Pooling isolates to address the diversity in antimicrobial susceptibility of Pseudomonas aeruginosa in cystic fibrosis.

IMPORTANCE: People with cystic fibrosis (pwCF) often suffer from chronic lung infections with Pseudomonas aeruginosa. While antibiotics are still commonly used to treat P. aeruginosa infections, there is a high discordance between in vitro and in vivo antibiotic efficacy, which contributes to suboptimal antibiotic therapy. In the present study, we found that isolates from the same sputum sample had highly diverse antibiotic resistance profiles [based on the minimal inhibitory concentration (MIC)], which may explain the reported discrepancy between in vitro and in vivo antibiotic efficacy. Through systematic analysis, we report that pooling nine isolates per sputum sample significantly decreased intrasample diversity in MIC and influenced clinical interpretation of antibiotic susceptibility tests compared to single isolate testing. Hence, pooling of isolates may offer a solution to obtain a consistent MIC test result and could lead to optimizing antibiotic therapy in pwCF and other infectious diseases where diversity in antibiotic resistance is observed.

Comparison between the Roche Cobas 4800 Human Papillomavirus (HPV), Abbott RealTime High-Risk HPV, Seegene Anyplex II HPV28, and Novel Seegene Allplex HPV28 Assays for High-Risk HPV Detection and Genotyping in Mocked Self-Samples.

Infection with high-risk human papillomavirus (hrHPV) is well recognized as the main cause of cervical cancer. The recently developed Seegene Allplex HPV28 assay is a novel quantitative PCR (qPCR) assay designed to separately detect and quantify 28 distinct HPV genotypes in a fully automated and user-friendly manner. This study evaluated and compared the performance of this new assay with the performance of the Roche Cobas 4800, the Abbott RealTime high-risk HPV, and the Seegene Anyplex II HPV28 assays. A total of 114 mocked self-samples, i.e., semicervical samples collected by gynecologists using the Viba-Brush, were analyzed with all four HPV assays. Agreement in terms of detecting and genotyping HPV was assessed by the mean of the Cohen's kappa (κ) coefficient. Results of all four HPV assays agreed in 85.9% of the cases when using the Abbott RealTime manufacturer's recommended quantification cycle (Cq) cutoff for positivity (<32.00) and 91.2% when using an adapted range (32.00 to 36.00). An intercomparison of the included assays demonstrated an overall agreement ranging from 85.9 to 100.0% (κ = 0.42 to 1.00) when using the manufacturer's guidelines and 92.9 to 100.0% (κ = 0.60 to 1.00) with the adapted range. For all assays, highly significant, strongly positive Pearson correlations were shown between the Cq values of positive test results. This study thereby shows high concordance between results of the included HPV assays on mocked self-samples. Based on these findings, we imply that the novel Allplex HPV28 assay demonstrates a comparable performance to those of available qPCR HPV assays, potentially providing opportunities for the simplification and standardization of future large-scale testing. IMPORTANCE This study proves that the novel Allplex HPV28 assay has a good diagnostic performance in comparison with the well-known, validated, and frequently used Roche Cobas 4800, Abbott RealTime, and Anyplex II HPV28 assays. According to our experience, the novel Allplex HPV28 assay had a user-friendly and automated workflow with short hands-on time, had an open platform which facilitates the use of add-on assays, and provided quick and easy-to-interpret results. Together with its ability to detect and quantify 28 HPV genotypes, the Allplex HPV28 assay could therefore potentially provide opportunities for the simplification and standardization of future diagnostic testing programs.

GRACE-trial: a randomised active-controlled trial for vulvovaginal atrophy in patients with breast cancer on endocrine therapy - study protocol.

