A multicenter evaluation of the QIAstat-Dx meningitis-encephalitis syndromic test kit as compared to the conventional diagnostic microbiology workflow.

PURPOSE: Rapid diagnosis and treatment of infectious meningitis and encephalitis (ME) is critical to minimize morbidity and mortality. Recently, Qiagen introduced the CE-IVD QIAstat-Dx ME panel (QS-ME) for syndromic diagnostic testing of meningitis and encephalitis. Some data on the performance of the QS-ME in comparison to the BioFire FilmArray ME panel are available. In this study, the performance of the QS-ME is compared to the current diagnostic workflow in two academic medical centers in the Netherlands. METHODS: A total of 110 cerebrospinal fluid samples were retrospectively tested with the QS-ME. The results obtained were compared to the results of laboratory-developed real-time PCR assays (LDTs), IS-pro, bacterial culture, and cryptococcal antigen (CrAg) testing. In addition, the accuracy of the QS-ME was also investigated using an external quality assessment (EQA) panel consisting of ten samples. RESULTS: Four of the 110 samples tested failed to produce a valid QS-ME result. In the remaining 106 samples, the QS-ME detected 53/53 viral targets, 38/40 bacterial targets, and 7/13 Cryptococcus neoformans targets. The discrepant bacterial results consisted of two samples that were previously tested positive for Listeria monocytogenes (CT 35.8) and Streptococcus pneumoniae (CT 40), respectively. The QS-ME detected one additional result, consisting of a varicella-zoster virus signal (CT 35.9), in a sample in which both techniques detected Streptococcus pyogenes. Finally, 100% concordance was achieved in testing a blinded bacterial ME EQA panel. CONCLUSION: The QS-ME is a relevant addition to the syndromic testing landscape to assist in diagnosing infectious ME.

Rapid Diagnostics of Joint Infections Using IS-Pro.

Diagnosis of bone and joint infections (BJI) relies on microbiological culture which has a long turnaround time and is challenging for certain bacterial species. Rapid molecular methods may alleviate these obstacles. Here, we investigate the diagnostic performance of IS-pro, a broad-scope molecular technique that can detect and identify most bacteria to the species level. IS-pro additionally informs on the amount of human DNA present in a sample, as a measure of leukocyte levels. This test can be performed in 4 h with standard laboratory equipment. Residual material of 591 synovial fluid samples derived from native and prosthetic joints from patients suspected of joint infections that were sent for routine diagnostics was collected and subjected to the IS-pro test. Bacterial species identification as well as bacterial load and human DNA load outcomes of IS-pro were compared to those of culture. At sample level, percent positive agreement (PPA) between IS-pro and culture was 90.6% (95% CI 85.7- to 94%) and negative percent agreement (NPA) was 87.7% (95% CI 84.1 to 90.6%). At species level PPA was 80% (95% CI 74.3 to 84.7%). IS-pro yielded 83 extra bacterial detections over culture for which we found supporting evidence for true positivity in 40% of the extra detections. Missed detections by IS-pro were mostly related to common skin species in low abundance. Bacterial and human DNA signals measured by IS-pro were comparable to bacterial loads and leukocyte counts reported by routine diagnostics. We conclude that IS-pro showed an excellent performance for fast diagnostics of bacterial BJI.

Bacterial and Viral Respiratory Tract Microbiota and Host Characteristics in Adults With Lower Respiratory Tract Infections: A Case-Control Study.

