We evaluated the importance of measuring early vaginal levels of eight bacterial vaginosis (BV)-associated bacteria, at two points in pregnancy, and the risk of spontaneous preterm delivery (SPTD) among pregnant women and the subgroup of pregnant women with a history of preterm delivery (PTD).
Approximately 45% of nongonococcal urethritis cases have no identified etiology. Novel bacteria recently associated with bacterial vaginosis (BV) in women may be involved. We evaluated the association of idiopathic nongonococcal urethritis and 5 newly described BV-associated bacteria (BVAB).
Among urban, primarily African American pregnant women, 74% were identified with Nugent score bacterial vaginosis (BV). All BV-associated bacteria were more prevalent among women with Nugent score BV. Bacterial vaginosis-associated bacteria 3 (BVAB3) had the highest positive predictive value, whereas Gardnerella vaginalis and Atopobium spp. had the highest sensitivity. Atopobium spp. levels had the most significant area under the curve.
ABSTRACT: BACKGROUND: Voriconazole is approved for treatment of invasive aspergillosis and other invasive fungal infections, but the role for therapeutic drug monitoring (TDM) is not clear. METHODS: We performed a retrospective cohort study of patients at the University of Washington Medical Center and Fred Hutchinson Cancer Research Center from 2007--2009. We compared the effect of therapeutic levels on clinical outcomes and evaluated the relationship between drug levels and adverse events. RESULTS: A total of 108 patients had voriconazole TDM performed, of whom 84 (77.8%) had a hematologic malignancy and 47 (43.5%) had undergone hematopoietic stem cell transplantation. The primary reasons for treatment were presumed pulmonary aspergillosis (n = 83, 76.8%), other invasive mould infections (n = 13, 12.0%) and candidiasis (n = 9, 8.3%). There was a high degree of variability in voriconazole drug levels among patients (r2 = 0.01; range, <0.10 - 20 mg/L). Of the 46 patients with proven or probable invasive fungal disease, 25 (54.3%) achieved partial or complete response to therapy. There was no significant relationship between therapeutic drug levels and achievement of complete or partial response at 12 weeks (OR 0.29, 95% CI: 0.05-1.34) or radiologic response (OR 1.46, 95% CI: 0.32-7.83). Overall, 45 (41.7%) patients experienced adverse events. Voriconazole levels > 5.5 mg/L were not associated with increased incidence of encephalopathy (OR 3.08, 95% CI 0.79-11.0) or hepatotoxicity (OR 2.45, 95% CI 0.49-10.1). CONCLUSIONS: Voriconazole therapeutic drug levels were not associated with improvement in clinical outcomes among patients with proven or probable invasive fungal disease. We also did not find an association between supratherapeutic drug levels and hepatoxicity or encephalopathy. It is possible that the utility of voriconazole therapeutic drug monitoring to improve clinical efficacy or decrease adverse events may be limited to a subset of high-risk patients.
Bacterial vaginosis (BV) is a highly prevalent condition associated with adverse health outcomes. Gram stain analysis of vaginal fluid is the standard for confirming the diagnosis of BV, wherein abundances of key bacterial morphotypes are assessed. These Lactobacillus, Gardnerella, Bacteroides, and Mobiluncus morphotypes were originally linked to particular bacterial species through cultivation studies, but no studies have systematically investigated associations between uncultivated bacteria detected by molecular methods and Gram stain findings. In this study, 16S-rRNA PCR/pyrosequencing was used to examine associations between vaginal bacteria and bacterial morphotypes in 220 women with and without BV. Species-specific quantitative PCR (qPCR) and fluorescence in Situ hybridization (FISH) methods were used to document concentrations of two bacteria with curved rod morphologies: Mobiluncus and the fastidious BV-associated bacterium-1 (BVAB1). Rank abundance of vaginal bacteria in samples with evidence of curved gram-negative rods showed that BVAB1 was dominant (26.1%), while Mobiluncus was rare (0.2% of sequence reads). BVAB1 sequence reads were associated with Mobiluncus morphotypes (p<0.001). Among women with curved rods, mean concentration of BVAB1 DNA was 2 log units greater than Mobiluncus (p<0.001) using species-specific quantitative PCR. FISH analyses revealed that mean number of BVAB1 cells was 2 log units greater than Mobiluncus cells in women with highest Nugent score (p<0.001). Prevotella and Porphyromonas spp. were significantly associated with the "Bacteroides morphotype," whereas Bacteroides species were rare. Gram-negative rods designated Mobiluncus morphotypes on Gram stain are more likely BVAB1. These findings provide a clearer picture of the bacteria associated with morphotypes on vaginal Gram stain.
