Molecular characterization of vaginal microbiota using a new 22-species qRT-PCR test to achieve a relative-abundance and species-based diagnosis of bacterial vaginosis.

Background: Numerous bacteria are involved in the etiology of bacterial vaginosis (BV). Yet, current tests only focus on a select few. We therefore designed a new test targeting 22 BV-relevant species. Methods: Using 946 stored vaginal samples, a new qPCR test that quantitatively identifies 22 bacterial species was designed. The distribution and relative abundance of each species, α- and ß-diversities, correlation, and species co-existence were determined per sample. A diagnostic index was modeled from the data, trained, and tested to classify samples into BV-positive, BV-negative, or transitional BV. Results: The qPCR test identified all 22 targeted species with 95 - 100% sensitivity and specificity within 8 hours (from sample reception). Across most samples, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii, Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae, Prevotella bivia, and Megasphaera sp. type 1 were relatively abundant. BVAB-1 was more abundant and distributed than BVAB-2 and BVAB-3. No Mycoplasma genitalium was found. The inter-sample similarity was very low, and correlations existed between key species, which were used to model, train, and test a diagnostic index: MDL-BV index. The MDL-BV index, using both species and relative abundance markers, classified samples into three vaginal microbiome states. Testing this index on our samples, 491 were BV-positive, 318 were BV-negative, and 137 were transitional BV. Although important differences in BV status were observed between different age groups, races, and pregnancy status, they were statistically insignificant. Conclusion: Using a diverse and large number of vaginal samples from different races and age groups, including pregnant women, the new qRT-PCR test and MDL-BV index efficiently diagnosed BV within 8 hours (from sample reception), using 22 BV-associated species.

Case Report: Post-Partum Complications of NFκB1 Deficiency Underscore a Need to Better Understand Primary Immunodeficiency Management During Pregnancy.

Nuclear factor κappa-B (NFκB) is a family of transcription factors involved in regulating inflammation and immunity. Mutations in the NFκB1 pathway are associated with primary immune defects and underlie the most common monogenic etiology of common variable immunodeficiency (CVID). However, little is known about how NFκB1 defects or primary immunodeficiency (PID) complicate pregnancy. We present a previously healthy 34-year-old patient who suffered from poor wound healing and sterile sepsis during the post-partum period of each of her three pregnancies. She was otherwise asymptomatic, but her daughter developed Evans Syndrome (ES) with hypogammaglobulinemia prompting expanded genetic testing which revealed a novel monoallelic variant in NFκB1. This case highlights that pregnancy-related complications of PID can be difficult to recognize and may portend adverse patient outcomes. For these reasons, guidance regarding diagnosis and management of women of childbearing age with PID is warranted.

Development of a Novel Test for Simultaneous Bacterial Identification and Antibiotic Susceptibility.

Background. Elucidation of a pathogen's antimicrobial susceptibility requires subculture after the organism is first isolated. This takes several days, requiring patients to be treated with broad-spectrum antibiotics. This approach contributes to the development of bacterial resistance. Methods. Microtiter wells were coated with a polyclonal antibody targeting the pathogen of interest. Bacterial suspensions were added in the presence/absence of selected antibiotics. After washing, captured bacteria were detected. Findings. Group B streptococcus (GBS), Enterococcus faecalis, and Neisseria gonorrhoeae were each detected at 105 bacteria/mL following a 20-minute incubation period. Susceptibility to select antibiotics was discernable following a 6-hour incubation period (GBS and Enterococcus). Sensitivity was increased to 10-2 bacteria/mL for GBS, 10-1 bacteria/mL for E. faecalis, and 101 bacteria/mL for N. gonorrhoeae following 18-24-hour culture. Conclusion. This novel assay allows for the highly sensitive and specific identification of a pathogen and simultaneous determination of its antimicrobial susceptibility in a reduced time.

Repair of a recurrent rectovaginal fistula with a biological graft.

This case involves a patient with the congenital absence of the lower third of the vagina. While undergoing surgical restoration of the vagina, she sustained a laceration, which ultimately led to the development of a rectovaginal fistula. After two unsuccessful attempts at repair, the recommendation was for a diverting colostomy with another attempted repair, and she presented to our clinic to discuss other possible surgical options. The patient underwent repair of the fistula using a porcine-derived small intestinal submucosal extracellular matrix graft, which resulted in the repair of the rectovaginal fistula without recurrence at 18 months' follow-up.

Accuracy of an accelerated, culture-based assay for detection of group B streptococcus.