BACKGROUND: Viruses and bacteria from the nasopharynx are capable of causing community-acquired pneumonia (CAP), which can be difficult to diagnose. We aimed to investigate whether shifts in the composition of these nasopharyngeal microbial communities can be used as diagnostic biomarkers for CAP in adults. METHODS: We collected nasopharyngeal swabs from adult CAP patients and controls without infection in a prospective multicenter case-control study design. We generated bacterial and viral profiles using 16S ribosomal RNA gene sequencing and multiplex polymerase chain reaction (PCR), respectively. Bacterial, viral, and clinical data were subsequently used as inputs for extremely randomized trees classification models aiming to distinguish subjects with CAP from healthy controls. RESULTS: We enrolled 117 cases and 48 control subjects. Cases displayed significant beta diversity differences in nasopharyngeal microbiota (P = .016, R2 = .01) compared to healthy controls. Our extremely randomized trees classification models accurately discriminated CAP caused by bacteria (area under the curve [AUC] .83), viruses (AUC .95) or mixed origin (AUC .81) from healthy control subjects. We validated this approach using a dataset of nasopharyngeal samples from 140 influenza patients and 38 controls, which yielded highly accurate (AUC .93) separation between cases and controls. CONCLUSIONS: Relative proportions of different bacteria and viruses in the nasopharynx can be leveraged to diagnose CAP and identify etiologic agent(s) in adult patients. Such data can inform the development of a microbiota-based diagnostic panel used to identify CAP patients and causative agents from nasopharyngeal samples, potentially improving diagnostic specificity, efficiency, and antimicrobial stewardship practices.

Comparison of fast Fourier transform infrared spectroscopy biotyping with whole genome sequencing-based genotyping in common nosocomial pathogens.

Early detection of bacterial transmission and outbreaks in hospitals is important because nosocomial infections can result in health complications and longer hospitalization. Current practice to detect outbreaks uses genotyping methods amplified fragment length polymorphism (AFLP) and whole genome sequencing (WGS), which are not suitable methods for real-time transmission screening of both susceptible and resistant bacteria. The aim was to assess the typing technique Fourier transform infrared (FTIR) spectroscopy as real-time screening method to discriminate large amounts of susceptible and resistant bacteria at strain level when there is no evident outbreak in comparison with the WGS reference. Isolates of past hospital outbreak strains of Acinetobacter baumannii/calcoaceticus complex (n = 25), Escherichia coli (n = 31), Enterococcus faecium (n = 22), Staphylococcus aureus (n = 37) and Pseudomonas aeruginosa (n = 30) were used for validation of FTIR. Subsequently, Enterococcus faecalis (n = 106) and Enterococcus faecium (n = 104) isolates from weekly routine screening samples when no potential outbreak was present were analysed. FTIR showed reproducibility and congruence of cluster composition with WGS for A. baumannii/calcoaceticus complex and E. faecium outbreak isolates. The FTIR results of E. faecalis and E. faecium isolates from routine samples showed reproducibility, but the congruence of cluster composition with WGS was low. For A. baumannii/calcoaceticus complex and E. faecium outbreak isolates, FTIR appears to be a discriminatory typing tool. However, our study shows the discriminatory power is too low to screen real-time for transmission of E. faecium and E. faecalis at patient wards based on isolates acquired in routine surveillance cultures when there is no clear suspicion of an ongoing outbreak.

Efficiency Correction Is Required for Accurate Quantitative PCR Analysis and Reporting.

BACKGROUND: Quantitative PCR (qPCR) aims to measure the DNA or RNA concentration in diagnostic and biological samples based on the quantification cycle (Cq) value observed in the amplification curves. Results of qPCR experiments are regularly calculated as if all assays are 100% efficient or reported as just Cq, ΔCq, or ΔΔCq values. CONTENTS: When the reaction shows specific amplification, it should be deemed to be positive, regardless of the observed Cq. Because the Cq is highly dependent on amplification efficiency that can vary among targets and samples, accurate calculation of the target quantity and relative gene expression requires that the actual amplification efficiency be taken into account in the analysis and reports. PCR efficiency is frequently derived from standard curves, but this approach is affected by dilution errors and hampered by properties of the standard and the diluent. These factors affect accurate quantification of clinical and biological samples used in diagnostic applications and collected in challenging conditions. PCR efficiencies determined from individual amplification curves avoid these confounders. To obtain unbiased efficiency-corrected results, we recommend absolute quantification with a single undiluted calibrator with a known target concentration and efficiency values derived from the amplification curves of the calibrator and the unknown samples. SUMMARY: For meaningful diagnostics or biological interpretation, the reported results of qPCR experiments should be efficiency corrected. To avoid ambiguity, the Minimal Information for Publications on Quantitative Real-Time PCR Experiments (MIQE) guidelines checklist should be extended to require the methods that were used (1) to determine the PCR efficiency and (2) to calculate the reported target quantity and relative gene expression value.