INTRODUCTION: Breast cancer is the most common cancer type in women worldwide. Due to hormone receptor positivity in the majority of the breast cancer tumours is endocrine therapy a crucial part in the treatment landscape of breast cancer. Endocrine therapy consists of the use of selective oestrogen-receptor modulators or aromatase inhibitors. These medicines generate a hypoestrogenic environment by reducing circulating oestrogen or by altering the effect of oestrogen on tissue cells by receptor blockade. As a common side effect, vulvovaginal atrophy occurs in the majority of patients with breast cancer using endocrine therapy. Vulvovaginal atrophy has a significant impact on physical and psychological well-being due to negative influence on quality-of-life, self-esteem and sexuality. As a consequence, adherence to endocrine therapy for the standard duration of 5-10 years is challenging, resulting in higher rates of therapy interruption, leading to poorer prognosis with shorter distant disease-free survival. The standard treatment for vulvovaginal atrophy in postmenopausal women is based on the use of local hormonal treatment. However, when a patient has a history of breast cancer, delay of treatment and undertreatment are ubiquitous. METHODS AND ANALYSIS: In this first ever prospective randomised trial patients with breast cancer on endocrine therapy with vulvovaginal atrophy will be treated with the available local treatment modalities with a 1:1:1:1 randomisation: oestrogen, dehydroepiandrosterone, moisturisers and a co-treatment of oestrogen and probiotics. Patient-reported outcomes measurements will be implemented to investigate the efficacy of the implemented treatments. Safety of the treatments will be evaluated by assessing systemic sex hormones concentrations. ETHICS AND DISSEMINATION: This study was approved by the Ethical Committee of Ghent University Hospital and by the Federal Agency for Medicines and Health Products. Results will be published in peer-reviewed journals and released in international conferences. TRIAL REGISTRATION NUMBER: 2021-001921-31.

Visual diagnosis of female genital schistosomiasis in Zambian women from hand-held colposcopy: agreement of expert image review.

Female genital schistosomiasis (FGS) can occur in S. haematobium infection and is caused by parasite egg deposition in the genital tract. Confirming a diagnosis of FGS is challenging due to the lack of a diagnostic reference standard. A 2010 expert-led consensus meeting proposed visual inspection of the cervicovaginal mucosa as an adequate reference standard for FGS diagnosis. The agreement of expert human reviewers for visual-FGS has not been previously described. Methods: In two Zambian communities, non-menstruating, non-pregnant, sexually-active women aged 18-31 years participating in the HPTN 071 (PopART) Population-Cohort were enrolled in a cross-sectional study. Self-collected genital swabs and a urine specimen were collected at a home visit; trained midwives performed CVL and hand-held colposcopy at a clinic visit. S. haematobium eggs and circulating anodic antigen (CAA) were detected from urine. Two expert reviewers independently diagnosed visual-FGS as the presence of sandy patches, rubbery papules or abnormal blood vessels in digital cervicovaginal images obtained by hand-held colposcopy. PCR-FGS was defined as Schistosoma DNA detected by real-time PCR in any genital specimen (CVL or genital swab). Results: Of 527 women with cervicovaginal colposcopic images, 468/527 (88.8%) were deemed interpretable by Reviewer 1 and 417/527 (79.1%) by Reviewer 2. Visual-FGS was detected in 35.3% (165/468) of participants by expert review of colposcopic images by Reviewer 1 and in 63.6% (265/417) by Reviewer 2. Cohen's kappa statistic for agreement between the two expert reviewers was 0.16, corresponding to "slight" agreement. The reviewers made concordant diagnoses in 38.7% (204/527) participants (100 negative, 104 positive) and discordant diagnoses in 31.8% (168/527) participants. Conclusions: The unexpectedly low level of correlation between expert reviewers highlights the imperfect nature of visual diagnosis for FGS based on cervicovaginal images obtained with a hand-held colposcope. This finding is a call to action for improved point-of-care diagnostics for female genital schistosomiasis.

The Presence of Hemoglobin in Cervicovaginal Lavage Is Not Associated With Genital Schistosomiasis in Zambian Women From the BILHIV Study.