OBJECTIVE: To determine the validity of a novel Group B Streptococcus (GBS) diagnostic assay for the detection of GBS in antepartum patients. STUDY DESIGN: Women were screened for GBS colonization at 35 to 37 weeks of gestation. Three vaginal-rectal swabs were collected per patient; two were processed by traditional culture (commercial laboratory versus in-house culture), and the third was processed by an immunoblot-based test, in which a sample is placed over an antibody-coated nitrocellulose membrane, and after a six-hour culture, bound GBS is detected with a secondary antibody. RESULTS: 356 patients were evaluated. Commercial processing revealed a GBS prevalence rate of 85/356 (23.6%). In-house culture provided a prevalence rate of 105/356 (29.5%). When the accelerated GBS test result was compared to the in-house GBS culture, it demonstrated a sensitivity of 97.1% and a specificity of 88.4%. Interobserver reliability for the novel GBS test was 88.2%. CONCLUSIONS: The accelerated GBS test provides a high level of validity for the detection of GBS colonization in antepartum patients within 6.5 hours and demonstrates a substantial agreement between observers.

Necrotizing soft-tissue infections in obstetric and gynecologic patients.

For the clinician, necrotizing soft-tissue infections have remained a daunting opponent since the first writings on the subject over 2000 years ago. Early disease may be incorrectly diagnosed as cellulitis, and this delay in correctly diagnosing and expeditiously proceeding to radical surgical debridement may lead to a high degree of mortality. Although several inciting events and risk factors have been described that allow for the development and progression of this disease, the diagnosis is still made clinically. Only aggressive surgical management in combination with broad-spectrum antibiotics will offer a chance at improving patient outcomes.

Optimization of a rapid diagnostic test for detection of group B streptococcus from antepartum patients.

We analyzed the performance of a new rapid diagnostic test for use in determining group B streptococcus colonization in pregnancy. Vaginal-rectal specimens were compared by the rapid test, a commercial laboratory culture result, and an in-house culture. Of 150 patient samples, 72 were positive by the rapid test, giving a prevalence of 48.0% versus 24.7% by traditional culture. Characterization of these results showed cross-reactivity with Enterococcus. The addition of bacitracin reduced this interference, and when reanalyzed, a colonization rate of 31.3% was found (P = 0.3961, chi-square), as well as a sensitivity of 100% (95% confidence interval [CI] 89.1-100) and a specificity of 93.6% (95% CI 86.9-97.2). The addition of bacitracin greatly improves the reliability of this diagnostic test and demonstrates a novel approach to reduce interference. An accurate determination of the test's sensitivity and specificity, however, awaits enrollment of the remaining subjects.

Rapid diagnostic test for identifying group B streptococcus.

Neonatal infection with Streptococcus agalactiae (group B streptococcus [GBS]) causes significant morbidity and mortality. A truly rapid diagnostic test for identifying GBS would allow for more timely initiation of antibiotic prophylaxis and also reduce the administration of antibiotics for the prevention of early onset neonatal GBS infection. A stock culture was formed from a laboratory reference strain of GBS and was diluted from 10 (7) to 10 (1) bacteria/mL. Specific concentrations were used to inoculate nitrocellulose membranes (NCMs) that had been coated previously with polyclonal rabbit antibody against GBS. After specific times, the NCMs were removed from the sheep blood agar medium, and horseradish-peroxidase conjugate polyclonal antibody against GBS was added. Bound antibody was detected with diaminobenzidine. After 6 hours of incubation, GBS was detected at concentrations from 10 (7) through 10 (4) bacterial/mL. After 4 hours of incubation, GBS was detected at concentrations from 10 (7) through 10 (5) bacteria/mL. GBS was not detected at 2 hours of incubation. Rapid growth and detection of GBS can be performed, and the results can be reliably attained as early as 4 hours. This is in marked contrast to the 48 to 72 hours required by current methods.

Screening for group B streptococcus: a private hospital's experience.

OBJECTIVE: To assess the effect of universal screening and administration of intrapartum antibiotic prophylaxis to prevent early-onset neonatal GBS sepsis at a private tertiary care hospital since issuance of the 2002 CDC guidelines for preventing perinatal GBS disease. METHODS: Retrospective analysis of women delivering between January 1, 2003 and December 31, 2004 at a private tertiary care hospital in Houston, Texas. The percentage of women screened, GBS positive women receiving intrapartum antibiotic prophylaxis, and infants developing early-onset GBS sepsis were determined. RESULTS: 2,108 women delivered 2,135 infants with 1,874 (89%) screened for GBS. Of those screened, 1,322 (71%) tested negative and 552 (29%) tested positive for GBS. In this analysis of 2,135 infants, 3 (0.94 cases/1,000 live births) were diagnosed with invasive GBS sepsis. CONCLUSION: High rates of screening of pregnant women for GBS colonization and use of intrapartum antibiotic prophylaxis for GBS carriers can be achieved in a private tertiary care hospital setting. " SYNOPSIS: High screening rates for group B streptococcus in a private tertiary care hospital reduce the incidence of maternal and early onset neonatal GBS infection."