Frequent delayed spontaneous seroclearance of hepatitis B virus after incident HBV infection among adult high-risk groups.

High rates (~25%) of developing chronic hepatitis B virus (HBV) infection (hepatitis B surface antigen (HBsAg)-positive for > 6 months following infection) have been observed in people who use drugs (PWUD) and men who have sex with men (MSM). We aimed to estimate the frequency of delayed HBsAg seroclearance, along with its determinants, and time to delayed HBsAg seroclearance. Data were used from MSM and PWUD enrolled in the Amsterdam Cohort Studies (1985-2002) who had anti-hepatitis B core antibody seroconversion. Potential determinants for standard HBsAg seroclearance, delayed HBsAg seroclearance and chronic HBV were examined using multinominal logistic regression. Time to HBsAg seroclearance was estimated using Kaplan-Meier curves. A total of 147 incident HBV infections occurred during follow-up. On initial HBsAg testing after infection (6-12 months), 42 (29%) were HBsAg-positive and 105 (71%) were HBsAg-negative ('standard HBsAg seroclearance'). Of the 42 initially HBsAg-positive individuals, 22 subsequently tested HBsAg-negative (of whom 7 (31.8%) were HBV DNA positive at last visit, suggesting occult HBV). Overall, 15 became HBsAg-negative and HBV DNA-negative ('delayed HBsAg seroclearance'), while 27 remained HBsAg and/or HBV DNA-positive ('chronic HBV'). The 5-year cumulative probability of delayed HBsAg seroclearance was 41.6% for initially HBsAg-positive individuals. Delayed HBsAg seroclearance and remaining chronically infected were associated with younger age and HIV/hepatitis C virus (HCV)-co-infection. In conclusion, delayed HBsAg seroclearance is common in these key adult populations at-risk for HBV, while proportion developing HBV chronicity (18%) is still higher compared to the general population (~5%). Given the proportion of individuals with occult HBV infection and that HCV direct-acting antivirals can lead to HBV reactivation, HBV DNA testing in HCV co-infected MSM/PWUD are warranted prior to treatment initiation.

Implementing a Clostridium difficile testing algorithm and its effect on isolation duration and treatment initiation: a pre- and post-implementation study.

A proportion of patients suspected of Clostridium difficile infection are unnecessarily placed in contact isolation. By introducing a random-access glutamate dehydrogenase (GDH) test for C. difficile, we aimed to reduce isolation time. In addition, we investigated whether the result of the toxin A&B enzyme immunoassay (EIA) was associated with the decision to initiate antibiotic treatment against C. difficile. This retrospective pre- and post-implementation study was from June 3, 2016, to June 4, 2018. Pre-implementation, only a NAAT was performed. In the post-implementation period, a GDH test was performed; if positive, a toxin A&B EIA followed the same day and subsequently a NAAT. Contact isolation for CDI was discontinued when the GDH test was negative. Median time in isolation was 50.8 h pre-implementation (n = 189) versus 28.0 h post-implementation (n = 119), p < 0.001. The GDH test had a negative predictive value of 98.8% (95% CI 97.9-99.4). In 7/31 (22.6%) patients with a positive NAAT and GDH test and a negative toxin A&B EIA, no antibiotics against C. difficile were initiated versus 4/28 (14.3%) patients who were NAAT, GDH and toxin A&B EIA positive. Introducing a random-access screening test resulted in a significant decrease in patient isolation time. The GDH test had a high negative predictive value making it suitable to determine whether contact isolation can be discontinued. Furthermore, the result of a toxin A&B EIA had limited added value on the percentage of patients in whom antibiotic treatment against C. difficile was initiated.

Lactobacillus iners-dominated vaginal microbiota is associated with increased susceptibility to Chlamydia trachomatis infection in Dutch women: a case-control study.