Background: Female genital schistosomiasis (FGS) occurs when Schistosoma haematobium eggs are deposited in reproductive tissue. Female genital schistosomiasis in the cervical mucosa is associated with increased vascularity. If FGS is associated with the presence of hemoglobin in cervicovaginal lavage (CVL), the use of urinary reagent strips to detect hemoglobin in CVL could supplement FGS diagnosis. Methods: Nonmenstruating, nonpregnant, sexually active women aged 18-31 participating in the HPTN 071 (PopART) Population-Cohort were invited in 2 Zambian communities. Genital self-swabs and a urine specimen were collected at a home visit, and CVL and hand-held colposcopy were performed at a midwife led clinic visit. Urinary reagent strips were used to identify hemoglobin in CVL. Eggs and circulating anodic antigen (CAA) were detected from urine. Visual-FGS was defined as the presence of sandy patches, rubbery papules, or abnormal blood vessels. Polymerase chain reaction (PCR)-FGS was defined as Schistosoma deoxyribonucleic acid detected by real-time PCR on CVL or cervical or vaginal swab. Results: Of 209 women with home genital swabs and companion CVL specimens, 66% (138 of 209) had detectable CVL hemoglobin, 13.4% (28 of 209) had PCR-defined FGS, and 17.2% (36 of 209) had visual-FGS. Active Schistosoma infection, diagnosed by CAA or urine microscopy, was present in 21.0% (44 of 209) participants. Active Schistosoma infection (P = .4), PCR-FGS (P = 0.7), and visual-FGS (P = 0.3) were not associated with CVL hemoglobin presence. Results did not differ in subgroups with high infection burden (cycle threshold < 35 or 2-3 positive genital PCR). Conclusions: Polymerase chain reaction-FGS, visual-FGS, and active Schistosoma infection were not associated with the presence of CVL hemoglobin. Further research is needed to establish accessible community-based FGS diagnostics.

Prevalence, risk factors and adverse pregnancy outcomes of second trimester bacterial vaginosis among pregnant women in Bukavu, Democratic Republic of the Congo.

BACKGROUND: Bacterial vaginosis (BV) is the most common gynecological condition in women of reproductive age and associated with adverse pregnancy outcomes. In the Democratic Republic of the Congo (DRC), neonatal mortality rate is as high as 2.8 percent with preterm birth (PTB) and low birth weight (LBW) as leading causes. Because no studies have addressed BV in DRC, we aimed to investigate the prevalence of BV, the risk factors and the association between BV and adverse pregnancy outcomes in a population of pregnant women from Bukavu, DRC. METHODS: A total of 533 pregnant women in the second trimester of pregnancy were recruited in the Provincial Reference Hospital of Bukavu, DRC, between January and October 2017, and followed until delivery. Clinical and sociodemographic data of mother and newborn, and data on (vaginal) hygiene practices, sexual behavior and reproductive history were collected. BV was diagnosed by Nugent scoring of Gram-stained vaginal smears. Two multivariate regression models were built to identify risk factors for BV and to investigate BV as a risk factor for adverse pregnancy outcomes. RESULTS: The prevalence of BV was 26.3% and approximately half of the women with BV were asymptomatic. Independent risk factors for BV were the use of alternatives to water for intravaginal washing, concurrent partners, unemployed status, the presence of vaginal Candida and clay consumption. BV was independently associated with both LBW and PTB of an infant with LBW. CONCLUSION: The prevalence of BV in Bukavu is high but in line with the global average. BV was associated with adverse pregnancy outcomes in our study population. Hence, research on modifiable risk factor-based interventions to reduce the prevalence of BV, and on screening/treatment of BV during antenatal care should be explored to reduce neonatal mortality and morbidity.

Cervicovaginal Immune Activation in Zambian Women With Female Genital Schistosomiasis.

