Pathogens in Urine from a Female Patient with Overactive Bladder Syndrome Detected by Culture-independent High Throughput Sequencing: A Case Report.

INTRODUCTION: Overactive bladder syndrome (OAB) is described as urgency, with or without urgency incontinence. A range of medical conditions shares the symptoms of OAB, however the diagnosis is contingent on the exclusion of urinary tract infection (UTI). Knowing that urine dipstick and routine culture of bacteria can miss UTI diagnosis caused by low-count bacteriuria or "difficult-to-culture" pathogens, we examined a case of OAB with a culture-independent approach. CASE PRESENTATION: A 61-year-old Norwegian female with a long history of urinary symptoms and a diagnosis of OAB was selected as a suitable subject for a culture-independent 16S rDNA analysis on the patient´s urine. The patient's medical records showed no history of recurrent UTI, however, when the urine specimen was sent to routine culture at the time of study it showed a significant bacteriuria caused by a single bacterium, and the patient was prescribed antibiotics. The 16S rDNA analysis revealed not one, but many different bacteria, including a considerable amount of fastidious bacteria, indicating a polymicrobial state. One year later, the subject was still experiencing severe symptoms, and a follow-up analysis was performed. This time the urine-culture was negative, however, the 16S rDNA profile was quite similar to that of the first sample, again displaying a complex bacterial profile. CONCLUSION: The use of 16S rDNA pyrosequencing and sequence analysis to uncover "difficult-to-culture" bacteria should be considered when examining patients with chronic urinary symptoms. These methods may contribute to further elucidation of the etiology of overactive bladder syndrome and other urinary syndromes.

Alterations of microbiota in urine from women with interstitial cystitis.

BACKGROUND: Interstitial Cystitis (IC) is a chronic inflammatory condition of the bladder with unknown etiology. The aim of this study was to characterize the microbial community present in the urine from IC female patients by 454 high throughput sequencing of the 16S variable regions V1V2 and V6. The taxonomical composition, richness and diversity of the IC microbiota were determined and compared to the microbial profile of asymptomatic healthy female (HF) urine. RESULTS: The composition and distribution of bacterial sequences differed between the urine microbiota of IC patients and HFs. Reduced sequence richness and diversity were found in IC patient urine, and a significant difference in the community structure of IC urine in relation to HF urine was observed. More than 90% of the IC sequence reads were identified as belonging to the bacterial genus Lactobacillus, a marked increase compared to 60% in HF urine. CONCLUSION: The 16S rDNA sequence data demonstrates a shift in the composition of the bacterial community in IC urine. The reduced microbial diversity and richness is accompanied by a higher abundance of the bacterial genus Lactobacillus, compared to HF urine. This study demonstrates that high throughput sequencing analysis of urine microbiota in IC patients is a powerful tool towards a better understanding of this enigmatic disease.

Assessing diversity of the female urine microbiota by high throughput sequencing of 16S rDNA amplicons.

BACKGROUND: Urine within the urinary tract is commonly regarded as "sterile" in cultivation terms. Here, we present a comprehensive in-depth study of bacterial 16S rDNA sequences associated with urine from healthy females by means of culture-independent high-throughput sequencing techniques. RESULTS: Sequencing of the V1V2 and V6 regions of the 16S ribosomal RNA gene using the 454 GS FLX system was performed to characterize the possible bacterial composition in 8 culture-negative (<100,000 CFU/ml) healthy female urine specimens. Sequences were compared to 16S rRNA databases and showed significant diversity, with the predominant genera detected being Lactobacillus, Prevotella and Gardnerella. The bacterial profiles in the female urine samples studied were complex; considerable variation between individuals was observed and a common microbial signature was not evident. Notably, a significant amount of sequences belonging to bacteria with a known pathogenic potential was observed. The number of operational taxonomic units (OTUs) for individual samples varied substantially and was in the range of 20-500. CONCLUSIONS: Normal female urine displays a noticeable and variable bacterial 16S rDNA sequence richness, which includes fastidious and anaerobic bacteria previously shown to be associated with female urogenital pathology.

Serum level of immunoglobulin E during pregnancy - does offspring sex matter?