INTRODUCTION: This prospective study aimed to study the composition and structure of the vaginal microbiota prior to Chlamydia trachomatis infection. METHODS: A nested case-control study was performed in 122 women, half of which acquired C. trachomatis within a year after their first visit. At the first visit, the composition and structure of vaginal microbial communities were analysed using 16S rRNA sequencing in the context of the sociodemographic and sexual risk behaviour information using logistic regression. RESULTS: Five vaginal community state types (CSTs) were identified. Four CSTs were dominated by Lactobacillus spp., of which L. crispatus (37%) and L. iners (33%) were the most common. One CST was characterised by the absence of Lactobacillus spp. (25%) and the presence of an array of strict and facultative anaerobes. Multivariate logistic regression analysis revealed that women with a L. iners-dominated CST had an increased risk of C. trachomatis infection (p=0.04; OR: 2.6, 95% CI 1.0 to 6.6). CONCLUSIONS: The distribution of CSTs dominated by Lactobacillus spp. agreed with previous studies. However, the frequency of dysbiosis among Caucasian women was relatively high (24%). Having vaginal microbiota dominated by L. iners was associated with an increased risk for C. trachomatis infection. Therefore, we hypothesise that specific signatures of vaginal microbiota are indicative of increased host predisposition to acquiring STIs.

The Cervicovaginal Microbiota in Women Notified for Chlamydia trachomatis Infection: A Case-Control Study at the Sexually Transmitted Infection Outpatient Clinic in Amsterdam, The Netherlands.

BACKGROUND: Increasing evidence suggests that the cervicovaginal microbiota (CVM) plays an important role in acquiring sexually transmitted infections (STIs). Here we study the CVM in a population of women notified by a sex partner for Chlamydia trachomatis infection. METHODS: We included 98 women who were contact-traced by C. trachomatis-positive sex partners at the STI outpatient clinic in Amsterdam, the Netherlands, and analyzed their cervicovaginal samples and clinical data. CVMs were characterized by sequencing the V3/V4 region of the 16S ribosomal RNA gene and by hierarchical clustering. Characteristics associating with C. trachomatis infection were examined using bivariable and multivariable logistic regression analysis. RESULTS: The CVM was characterized for 93 women, of whom 52 tested C. trachomatis positive and 41 C. trachomatis negative. We identified 3 major CVM clusters. Clustered CVM predominantly comprised either diverse anaerobic bacteria (n = 39 [42%]), Lactobacillus iners (n = 32 [34%]), or Lactobacillus crispatus (n = 22 [24%]). In multivariable analysis, we found that CVM was significantly associated with C. trachomatis infection (odds ratio [OR], 4.2 [95% confidence interval {CI}, 1.2-15.4] for women with diverse anaerobic CVM and OR, 4.4 [95% CI, 1.3-15.6], for women with L. iners-dominated CVM, compared with women with L. crispatus-dominated CVM), as was younger age (OR, 3.1 [95% CI, 1.1-8.7] for those ≤21 years old) and reporting a steady sex partner (OR, 3.6 [95% CI, 1.4-9.4]). CONCLUSIONS: Women who tested positive for Chlamydia trachomatis infection after having been contact-traced by a chlamydia-positive partner were more likely to have CVM dominated by L. iners or by diverse anaerobic bacteria, than by L. crispatus.

Vaginal microbiota and spontaneous preterm birth in pregnant women at high risk of recurrence.

We describe vaginal microbiota, including Gardnerella species and sexually transmitted infections (STIs), during pregnancy and their associations with recurrent spontaneous preterm birth (sPTB). We performed a prospective cohort study in a tertiary referral centre in the Netherlands, among pregnant women with previous sPTB <34 weeks' gestation. Participants collected three vaginal swabs in the first and second trimester. Vaginal microbiota was profiled with 16S rDNA sequencing. Gardnerella species and STI's were tested with qPCR. Standard care was provided according to local protocol, including screening and treatment for bacterial vaginosis (BV), routine progesterone administration and screening for cervical length shortening. Of 154 participants, 26 (16.9 %) experienced recurrent sPTB <37 weeks' gestation. Microbiota composition was not associated with sPTB. During pregnancy, the share of Lactobacillus iners-dominated microbiota increased at the expense of diverse microbiota between the first and second trimester. This change coincided with treatment for BV, demonstrating a similar change in microbiota composition after treatment. In this cohort of high-risk women, we did not find an association between vaginal microbiota composition and recurrent sPTB. This should be interpreted with care, as these women were offered additional preventive therapies to reduce sPTB according to national guidelines including progesterone and BV treatment. The increase observed in L. iners dominated microbiota and the decrease in diverse microbiota mid-gestation was most likely mediated by BV treatment. Our findings suggest that in recurrent sPTB occurring despite several preventive therapies, the microbe-related etiologic contribution might be limited.