HIV-1 infection disproportionately affects women in sub-Saharan Africa, where areas of high HIV-1 prevalence and Schistosoma haematobium endemicity largely overlap. Female genital schistosomiasis (FGS), an inflammatory disease caused by S. haematobium egg deposition in the genital tract, has been associated with prevalent HIV-1 infection. Elevated levels of the chemokines MIP-1α (CCL-3), MIP-1ß (CCL-4), IP-10 (CXCL-10), and IL-8 (CXCL-8) in cervicovaginal lavage (CVL) have been associated with HIV-1 acquisition. We hypothesize that levels of cervicovaginal cytokines may be raised in FGS and could provide a causal mechanism for the association between FGS and HIV-1. In the cross-sectional BILHIV study, specimens were collected from 603 female participants who were aged 18-31 years, sexually active, not pregnant and participated in the HPTN 071 (PopART) HIV-1 prevention trial in Zambia. Participants self-collected urine, and vaginal and cervical swabs, while CVLs were clinically obtained. Microscopy and Schistosoma circulating anodic antigen (CAA) were performed on urine. Genital samples were examined for parasite-specific DNA by PCR. Women with FGS (n=28), defined as a positive Schistosoma PCR from any genital sample were frequency age-matched with 159 FGS negative (defined as negative Schistosoma PCR, urine CAA, urine microscopy, and colposcopy imaging) women. Participants with probable FGS (n=25) (defined as the presence of either urine CAA or microscopy in combination with one of four clinical findings suggestive of FGS on colposcope-obtained photographs) were also included, for a total sample size of 212. The concentrations of 17 soluble cytokines and chemokines were quantified by a multiplex bead-based immunoassay. There was no difference in the concentrations of cytokines or chemokines between participants with and without FGS. An exploratory analysis of those women with a higher FGS burden, defined by ≥2 genital specimens with detectable Schistosoma DNA (n=15) showed, after adjusting for potential confounders, a higher Th2 (IL-4, IL-5, and IL-13) and pro-inflammatory (IL-15) expression pattern in comparison to FGS negative women, with differences unlikely to be due to chance (p=0.037 for IL-4 and p<0.001 for IL-5 after adjusting for multiple testing). FGS may alter the female genital tract immune environment, but larger studies in areas of varying endemicity are needed to evaluate the association with HIV-1 vulnerability.

Urogenital pathogens, associated with Trichomonas vaginalis, among pregnant women in Kilifi, Kenya: a nested case-control study.

BACKGROUND: Screening of curable sexually transmitted infections is frequently oriented towards the diagnosis of chlamydia, gonorrhea, syphilis and trichomoniasis, whereas other pathogens, sometimes associated with similar urogenital syndromes, remain undiagnosed and/or untreated. Some of these pathogens are associated with adverse pregnancy outcomes. METHODS: In a nested case-control study, vaginal swabs from 79 pregnant women, i.e., 28 T. vaginalis-positive (cases) and 51 T. vaginalis-negative (controls), were screened by quantitative PCR for Adenovirus 1 and 2, Cytomegalovirus, Herpes Simplex Virus 1 and 2, Chlamydia trachomatis, Escherichia coli, Haemophilus ducreyi, Mycoplasma genitalium, M. hominis, candidatus M. girerdii, Neisseria gonorrhoeae, Streptococcus agalactiae, Treponema pallidum, Ureaplasma parvum, U. urealyticum, and Candida albicans. Additionally, we determined whether women with pathogens highly associated with T. vaginalis had distinct clinical signs and symptoms compared to women with T. vaginalis mono-infection. RESULTS: M. hominis was independently associated with T. vaginalis (adjusted odds ratio = 6.8, 95% CI: 2.3-19.8). Moreover, M. genitalium and Ca M. girerdii were exclusively detected in women with T. vaginalis (P = 0.002 and P = 0.001), respectively. Four of the six women co-infected with T. vaginalis and Ca M. girerdii complained of vaginal itching, compared to only 4 out of the 22 women infected with T. vaginalis without Ca M. girerdii (P = 0.020). CONCLUSION: We confirm M. hominis as a correlate of T. vaginalis in our population, and the exclusive association of both M. genitalium and Ca. M. girerdii with T. vaginalis. Screening and treatment of these pathogens should be considered.