We assessed maternal serum levels of total immunoglobulin E (IgE) in the first, second and third trimester and changes in total IgE levels from first to third trimester in relation to offspring sex. Within a cohort of 29 948 pregnant women, 392 women without a history of pre-eclampsia and with a liveborn child were randomly selected. Information on offspring sex was obtained through linkage to the Medical Birth Registry of Norway. Blood samples from each trimester were analysed for total IgE concentration. Differences in mean levels according to offspring sex were estimated and changes in total IgE levels from first to third trimester were assessed. In all three trimesters there was a tendency of women carrying a male fetus to have a higher mean total IgE level, but significant statistical differences were not reached. The total IgE concentration decreased during pregnancy, but the decrement was less in women carrying a male fetus compared with those who carried a female fetus.

Bacteria, biofilm and honey: a study of the effects of honey on 'planktonic' and biofilm-embedded chronic wound bacteria.

Chronically infected wounds are a costly source of suffering. An important factor in the failure of a sore to heal is the presence of multiple species of bacteria, living cooperatively in highly organized biofilms. The biofilm protects the bacteria from antibiotic therapy and the patient's immune response. Honey has been used as a wound treatment for millennia. The components responsible for its antibacterial properties are now being elucidated. The study aimed to determine the effects of different concentrations of 'Medihoney' therapeutic honey and Norwegian Forest Honey 1) on the real-time growth of typical chronic wound bacteria; 2) on biofilm formation; and 3) on the same bacteria already embedded in biofilm. Reference strains of MRSE, MRSA, ESBL Klebsiella pneumoniae and Pseudomonas aeruginosa were incubated with dilution series of the honeys in microtitre plates for 20 h. Growth of the bacteria was assessed by measuring optical density every 10 min. Growth curves, biofilm formation and minimum bactericidal concentrations are presented. Both honeys were bactericidal against all the strains of bacteria. Biofilm was penetrated by biocidal substances in honey. Reintroduction of honey as a conventional wound treatment may help improve individual wound care, prevent invasive infections, eliminate colonization, interrupt outbreaks and thereby preserve current antibiotic stocks.

Prevalence of sexually transmitted infections among pregnant women with known HIV status in northern Tanzania.

OBJECTIVES: To determine the prevalence of sexually transmitted infections (STIs) and other reproductive tract infections (RTIs) among pregnant women in Moshi, Tanzania and to compare the occurrence of STIs/RTIs among human immunodeficiency virus (HIV)-infected and uninfected women. METHODS: Pregnant women in their 3rd trimester (N = 2654) were recruited from two primary health care clinics between June 2002 and March 2004. They were interviewed, examined and genital and blood samples were collected for diagnosis of STIs/RTIs and HIV. RESULTS: The prevalence of HIV, active syphilis and herpes simplex virus - type 2 (HSV-2) were 6.9%, 0.9% and 33.6%, respectively, while 0.5% were positive for N gonorrhoeae, 5.0% for T vaginalis and 20.9% for bacterial vaginosis. Genital tract infections were more prevalent in HIV-seropositive than seronegative women, statistically significant for syphilis (3.3% vs 0.7%), HSV-2 (43.2% vs 32.0%), genital ulcers (4.4% vs 1.4%) and bacterial vaginosis (37.2% vs 19.6%). In comparison with published data, a declining trend for curable STIs/RTIs (syphilis, trichomoniasis and bacterial vaginosis) was noted. CONCLUSION: Rates of STIs and RTIs are still high among pregnant women in Moshi. Where resources allow, routine screening and treatment of STIs/RTIs in the antenatal care setting should be offered. Higher STIs/RTIs in HIV-seropositive women supports the expansion of HIV-counseling and testing services to all centers offering antenatal care. After identification, STIs/RTIs need to be aggressively addressed in HIV-seropositive women, both at antenatal and antiretroviral therapy care clinics.

Levels of angiogenic factors in pregnancy and post-partum bleeding.