Hepatitis B vaccination of men who have sex with men in the Netherlands: should we vaccinate more men, younger men or high-risk men?

OBJECTIVES: The selective vaccination programme against hepatitis B virus (HBV) was introduced in the Netherlands in 2002 targeting high-risk groups, including men who have sex with men (MSM). Despite the high average age of vaccination in MSM, the number of notifications of acute HBV recently declined. We investigate whether this can be attributed to the selective vaccination programme. We examine how vaccination strategies could be improved and the impact of universal infant vaccination introduced in 2011. METHODS: We use a mathematical model for HBV transmission among MSM. The incidence of HBV was calculated from the model and from notification data of acute HBV. RESULTS: A decline was observed in the incidence of HBV since 2006, as calculated from the model; this decline was smaller than that observed in data if all MSM were equally likely to be vaccinated. Assuming that high-risk MSM were more likely to be vaccinated than low-risk MSM resulted in a steeper decline in modelled incidence and better agreement with observed incidence. Vaccinating MSM at a younger age or doubling the vaccination rate would increase the impact of selective vaccination, but is less effective than vaccinating high-risk MSM. CONCLUSIONS: Selective HBV vaccination of MSM in the Netherlands has had a substantial impact in reducing HBV incidence. The reduction suggests that vaccination rates among high-risk MSM were higher than those among low-risk MSM. Countries that have not yet reached 35-year cohorts with universal childhood vaccination should actively implement or continue selective high-risk MSM vaccination.

Screening for hepatitis B and C in first-generation Egyptian migrants living in the Netherlands.

BACKGROUND: Egypt has high prevalence of hepatitis C virus (HCV) infection and intermediate prevalence of hepatitis B virus (HBV) infection; however, infection prevalence among Egyptian migrants is unknown. Considering the asymptomatic onset and development of disease in chronically-infected patients, many may remain undiagnosed. AIMS: To evaluate an HCV- and HBV-screening programme designed to identify undetected infections among first-generation Egyptian migrants in Amsterdam, the Netherlands. METHODS: In 2009 and 2010, viral hepatitis educational and screening sessions were established at Egyptian meeting places. Data regarding demographics and HCV risk factors were collected. Chronically infected participants were referred and followed up. Phylogenetic analyses were used to ascertain the geographic origin of infections. RESULTS: Eleven of 465 (2.4%; 95% CI = 1.3-4.2%) migrants had HCV antibodies; 10/11 were HCV RNA positive. All had genotype 4a, and strains were typical of those of Egypt and the Middle East. Older age and exposure to parenteral antischistosomal therapy (PAT) were significantly associated with HCV. Anti-HBc prevalence was 16.8% (95% CI = 13.7-20.4%); HBsAg prevalence was 1.1% (95% CI = 0.5-2.5%). All had genotype D, typical of those of the Middle East. Most (9/10 HCV; 3/5 HBV) chronic infections were newly diagnosed; four of the HCV-infected individuals started treatment. CONCLUSIONS: Anti-HCV and HBsAg prevalence among Egyptian migrants was lower compared with the general Egyptian population, but higher than the general population of Western countries. Phylogenetic analyses suggest that all infections were from the region of origin. HCV-screening programmes should target first-generation Egyptian migrants, especially those of older age and those who received PAT.

Unexpectedly high proportion of drug users and men having sex with men who develop chronic hepatitis B infection.