Adeno-associated virus (AAV) is widely considered to be nonpathogenic, but the clinical epidemiology of this virus is limited. By use of polymerase chain reaction assays, we investigated the incidence and clinical significance of AAV viremia in a population of consecutive recipients of a hematopoietic cell transplant (HCT). Four (2.8%) of 145 patients developed AAV viremia after HCT. Viremia was low level and transient in all patients. Two patients were admitted to the hospital and died in proximity to AAV viremia (<7 weeks between diagnosis and death); however, AAV was not detected in tissue specimens obtained at autopsy. Thus, AAV does not appear to play a pathogenic role in organ-specific illness, even in a highly immunocompromised population.
Several fastidious bacteria have been associated with bacterial vaginosis (BV), but their role in lactobacilli recolonization failure is unknown. We studied the effect of 7 BV-associated bacterial species and 2 Lactobacillus species on vaginal colonization with Lactobacillus crispatus CTV-05 (LACTIN-V).
Host iron availability is fundamental to mucormycosis pathogenesis. The combination of liposomal amphotericin B (LAmB) and deferasirox iron chelation therapy synergistically improved survival in diabetic mice with mucormycosis. To determine the safety of combination deferasirox plus LAmB therapy for mucormycosis, a multicentred, placebo-controlled, double-blinded clinical trial was conducted.
Urinary tract infections (UTIs) are common among women and frequently recur. Depletion of vaginal lactobacilli is associated with UTI risk, which suggests that repletion may be beneficial. We conducted a double-blind placebo-controlled trial of a Lactobacillus crispatus intravaginal suppository probiotic (Lactin-V; Osel) for prevention of recurrent UTI in premenopausal women.
Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients.
The human vagina hosts a collection of microbes that is distinct from other human surfaces and mucosal sites, with reduced microbial diversity that is likely driven by the acidic environment. The microbial ecosystem of the vagina is dominated by lactobacilli in women without bacterial vaginosis (BV), and is characterize by increased species richness, diversity, and evenness in women with BV. The use of molecular, cultivation-independent methods to describe the bacterial biota of the human vagina has revealed many novel putative anaerobes in women with BV, and has demonstrated the almost ubiquitous nature of Lactobacillus iners which is found in most women regardless of BV status. A variety of molecular tools are being employed to study the vaginal microbiota, and each approach has distinct advantages and disadvantages that are reviewed. Longitudinal studies have demonstrated that the vaginal microbiota can be highly dynamic, with dramatic shifts in bacterial composition and concentrations in response to numerous endogenous and exogenous factors.
The microbiota of the human vagina can affect the health of women, their fetuses, and newborns. Bacterial vaginosis (BV) is the most prevalent form of vaginal infection in women of reproductive age, affecting 8% to 23%, and is the most common etiology of vaginal symptoms prompting women to seek medical care. While traditional cultivation has identified numerous BV-associated bacteria involved in these processes, recent advances in molecular biology have facilitated the detection and identification of bacteria without cultivation, some of which have not previously been described or well characterized. A more complete understanding of vaginal microbial populations resulting from the adoption of molecular tools may lead to better strategies to maintain healthy vaginal microbial communities-thus enhancing womens health-and will create opportunities to explore the role of novel bacteria in reproductive tract diseases. On November 19-20, 2008, the NIH convened a workshop of experts in the field of research and clinical practice related to BV in order to discuss how these new advances should be interpreted and applied to research in progress and collaborations between relevant disciplines. This paper summarizes the presentations of this workshop and outlines general recommendations arising from the related discussions. Future studies of BV and its associated adverse outcomes should determine if specific combinations of organisms are more pathogenic than others, and causally associated with different adverse events. Moreover, determination of causality will depend not only on more precise categorization of the vaginal microbiota, but also on variations in the host environment that may be associated with changes in bacterial communities over time. In this report, we offer suggestions and recommendations that we hope will facilitate conduct of consistent approaches to collaborative efforts towards advancing our understanding of the vaginal microbiota and its impact on human health.