Lactocin 160, a Bacteriocin Produced by Vaginal Lactobacillus rhamnosus, Targets Cytoplasmic Membranes of the Vaginal Pathogen, Gardnerella vaginalis.

Bacterial vaginosis (BV) is a commonly occurring vaginal infection that is associated with a variety of serious risks related to the reproductive health of women. Conventional antibiotic treatment for this condition is frequently ineffective because the antibiotics tend to inhibit healthy vaginal microflora along with the pathogens. Lactocin 160, a bacteriocin produced by healthy vaginal lactobacilli, is a promising alternative to antibiotics; this compound specifically inhibits the BV-associated vaginal pathogens such as Gardnerella vaginalis and Prevotella bivia without affecting the healthy microflora. This study investigates the molecular mechanism of action for lactocin 160 and reveals that this compound targets the cytoplasmic membrane of G. vaginalis, causing the efflux of ATP molecules and dissipation of the proton motive force.

Prevotella bivia as a source of lipopolysaccharide in the vagina.

OBJECTIVES: To compare vaginal lipopolysaccharides (LPS) concentrations between patients with and without bacterial vaginosis (BV), to evaluate the correlation between Prevotella bivia colonization density and LPS concentration, and to determine the impact of LPS on loss of dopamine neurons (DA). METHODS: Vaginal washes obtained from patients with (n=43) and without (n=59) BV were tested for quantity of P. bivia cells using quantitative PCR and for concentrations of LPS using the Limulus Amebocyte Lysate gel clot method. Prevotella bivia, Gardnerella vaginalis and Escherichia coli sonicated cell extracts were also tested for LPS production. DA neuron cells obtained from embryonic day (E) 14.5 pregnant rats were exposed to fluid from eight vaginal washes; tyrosine hydrolase immunoreactive staining was applied for visualization and cell counts. RESULTS: The median LPS concentrations were dramatically higher among patients who had symptoms of BV compared to those who did not have symptoms (3235.0 vs 46.4 EU/ml, respectively, P<0.001); patients who had BV also had much higher colonization densities of P. bivia (0.06+/-0.36 vs 5.4+/-2.2 log(10) CFU/ml, respectively, P<0.001). Prevotella bivia cell lysates resulted in a higher LPS concentration (10,713.0+/-306.6 EU/ml) than either E. coli (4679.0+/-585.3 EU/ml) or G. vaginalis (0.07+/-0.01 EU/ml of LPS). The loss of DA neuron was 20-27% in cultures treated with vaginal washes from BV-negative patients and 58-97% in cultures treated with vaginal washes from patients with BV. CONCLUSION: P. bivia produces high LPS concentration, which may create a toxic vaginal environment that damages DA neurons.

Spermicidal activity of the safe natural antimicrobial peptide subtilosin.

Bacterial vaginosis (BV), a condition affecting millions of women each year, is primarily caused by the gram-variable organism Gardnerella vaginalis. A number of organisms associated with BV cases have been reported to develop multidrug resistance, leading to the need for alternative therapies. Previously, we reported the antimicrobial peptide subtilosin has proven antimicrobial activity against G. vaginalis, but not against the tested healthy vaginal microbiota of lactobacilli. After conducting tissue sensitivity assays using an ectocervical tissue model, we determined that human cells remained viable after prolonged exposures to partially-purified subtilosin, indicating the compound is safe for human use. Subtilosin was shown to eliminate the motility and forward progression of human spermatozoa in a dose-dependent manner, and can therefore be considered a general spermicidal agent. These results suggest subtilosin would be a valuable component in topical personal care products aimed at contraception and BV prophylaxis and treatment.

Postoperative pelvic infections.

Infectious morbidity affecting the postoperative course has long been a concern for obstetricians and gynecologists. The incidence of postoperative infections approaches 38%. The third most common nosocomial infection is surgical site infection. The realm of postoperative infections includes obstetric and gynecologic sources. An understanding of the basic fundamentals of the vaginal flora and addressing host and surgical risk factors can aid in prevention of postoperative infections, which result in significant morbidity and mortality.

The efficacy and safety of a single dose of Clindesse vaginal cream versus a seven-dose regimen of Cleocin vaginal cream in patients with bacterial vaginosis.