BACKGROUND & AIMS: In low endemic countries, most hepatitis B virus (HBV) infections are found in adult behavioural risk groups, such as drug users (DU) and men having sex with men (MSM). These risk groups are frequently exposed to HBV, which might induce a different rate of viral clearance compared with the general adult population, in whom the chronicity rate is estimated to be 5-10%. Our aim was to obtain insights into the proportion of MSM and DU developing chronic infection after a primary HBV infection, and the underlying risk factors. METHODS: From 1984 to 2002, sera of 1862 MSM and 1268 DU of the Amsterdam Cohort Studies were retrospectively tested for anti-HBc, HBsAg, and HBV DNA. As of 2003, all of the cohort participants were vaccinated, making further testing redundant. RESULTS: Hundred and forty seven participants seroconverted for anti-HBc during follow-up. The median age at the moment of the acute infection was 31 years. The proportion of those becoming chronically infected was 23% and 28% for MSM and DU, respectively. In both cohorts, being younger was a risk factor for developing chronic infection (OR: 0.9; 95% CI: 0.82-0.99). HIV/HCV co-infection was associated with developing chronic HBV infection in the DU cohort (OR: 32.1, 95% CI: 3.1-334.5). CONCLUSIONS: Compared with the general population, MSM and DU had an unanticipated high rate of developing chronic HBV infections. HIV/HCV co-infection proved to be an important risk factor for developing chronic HBV infections in DU. The reason for the high rate of MSM becoming chronically infected remains unclear.

Recurrent E. coli Urinary Tract Infections in Nursing Homes: Insight in Sequence Types and Antibiotic Resistance Patterns.

INTRODUCTION: Recurrent urinary tract infections (rUTI) largely contribute to antibiotic use in older adults. Understanding the genetic characteristics of Escherichia coli (E.coli) is needed to identify patients at risk for recurrence. The aim of this study was to obtain a greater understanding of the genetics of E. coli rUTI in nursing home residents. METHODS: This is a secondary analysis of a multicenter Dutch nursing home study (PROGRESS). E. coli strains from residents with a suspected UTI and positive urine culture were analyzed using antimicrobial susceptibility testing and whole-genome sequencing (WGS). Same-strain recurrences were identified by single-nucleotide polymorphism (SNP) analysis. RESULT: In total, 121 E. coli strains were analyzed using WGS, of which 54 belonged to a rUTI episode. One third of E. coli rUTI episodes were caused by the same strain (n = 18, 33.3%). Same-strain recurrence occurred anywhere between 30 and 434 days after the index UTI, caused by sequence types (ST): ST12, ST23, ST73, ST131, ST453, ST538 and ST2522, in seven nursing home residents. In both single UTI and rUTI, antimicrobial resistance rates were low. CONCLUSION: Recurrent UTI in nursing home residents are caused by same-strain E. coli as well as due to different E. coli strains or other uropathogens. Same-strain recurrence can occur over 400 days after the index UTI, suggesting that some strains have the ability to colonize the bladder or gut for longer periods.

The impact of the updated EORTC/MSG criteria on the classification of hematological patients with suspected invasive pulmonary aspergillosis.

OBJECTIVES: Our aim was to evaluate the effect of the updated European Organization for Research and Treatment of Cancer (EORTC) and Mycoses Study Group 2019 definitions for invasive pulmonary aspergillosis (IPA) on patient classification and the related all-cause 12-week mortality. METHODS: In this retrospective cohort study from our tertiary care centre, we reclassified patients with haematological malignancy who underwent bronchoalveolar lavage between 2014 and 2019 for suspected IPA using the novel EORTC 2019 criteria. We performed receiver operating characteristic curve analysis to define the optimal cut-off for positive PCR and galactomannan and present survival analyses and their possible association with these diagnostic criteria through post hoc comparisons with log rank and Cox regression. RESULTS: From 323 episodes of suspected IPA in 282 patients, 73 were reclassified: 31 (42.5%) from possible to probable IPA, 5 (6.8%) from EORTC criteria not met to probable IPA, and 37 (50.7%) from EORTC criteria not met to possible IPA. Probable IPA increased therefore 11.1% (64/323, 19.8% to 100/323, 30.9%), mostly due to positive PCR (31/36, 86.1%). There was no difference in mortality between newly defined possible and probable IPA (log rank p = 0.950). Mortality was higher in probable cases with lower cycle thresholds (Ct values) versus higher Ct values (p = 0.004). Receiver operating characteristic curve analysis showed an optimal Ct value cut-off of 36.8 with a sensitivity of 75% (95% CI 64.9%-85.1%) and a specificity of 61.7% (95% CI 53.5-69.9) for 12-week mortality. DISCUSSION: The new EORTC criteria led to 11.1% more probable IPA diagnoses, mostly due to Aspergillus PCR. Restricting positive PCR to below a certain threshold might improve the discrimination of the new EORTC IPA categories for mortality.