Little is known about short-term bacterial fluctuations in the human vagina. This study used PCR to assess the variability in concentrations of key vaginal bacteria in healthy women and the immediate response to antibiotic treatment in women with bacterial vaginosis (BV).
Bacterial vaginosis (BV) is common in women who have sex with women. While cross-sectional data support a role for sexual transmission, risks for incident BV have not been prospectively studied in this group.
PCR is a very appealing technology for the detection of human pathogens, but the detection of fungal pathogens is particularly challenging. Fungi have cell walls that impede the efficient lysis of organisms and liberation of DNA, which can lead to false-negative PCR results. Conversely, some human pathogens are also ubiquitous environmental saprophytes that can contaminate PCR reagents and cause false-positive results. We examine the quality of PCR-based studies for fungal diagnostics using 42 variables within the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines. This review focuses on taxon-directed PCR assays for the diagnosis of invasive aspergillosis, candidiasis and Pneumocystis pneumonia. Finally, we evaluate broad-range fungal PCR assays capable of detecting a wide spectrum of human pathogens.
Bacterial vaginosis (BV) affects millions of women, is extremely prevalent and is frequently chronic. We recognize numerous microbiologic variations among women with BV and this variability may explain the limited effectiveness of metronidazole in curing BV and/or reducing the risk of spontaneous preterm birth (SPTB) among BV-positive pregnant women. We assessed the independent role of seven common BV-associated bacteria on the risk of spontaneous preterm birth (SPTB) among urban pregnant women.
Bacterial vaginosis (BV) is a common condition that is associated with preterm birth and acquisition of complex communities of vaginal bacteria that include several fastidious species. Treatment of BV in pregnancy has mixed effects on the risk of preterm delivery, which some hypothesize is due to variable antibiotic efficacy for the fastidious bacteria. Both oral and intravaginal metronidazole can be used to treat bacterial vaginosis in pregnancy, but little is known about the impact of different routes of antibiotic administration on concentrations of fastidious vaginal bacteria.
The bacterial biota of the human vagina can have a profound impact on the health of women and their neonates. Changes in the vaginal microbiota have been associated with several adverse health outcomes including premature birth, pelvic inflammatory disease, and acquisition of HIV infection. Cultivation-independent molecular methods have provided new insights regarding bacterial diversity in this important niche, particularly in women with the common condition bacterial vaginosis (BV). PCR methods have shown that women with BV have complex communities of vaginal bacteria that include many fastidious species, particularly from the phyla Bacteroidetes and Actinobacteria. Healthy women are mostly colonized with lactobacilli such as Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus iners, though a variety of other bacteria may be present. The microbiology of BV is heterogeneous. The presence of Gardnerella vaginalis and Atopobium vaginae coating the vaginal epithelium in some subjects with BV suggests that biofilms may contribute to this condition.