OBJECTIVE: To determine whether a single dose of Clindesse vaginal cream is comparable in efficacy and safety to Cleocin vaginal cream administered once daily for 7 days in the treatment of bacterial vaginosis. STUDY DESIGN: This multicenter, randomized, single-blind, parallel-group study enrolled 540 patients with BV infections. Treatment consisted of either a single intravaginal dose of Clindesse or 7 daily doses of Cleocin. Efficacy and safety were assessed 21-30 days after the start of treatment. The efficacy endpoints were Investigator Cure, Clinical Cure (a composite of all 4 Amsel's criteria and Investigator Cure), Nugent Cure (Nugent score < 4), and Therapeutic Cure (a composite of Clinical Cure and Nugent Cure). Resolution of individual Amsel's criteria was also evaluated. Treatment-emergent adverse events were monitored throughout the study. RESULTS: There were no significant differences in cure rates between the Clindesse and Cleocin treatment groups in Investigator Cure (P=0.702), Clinical Cure (P=0.945), Nugent Cure (P=0.788), or Therapeutic Cure (P=0.572). Results were also similar for 3 of 4 and 2 of 4 Amsel's criteria and for each individual Amsel's criterion (all P-values >0.200). Ninety-five percent confidence intervals for each endpoint were consistent with equivalence between the 2 products. There was no significant difference between the treatment groups in the incidence of treatment-emergent adverse events (P=0.386). CONCLUSIONS: A single dose of Clindesse vaginal cream is equivalent in safety and efficacy to a 7-dose regimen of Cleocin vaginal cream in the treatment of bacterial vaginosis. This represents a significant advance in the treatment of bacterial vaginosis.

Postpartum endometritis.

Postpartum endometritis or a surgical site infection should be suspected if the patient develops an elevated oral temperature, 100.4 degrees F or higher, with an associated tachycardia following the procedure. A tachycardia paralleling the temperature strongly indicates infection. A thorough examination should be performed. Patients failing to respond to initial antibiotic therapy should be thoroughly evaluated for the possible emergence of a resistant bacterium or the development of an abscess or septic pelvic thrombosis. Antibiotic therapy should be continued until the patient is afebrile for 24 to 48 hours, the white blood cell count returns to normal, and the patient is tolerating oral liquids and solids, and ambulating without difficulty.

Mode of action of lactocin 160, a bacteriocin from vaginal Lactobacillus rhamnosus.

OBJECTIVES: To determine the mechanism of antimicrobial action of lactocin 160, a bacteriocin produced by the healthy vaginal strain of Lactobacillus rhamnosus, using an established model, with Micrococcus luteus ATCC 10420 as a test organism. METHODS: Sensitivity of M. luteus to lactocin 160 was determined by the diffusion assay. Loss of cellular ATP in the lactocin-treated cells was elucidated using a commercially available ATP determination kit (luciferin-luciferase bioluminescence assay). Luminescence intensity as a reflection of ATP quantity was determined using a luminometer. Dissipation of membrane potential (Deltapsi) was studied using fluorophore DiSC3(5) with the fluorescence spectrum sensitive to changes in Deltapsi. RESULTS: Lactocin 160 inhibited growth of M. luteus ATCC 10420 at a concentration of 5 microg/ml. There were no significant changes in the intracellular ATP level of M. luteus upon the addition of 20 microg/ml of lactocin 160. However, the extracellular ATP level increased significantly. This means that the treatment of cells with lactocin 160 resulted in an efflux of ATP from inside the cells. Therefore, a partially purified lactocin 160 preparation (16 microg /ml of the bacteriocin in the sample) killed sensitive cells and dissipated 3.12 +/- 0.36% of Deltapsi. CONCLUSION: Lactocin 160 has a mode of action typical for bacteriocins. It disturbs the cellular membrane (Deltapsi dissipation) and induces ATP efflux, most likely because of the pore formation, which is a common mechanism of action for many bacteriocins.

Impact of treatment for bacterial vaginosis on prematurity among Brazilian pregnant women: a retrospective cohort study.