Role of HIV exposure and infection in relation to neonatal GBS disease and rectovaginal GBS carriage: a systematic review and meta-analysis.

Streptococcus agalactiae (GBS) is the leading cause worldwide of neonatal sepsis. We sought to assess to which extent HIV exposure of neonates is associated with GBS neonatal disease. Furthermore, we assessed to which extent HIV infection in women is associated with maternal rectovaginal GBS carriage, the single most important risk factor for GBS neonatal disease. We searched Pubmed, Embase, and Web of Science for studies assessing the association between neonatal GBS disease and HIV-status of the mother and studies that assessed the association between rectovaginal GBS colonization and HIV status in women. HIV-exposed uninfected neonates were more than twice as likely to have neonatal GBS disease compared to unexposed neonates. HIV-exposed neonates were not at increased risk for early-onset neonatal disease, but were 4.43 times more likely to have late-onset neonatal GBS disease. There was no significant association between HIV infection status and rectovaginal GBS carriage. Public health interventions preventing neonatal GBS disease are urgently needed for the increasing group of HIV-exposed neonates. A framework integrating and explaining our findings highlights opportunities for the clinical practice and global health policy to prevent disease. Well-designed studies should clarify the relation between HIV-status and GBS carriage.

A longitudinal analysis of the vaginal microbiota and vaginal immune mediators in women from sub-Saharan Africa.

In cross-sectional studies increased vaginal bacterial diversity has been associated with vaginal inflammation which can be detrimental for health. We describe longitudinal changes at 5 visits over 8 weeks in vaginal microbiota and immune mediators in African women. Women (N = 40) with a normal Nugent score at all visits had a stable lactobacilli dominated microbiota with prevailing Lactobacillus iners. Presence of prostate-specific antigen (proxy for recent sex) and being amenorrhoeic (due to progestin-injectable use), but not recent vaginal cleansing, were significantly associated with microbiota diversity and inflammation (controlled for menstrual cycle and other confounders). Women (N = 40) with incident bacterial vaginosis (Nugent 7-10) had significantly lower concentrations of lactobacilli and higher concentrations of Gardnerella vaginalis, Atopobium vaginae, and Prevotella bivia, at the incident visit and when concentrations of proinflammatory cytokines (IL-1ß, IL-12p70) were increased and IP-10 and elafin were decreased. A higher 'composite-qPCR vaginal-health-score' was directly associated with decreased concentrations of proinflammatory cytokines (IL-1α, IL-8, IL-12(p70)) and increased IP-10. This longitudinal study confirms the inflammatory nature of vaginal dysbiosis and its association with recent vaginal sex and progestin-injectable use. A potential role for proinflammatory mediators and IP-10 in combination with the vaginal-health-score as predictive biomarkers for vaginal dysbiosis merits further investigation.

Epidemic Achromobacter xylosoxidans strain among Belgian cystic fibrosis patients and review of literature.

BACKGROUND: Achromobacter xylosoxidans is increasingly being recognized as an emerging pathogen in cystic fibrosis. Recent severe infections with A. xylosoxidans in some of our cystic fibrosis (CF) patients led to a re-evaluation of the epidemiology of CF-associated A. xylosoxidans infections in two Belgian reference centres (Antwerp and Ghent). Several of these patients also stayed at the Rehabilitation Centre De Haan (RHC). In total, 59 A. xylosoxidans isolates from 31 patients (including 26 CF patients), collected between 2001 and 2014, were studied. We evaluated Matrix Assisted Laser Desorption Ionisation -Time of Flight mass spectrometry (MALDI-TOF) as an alternative for McRAPD typing. RESULTS: Both typing approaches established the presence of a major cluster, comprising isolates, all from 21 CF patients, including from two patients sampled when staying at the RHC a decade ago. This major cluster was the same as the cluster established already a decade ago at the RHC. A minor cluster consisted of 13 isolates from miscellaneous origin. A further seven isolates, including one from a non-CF patient who had stayed recently at the RHC, were singletons. CONCLUSIONS: Typing results of both methods were similar, indicating transmission of a single clone of A. xylosoxidans among several CF patients from at least two reference centres. Isolates of the same clone were already observed at the RHC, a decade ago. It is difficult to establish to what extent the RHC is the source of transmission, because the epidemic strain was already present when the first epidemiological study in the RHC was carried out. This study also documents the applicability of MALDI-TOF for typing of strains within the species A. xylosoxidans and the need to use the dynamic cutoff algorithm of the BioNumerics® software for correct clustering of the fingerprints.