Several fastidious bacteria have been associated with bacterial vaginosis (BV) using broad-range bacterial PCR methods such as consensus sequence 16S rRNA gene PCR, but their role in BV remains poorly defined. We describe changes in vaginal bacterial concentrations following metronidazole therapy for BV. Vaginal swabs were collected from women with BV diagnosed using Amsel clinical criteria, and vaginal fluid was assessed by Gram stain to generate Nugent scores. Follow-up swabs were collected 1 month after a 5-day course of vaginal 0.75% metronidazole gel and analyzed for 24 subjects with cured BV and 24 subjects with persistent BV. Changes in bacterial concentrations were measured using eight bacterium-specific 16S rRNA gene quantitative PCR assays. DNA from several fastidious BV-associated bacteria (BVAB) were present at high concentrations in the vagina prior to treatment. Successful antibiotic therapy resulted in 3- to 4-log reductions in median bacterial loads of BVAB1 (P=0.02), BVAB2 (P=0.0004), BVAB3 (P=0.03), a Megasphaera-like bacterium (P<0.0001), Atopobium species (P<0.0001), Leptotrichia/Sneathia species (P=0.0002), and Gardnerella vaginalis (P<0.0001). Median posttreatment bacterial levels did not change significantly in subjects with persistent BV except for a decline in levels of BVAB3. The presence or absence of BV is reflected by vaginal concentrations of BV-associated bacteria such as BVAB1, BVAB2, Leptotrichia/Sneathia species, Atopobium species, Gardnerella vaginalis, and a Megasphaera-like bacterium, suggesting that these bacteria play an important role in BV pathogenesis and may be suitable markers of disease and treatment response.
rRNA genes are attractive targets for developing PCR assays targeting human fungal pathogens. Most studies have focused on the 18S rRNA gene, internal transcribed spacers, and the 5 end of the 28S rRNA gene. An approximately 2,900-bp region of the 28S rRNA gene remains largely unexplored because sequences of many medically relevant fungi are either unavailable or undefined in genomic databases. The internal transcribed spacers and 28S rRNA gene of nine medically and phylogenetically important fungi were sequenced. In addition, 42 sequences from this region were acquired from public databases, resulting in an alignment of 51 fungal sequences spanning 30 fungal genera. For the nearly 3,950-bp region from the 3 end of 18S rRNA gene to the 3 end of the 28S rRNA gene, 27 broad-range PCR primers were designed such that their sequence homology with the human rRNA gene was minimal. All 62 possible amplicons in the size range from 75 to 400 bp from 27 primers were screened using fungal genomic DNA from 26 species spanning 14 genera. Eleven of the 62 amplicons did not cross-react with 1 microg/PCR human DNA but simultaneously amplified 10 fg of fungal DNA. Phylogenetic distance matrices were calculated for regions covered by these 11 amplicons based on 51 fungi. Two of these 11 amplicons successfully amplified 30 fg of fungal DNA from 25 of 26 fungi and provided the most phylogenetic information for species identification based on the distance matrices. These PCR assays hold promise for detection and identification of fungal pathogens in human tissues.
Fastidious bacteria have been associated with bacterial vaginosis (BV) using PCR methods. We assessed the prevalence of these bacteria in HIV-1 infected women and their relationship with vaginal pH and shedding of HIV-1 RNA.
Acidform gel, an acid-buffering product that inactivates spermatozoa, may be an effective topical non-hormonal contraceptive. This study was designed to evaluate the safety of vaginal dosing and effects of Acidform on mucosal immune mediators, antimicrobial properties of genital secretions, and vaginal microbiota.
Bacterial vaginosis has been associated with genital HIV-1 shedding; however, the effect of specific vaginal bacterial species has not been assessed. We tested cervicovaginal lavage from HIV-1-seropositive women for common Lactobacillus species: L. crispatus, L. jensenii, and seven BV-associated species: BVAB1, BVAB2, BVAB3, Leptotrichia, Sneathia, Megasphaera, and Atopobium spp. using quantitative PCR. We used linear and Poisson regression to evaluate associations between vaginal bacteria and genital HIV-1 RNA and DNA. Specimens from 54 U.S. (310 visits) and 50 Kenyan women (137 visits) were evaluated. Controlling for plasma viral load, U.S. and Kenyan women had similar rates of HIV-1 RNA (19% of visits vs. 24%; IRR=0.95; 95% CI 0.61, 1.49) and DNA shedding (79% vs. 76%; IRR=0.90; 0.78, 1.05). At visits during antiretroviral therapy (ART), the likelihood of detection of HIV-1 RNA shedding was greater with BVAB3 (IRR=3.16; 95% CI 1.36, 7.32), Leptotrichia, or Sneathia (IRR=2.13; 1.02, 4.72), and less with L. jensenii (IRR=0.39; 0.18, 0.84). At visits without ART, only L. crispatus was associated with a lower likelihood of HIV-1 RNA detection (IRR=0.6; 0.40, 0.91). Vaginal Lactobacillus species were associated with lower risk of genital HIV-1 shedding, while the presence of certain BV-associated species may increase that risk.