CONTEXT AND OBJECTIVE: Bacterial vaginosis has been associated with prematurity and other perinatal complications. However, the efficacy of the treatment for preventing such complications has not yet been well established. The objective of this study was to evaluate the impact of treatment for bacterial vaginosis on a low-risk population of Brazilian pregnant women, in order to prevent prematurity and other perinatal complications. DESIGN AND SETTING: Observational retrospective cohort study, at the Obstetric and Gynecology Department, Universidade Estadual de Campinas (Unicamp). METHODS: Vaginal bacterioscopy results from 785 low-risk pregnant women were studied. Three different groups of women were identified: 580 without bacterial vaginosis during pregnancy, 134 with bacterial vaginosis treated using imidazoles (metronidazole, tinidazole, or secnidazole) during pregnancy, and 71 with bacterial vaginosis not treated during pregnancy. The diagnosis of bacterial vaginosis was based on Nugent's criteria, from the vaginal bacterioscopy performed during the first prenatal care visit. RESULTS: The frequency of prematurity was 5.5% among the women without bacterial vaginosis, 22.5% among those with untreated bacterial vaginosis and 3.7% among those with treated bacterial vaginosis. The risk ratios for perinatal complications were significantly higher in the group with untreated bacterial vaginosis: premature rupture of membranes, 7.5 (95% CI: 1.9-34.9); preterm labor, 3.4 (95% CI: 1.4-8.1); preterm birth, 6.0 (95% CI: 1.9-19.7); and low birth weight, 4.2 (95% CI: 1.2-14.3). CONCLUSION: The treatment of bacterial vaginosis significantly reduced the rates of prematurity and other perinatal complications among these low-risk Brazilian pregnant women, regardless of the history of previous preterm delivery.

Antibiotic resistance patterns of group B streptococcal clinical isolates.

OBJECTIVES: To determine the in vitro resistance of group B streptococcus (GBS) to 12 antibiotics. To determine if there has been any decrease in sensitivity to the penicillins or other antibiotics currently used for GBS chemoprophylaxis in pregnant women. Find suitable alternative antibiotics to penicillin. Find an antibiotic that will have minimal selective pressure for resistance among the endogenous resident vaginal microflora. METHODS: The antibiotic susceptibility profiles of 52 clinical isolates of GBS were evaluated to 12 antibiotics: ampicillin, azithromycin, cefamandole, cefazolin, ceftriaxone, ciprofloxacin, clindamycin, erythromycin, nitrofurantoin, ofloxacin, penicillin and vancomycin. Antibiotic sensitivities were determined using disk diffusion and microdilution methods according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS). RESULTS: All isolates were sensitive to vancomycin, ofloxacin, ampicillin, ciprofloxacin, nitrofurantoin and penicillin. However, the following number of clinical isolates exhibited intermediate or decreased sensitivity, nine (17%) to ampicillin, eight (15%) to penicillin, 14 (32%) to ciprofloxacin and one (2%) to nitrofurantoin. Thirty-one percent of the isolates were resistant to azithromycin and ceftriaxone, 19% to clindamycin, 15% to cefazolin and 13% to cefamandole. Eighteen (35%) of the clinical isolates tested were resistant to 6 of the 12 antibiotics tested. CONCLUSIONS: The relatively high rates of resistance for 6 of the 12 antibiotics tested suggest that for women allergic to penicillin and colonized with GBS, antibiotic sensitivities to their isolates should be determined. The antibiotic selected for intrapartum chemoprophylaxis should be guided by the organism's antibiotic sensitivity pattern. Patients with GBS bacteriuria should be treated with nitrofurantoin.

Multicenter study of a rapid molecular-based assay for the diagnosis of group B Streptococcus colonization in pregnant women.

BACKGROUND: Current prevention of infection due to group B Streptococcus (GBS) involves giving intrapartum antibiotics to women on the basis of either antenatal culture colonization status or presence of risk factors. METHODS: We prospectively compared the performance characteristics of a rapid molecular diagnostic test (IDI-Strep B; Infectio Diagnostic) with culture for intrapartum GBS detection after 36 weeks' gestation in 5 North American centers during the period September 2001-May 2002. Antenatal GBS screening was done according to the usual practice of participating hospitals. Two combined vaginal/anal specimens were obtained from participants during labor by use of standard techniques and processed by the same laboratories that processed the antenatal specimens. Each swab sample was processed simultaneously by culture and with IDI-Strep B. The collected specimens were randomized for order of testing of the swab samples by culture or the rapid test. RESULTS: Of enrolled women, 803 (91.1%) were eligible for analysis. The overall intrapartum GBS colonization rate by culture was 18.6% (range, 9.1%-28.7%). Compared with intrapartum culture, the molecular test had a sensitivity of 94.0% (range, 90.1%-97.8%), specificity of 95.9% (range, 94.3%-97.4%), positive predictive value of 83.8% (range, 78.2%-89.4%), and negative predictive value of 98.6% (range, 97.7%-99.5%). The molecular test was superior to antenatal cultures (sensitivity, 94% vs. 54%; P<.0001) and prediction of intrapartum status on the basis of risk factors (sensitivity, 94% vs. 42%; P<.0001). CONCLUSION: Use of this test for determination of GBS colonization during labor is highly sensitive and specific and may lead to a further reduction in rates of neonatal GBS disease.