal CT is uncertain. In MSM, rectal CT is common, and most CT infections are at the rectal site, yet the risk of longer term complications is unknown. In both sexes, pharyngeal CT is uncommon and has no documented clinical impact. Controversy 3. In the treatment of rectal CT, doxycycline has superior effectiveness to azithromycin. Evidence has also accumulated on the harms of test-and-treat strategies.
Current practices vary widely, from widescale test-and-treat approaches to more individual patient- and partner-level case management. Choosing which asymptomatic people to test at what anatomic site, and whether to test or not, requires an urgent (re-)definition of the goals of testing and treating asymptomatic persons. Treatment guidelines are shifting toward universal doxycycline use, and clinical practice now faces the challenge of implementation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We investigated the effect of international travel on the gut resistome of 122 healthy travelers from the Netherlands by using a targeted metagenomic approach. Our results confirm high acquisition ...rates of the extended-spectrum β-lactamase encoding gene blaCTX-M, documenting a rise in prevalence from 9.0% before travel to 33.6% after travel (p<0.001). The prevalence of quinolone resistance encoding genes qnrB and qnrS increased from 6.6% and 8.2% before travel to 36.9% and 55.7% after travel, respectively (both p<0.001). Travel to Southeast Asia and the Indian subcontinent was associated with the highest acquisition rates of qnrS and both blaCTX-M and qnrS, respectively. Investigation of the associations between the acquisitions of the blaCTX-M and qnr genes showed that acquisition of a blaCTX-M gene was not associated with that of a qnrB (p = 0.305) or qnrS (p = 0.080) gene. These findings support the increasing evidence that travelers contribute to the spread of antimicrobial drug resistance.
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DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
For Chlamydia trachomatis (CT), a test of cure (TOC) within 3-5 weeks is not recommended. International guidelines differ in advising a Neisseria gonorrhoeae (NG) TOC. Retesting CT and NG positives ...within 3-12 months is recommended in international guidelines. We assessed TOC and retesting practices including extragenital testing in general practitioner (GP) practices located in different socioeconomic status (SES) areas to inform and optimize local test practices.
Laboratory data of 48 Dutch GP practices between January 2011 and July 2016 were used. Based on a patient's first positive CT or NG test, the proportion of TOC (<3 months) and retests (3-12 months) were calculated. Patient- and GP-related factors were assessed using multivariate logistic regression analyses.
For CT (n = 622), 20% had a TOC and 24% had a retest at the GP practice. GP practices in low SES areas were more likely to perform a CT TOC (OR:1.8;95%CI:1.1-3.1). Younger patients (<25 years) were more likely to have a CT TOC (OR:1.6;95%CI:1.0-2.4). For CT (n = 622), 2.4% had a TOC and 6.1% had a retest at another STI care provider. For NG (n = 73), 25% had a TOC and 15% had a retest at the GP practice. For NG (n = 73), 2.7% had a TOC and 12.3% had a retest at another STI care provider. In only 0.3% of the consultations patients were tested on extragenital sites.
Almost 20% of the patients returned for a CT TOC, especially at GP practices in low SES areas. For NG, 1 out of 4 patients returned for a TOC. Retesting rates were low for both CT (24%) and NG (15%), (re)infections including extragenital infections may be missed. Efforts are required to focus TOC and increase retesting practices of GPs in order to improve CT/NG control.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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