The presence of hydrogen peroxide (H(2)O(2)) producing Lactobacillus in the vagina may play a role in controlling genital HIV-1 shedding. Sensitive molecular methods improve our ability to characterize the vaginal microbiota; however, they cannot characterize phenotype. We assessed the concordance of H(2)O(2)-producing Lactobacillus detected by culture with quantitative PCR (qPCR) detection of Lactobacillus species commonly assumed to be H(2)O(2)-producers.
Genital secretions collected from adult women exhibit in vitro activity against herpes simplex virus (HSV) and Escherichia coli (E. coli), but prior studies have not investigated this endogenous antimicrobial activity or its mediators in adolescent females.
Bacterial vaginosis (BV) is a common condition that is associated with numerous adverse health outcomes and is characterized by poorly understood changes in the vaginal microbiota. We sought to describe the composition and diversity of the vaginal bacterial biota in women with BV using deep sequencing of the 16S rRNA gene coupled with species-level taxonomic identification. We investigated the associations between the presence of individual bacterial species and clinical diagnostic characteristics of BV.
Bacterial vaginosis (BV) represents shifts in microbiota from Lactobacillus spp. to diverse anaerobes. Although antibiotics relieve symptoms and temporarily eradicate BV-associated bacteria (BVAB), BV usually recurs. We investigated the role of extravaginal BVAB reservoirs in recurrence.
Optimal clinical care and clinical investigation of patients with mucormycosis are limited by absence of controlled trials, and absence of well-defined predictors of mortality or clinical response. The Deferasirox-AmBisome Therapy for mucormycosis (DEFEAT Mucor) study was the first randomized clinical trial conducted on patients with mucormycosis, and demonstrated that adjunctive deferasirox therapy did not improve outcomes of the disease. The current study describes clinical factors from the 20 patients enrolled to identify those associated with 90-day mortality of the 11 (55%) patients who died by day 90. Age, diabetes mellitus, transplant status, or antifungal therapy were not associated with mortality. However, active malignancy or neutropenia at enrollment were associated with increased mortality. Pulmonary infection was linked with lower Kaplan-Meier survival compared to non-pulmonary infection. Higher baseline serum concentrations of iron and ferritin were also associated with mortality. No patient who progressed clinically during the first 14 days of study therapy survived; however, many patients who clinically improved during that time did not survive to 90 days. In contrast, day 30 clinical response was predictive of 90-day survival. These factors may be useful in defining enrollment randomization stratification critieria for future clinical trials, and in supporting clinical care of patients with mucormycosis.
Determining the etiology of invasive fungal infections (IFI) is critical for patient management as fungi vary in their susceptibility to antifungals. However, the etiology remains obscure in many cases due to negative culture results. The identification of fungal DNA by PCR in pathology blocks and sequencing it is an alternative approach to determine the cause of IFI. Previous studies identified fungal DNA in only 50% of samples with positive histopathology results, probably due to DNA damage by tissue fixation. We used realtime PCR to quantify human and fungal DNA from formalin-fixed, paraffin-embedded tissue specimens in order to study the effect of thermal energy during extraction on the yield of amplifiable DNA and subsequent identification of fungal DNA. Tissue sections from eight patients with proven IFI were subjected to DNA extraction with varying exposure to thermal energy. Amplifiable DNA increased up to 76-fold by increasing the incubation temperature from 65°C to 90°C and an additional increase was documented by incubating samples for up to 6 hours at this temperature. The augmented amplification of fungal DNA was associated with improved species identification by the sequencing of the PCR amplicons. This may help illuminate the etiology of IFI and thereby improve patient management by guiding antifungal therapy.
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