Enhanced IgA coating of bacteria in women with Lactobacillus crispatus-dominated vaginal microbiota.

BACKGROUND: Immunoglobulin A (IgA) plays an important role in maintaining a healthy intestinal microbiome, but little is known about the interaction between local immunoglobulins and the vaginal microbiome. We assessed immunoglobulins (unbound and bound to bacteria), their association with vaginal microbiota composition and the changes over time in 25 healthy women of reproductive age. RESULTS: In both Lactobacillus crispatus-dominated and non-L. crispatus-dominated microbiota, IgA and IgG (unbound and bound to bacteria) were higher during menses (T = 1) compared to day 7­11 (T = 2) and day 17­25 (T = 3) after menses onset. The majority of vaginal bacteria are coated with IgA and/or IgG. Women with L. crispatus-dominated microbiota have increased IgA coating of vaginal bacteria compared to women with other microbiota compositions, but contained less IgA per bacterium. Presence of a dominantly IgA-coated population at T = 2 and/or T = 3 was also strongly associated with L. crispatus-dominated microbiota. In women with non-L. crispatus-dominated microbiota, more bacteria were uncoated. Unbound IgA, unbound IgG, and bound IgG levels were not associated with microbiota composition. CONCLUSIONS: In conclusion, L. crispatus-dominated vaginal microbiota have higher levels of bacterial IgA coating compared to non-L. crispatus-dominated vaginal microbiota. Similar to its regulating function in the intestinal tract, we hypothesize that IgA is involved in maintaining L. crispatus-dominated microbiota in the female genital tract. This may play a role in L. crispatus-associated health benefits. Video abstract.

Urine and vaginal microbiota compositions of postmenopausal and premenopausal women differ regardless of recurrent urinary tract infection and renal transplant status.

Postmenopausal women and renal transplant recipients are at increased risk of recurrent urinary tract infections (RUTI). Urine and vaginal microbiota of premenopausal controls (N = 18) and RUTI cases (18), and of postmenopausal controls (30) and RUTI cases (20) with and without a renal transplant, were characterized using 16S rRNA sequencing. Participants did not have UTI symptoms at the time of sampling. Gram-negative uropathobionts (predominantly Escherichia/Shigella, Pseudomonas, Klebsiella, and Acinetobacter) had a much higher mean relative abundance in urine than vaginal samples, especially in premenopausal women. No statistically significant differences in mean relative abundances of bacterial groups were found within the premenopausal group or within the postmenopausal group by RUTI or renal transplant status without chronic antibiotic use. Comparing postmenopausal to premenopausal women, mean relative abundances of lactobacilli (especially L. crispatus) in urine and vaginal samples and of Gram-negative uropathobionts in urine were lower, and of BV-anaerobes and Gram-positive uropathobionts in urine and vaginal samples were higher. While RUTI in premenopausal women is predominantly caused by Escherichia, the causative organisms in postmenopausal women are likely more diverse. The relative importance of individual organisms is currently unknown. We recommend that future studies, including intervention studies, include longitudinal microbiota assessments.

Simultaneous detection and ribotyping of Clostridioides difficile, and toxin gene detection directly on fecal samples.