Haemophilus influenzae biofilm formation in chronic otitis media with effusion.

Otitis media with effusion (OME) is a highly prevalent disease in children, but the exact pathogenesis and role of bacteria are still not well understood. This study aimed to investigate the presence of otopathogenic bacteria in the middle ear effusion (MEE) and adenoid of children with chronic OME (COME), and to investigate in vivo whether these bacteria, especially Haemophilus influenzae, are organized as a biofilm in the middle ear fluid. MEE and adenoid samples were collected from 21 patients with COME. Extensive bacterial culturing and genotyping was performed on all middle ear and adenoid samples. Fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) was used to visualize possible biofilm structures for a selection of middle ear effusion samples. 34 MEE samples were collected from 21 patients of which 64.7 % were culture positive for bacteria and 47.0 % were culture positive for Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and/or Streptococcus pneumoniae. All 21 adenoid samples were culture positive for one or more of these four otopathogens. H. influenzae (35.3 %) and S. pneumoniae (76.2 %) were the most frequently cultured bacteria in the MEE and adenoid samples, respectively. The same bacterial species was found in MEE and adenoid for 84.6 % of the patients and in 81.2 % of the cases where the same species was found in more than one site it involved the same bacterial genotype. FISH and CLSM demonstrated the presence of H. influenzae specific biofilm structures in five of the eight culture positive MEEs that were tested, but in none of the two culture negative MEEs. The findings in this study indicate that the adenoid acts as a reservoir for bacteria in MEE and confirms that biofilms, in at least half of the cases consisting of H. influenzae, are indeed present in the MEE of children with COME. Biofilms may thus play a crucial role in the pathogenesis of COME, which is important in the understanding of this disease and the development of potential future treatment options.

Longitudinal study of the dynamics of vaginal microflora during two consecutive menstrual cycles.

BACKGROUND: Although the vaginal microflora (VMF) has been well studied, information on the fluctuation of the different bacterial species throughout the menstrual cycle and the information on events preceding the presence of disturbed VMF is still very limited. Documenting the dynamics of the VMF during the menstrual cycle might provide better insights. In this study, we assessed the presence of different Lactobacillus species in relation to the BV associated species during the menstrual cycle, assessed the influence of the menstrual cycle on the different categories of vaginal microflora and assessed possible causes, such as menstruation and sexual intercourse, of VMF disturbance. To our knowledge, this is the first longitudinal study in which swabs and Gram stains were available for each day of two consecutive menstrual cycles, whereby 8 grades of VMF were distinguished by Gram stain analysis, and whereby the swabs were cultured every 7(th) day and identification of the bacterial isolates was carried out with a molecular technique. METHODS: Self-collected vaginal swabs were obtained daily from 17 non pregnant, menarchal volunteers, and used for daily Gram staining and weekly culture. Bacterial isolates were identified with tDNA-PCR and 16 S rRNA gene sequencing. RESULTS: Nine women presented with predominantly normal VMF and the 8 others had predominantly disturbed VMF. The overall VMF of each volunteer was characteristic and rather stable. Menses and antimicrobials were the major disturbing factors of the VMF. Disturbances were always accompanied by a rise in Gram positive cocci, which also appeared to be a significant group within the VMF in general. CONCLUSIONS: We observed a huge interindividual variability of predominantly stable VMF types. The importance of Gram positive cocci in VMF is underestimated. L. crispatus was the species that was most negatively affected by the menses, whereas the presence of the other lactobacilli was less variable.