BACKGROUND: Clostridioides difficile is the most common cause of nosocomial diarrhea. Ribotyping of cultured strains by a PCR-based test is used to study potential transmission between patients. We aimed to develop a rapid test that can be applied directly on fecal samples for simultaneous detection and ribotyping of C. difficile, as well as detection of toxin genes. METHODS: We developed a highly specific and sensitive primer set for simultaneous detection and ribotyping of C. difficile directly on total fecal DNA. Toxin genes were detected with primers adapted from Persson et al. (Clin Microbiol Infect 14(11):1057-1064). Our study set comprised 130 fecal samples: 65 samples with positive qPCR for C. difficile toxin A/B genes and 65 C. difficile qPCR negative samples. PCR products were analyzed by capillary gel electrophoresis. RESULTS: Ribosomal DNA fragment peak profiles and toxin genes were detected in all 65 C. difficile positive fecal samples and in none of the 65 C. difficile negative samples. The 65 samples were assigned to 27 ribotypes by the Dutch reference laboratory. Our peak profiles corresponded to these ribotypes, except for two samples. During a C. difficile outbreak, patients were correctly allocated to the outbreak-cluster based on the results of direct fecal ribotyping, before C. difficile isolates were cultured and conventionally typed. CONCLUSION: C. difficile ribotyping directly on fecal DNA is feasible, with sensitivity and specificity comparable to that of diagnostic toxin gene qPCR and with ribotype assignment similar to that obtained by conventional typing on DNA from cultured isolates. This supports simultaneous diagnosis and typing to recognize an outbreak.

Head-to-head validation of six immunoassays for SARS-CoV-2 in hospitalized patients.

BACKGROUND: Detecting SARS-CoV-2 antibodies may help to diagnose COVID-19. Head-to-head validation of different types of immunoassays in well-characterized cohorts of hospitalized patients remains needed. METHODS: We validated three chemiluminescence immunoassays (CLIAs) (Liaison, Elecsys, and Abbott) and one single molecule array assay (SIMOA) (Quanterix) for automated analyzers, one rapid immunoassay RIA (AllTest), and one ELISA (Wantai) in parallel in first samples from 126 PCR confirmed COVID-19 hospitalized patients and 158 pre-COVID-19 patients. Specificity of the AllTest was also tested in 106 patients with confirmed parasitic and dengue virus infections. Specificity of the Wantai assay was not tested due to limitations in sample volumes. RESULTS: Overall sensitivity in first samples was 70.6 % for the Liaison, 71.4 % for the Elecsys, 75.4 % for the Abbott, 70.6 % for the Quanterix, 77.8 % for the AllTest, and 88.9 % for the Wantai assay, respectively. Sensitivity was between 77.4 % (Liaison) and 94.0 % (Wantai) after 10 dpso. No false positive results were observed for the Elecsys and Abbott assays. Specificity was 91.1 % for the Quanterix, 96.2 % for the Liaison, and 98.1 % for the AllTest assay, respectively. CONCLUSION: We conclude that low sensitivity of all immunoassays limits their use early after onset of illness in diagnosing COVID-19 in hospitalized patients. After 10 dpso, the Wantai ELISA has a relatively high sensitivity, followed by the point-of-care AllTest RIA that compares favorably with automated analyzer immunoassays.

Molecular sequence data of hepatitis B virus and genetic diversity after vaccination.

The effect of vaccination programs on transmission of infectious disease is usually assessed by monitoring programs that rely on notifications of symptomatic illness. For monitoring of infectious diseases with a high proportion of asymptomatic cases or a low reporting rate, molecular sequence data combined with modern coalescent-based techniques offer a complementary tool to assess transmission. Here, the authors investigate the added value of using viral sequence data to monitor a vaccination program that was started in 1998 and was targeted against hepatitis B virus in men who have sex with men in Amsterdam, the Netherlands. The incidence in this target group, as estimated from the notifications of acute infections with hepatitis B virus, was low; therefore, there was insufficient power to show a significant change in incidence. In contrast, the genetic diversity, as estimated from the viral sequence collected from the target group, revealed a marked decrease after vaccination was introduced. Taken together, the findings suggest that introduction of vaccination coincided with a change in the target group toward behavior with a higher risk of infection. The authors argue that molecular sequence data provide a powerful additional monitoring instrument, next to conventional case registration, for assessing the impact of vaccination.