We have studied if serum levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and placenta growth factor (PlGF) in pregnancy could predict excess post-partum bleeding. In 392 normotensive singleton pregnancies, concentrations of sFlt-1 and PlGF in the first, second, and third trimester were compared between women with and without excess post-partum bleeding, defined as blood loss volume of at least 500 mL. Mean concentrations of sFlt-1 were consistently higher in all three trimesters among women who had excess post-partum bleeding compared to women without this, but significantly higher only in the second trimester. For PlGF, there were no significant differences between the groups. High concentrations of the anti-angiogenic factor sFlt-1 in maternal circulation during pregnancy may be associated with an increased risk of excess post-partum bleeding.

Serum levels of immunoglobulin E and the subsequent risk of pre-eclampsia: a population-based study.

OBJECTIVE: Women with allergy may be at increased risk of developing preeclampsia. We assessed serum levels of immunoglobulin E (IgE) in the first and second trimester, and changes in concentration between trimesters, in relation to risk of preeclampsia. MATERIALS AND METHODS: Population based case-control study within a cohort of 29,948 pregnant women. We included 154 preeclampsia cases with preterm delivery (before week 37) and 190 cases with delivery at term. As controls, 392 women without preeclampsia were randomly selected. Levels of IgE were in sera from the first and second trimester were measured and women who later developed preeclampsia were compared to controls with respect to IgE levels. RESULTS: Comparing the highest to the lowest quartile of IgE in the first trimester, the odds ratio for preterm preeclampsia was 1.7 (95% CI, 0.9-3.1), and in the second trimester, the odds ratio was 1.5 (95% CI, 0.8-2.9) for the highest compared to the lowest quartile of IgE. Change in IgE between trimesters was not associated with preeclampsia risk. CONCLUSION: There was a weak, however, not significant association between high levels of IgE in the first and second trimester and subsequent risk of preterm preeclampsia.

Changes in circulating level of IGF-I and IGF-binding protein-1 from the first to second trimester as predictors of preeclampsia.

OBJECTIVE: To assess whether circulating IGF-I and IGF-binding protein-1 (IGFBP-1) in the first and second trimester are associated with subsequent risk of preterm and term preeclampsia. METHODS: Nested case-control study within a cohort of 29 948 pregnant women. Cases were women, who later developed preeclampsia, and controls were randomly selected women, who did not develop preeclampsia. IGF-I and IGFBP-1 were measured with ELISA in maternal blood samples that were collected in the first and second trimesters. We assessed associations of IGF-I and IGFBP-1 concentrations with later development of preterm (before the 37th week of gestation) and term preeclampsia. RESULTS: An increase in IGF-I from the first to second trimester was associated with higher risk of preterm preeclampsia; the odds ratio (OR) for the highest compared with lowest quartile of increase was 4.9 (95% confidence interval, 1.1-21.8). Low concentrations of IGFBP-1, both in the first and in the second trimesters, were related to higher risk of term preeclampsia (OR 4.0, 95% confidence interval, 1.9-8.4) and moderately increased risk of preterm preeclampsia (OR 2.3, 95% confidence interval, 1.2-4.4). CONCLUSION: The higher risk of preterm preeclampsia related to IGF-I increase may reflect placental disease, whereas low concentrations of IGFBP-1 associated with term preeclampsia may reflect maternal metabolic aberrations, indicating different etiologies in preeclampsia.

CMV quantitative PCR in the diagnosis of CMV disease in patients with HIV-infection - a retrospective autopsy based study.

BACKGROUND: Patients with advanced HIV infection at the time of diagnosis and patients not responding to antiretroviral therapy are at risk of cytomegalovirus (CMV) disease. Earlier studies of patients with HIV infection have demonstrated that the diagnosis is often first made post-mortem. In recent years new molecular biological tests have become available for diagnosis of CMV disease. Although clinical evaluation of tests for diagnosis of CMV disease in HIV-infected individuals is suboptimal without autopsy, no results from such studies have been published. The aim of this study was to explore the diagnostic utility of CMV quantitative polymerase chain reaction (PCR) in plasma from HIV and CMV seropositive patients who died during the period 1991-2002 and in whom autopsy was performed. METHODS: Autopsy was performed in all cases, as part of routine evaluation of HIV-infected cases followed at Ullevaal University Hospital. Of 125 patients included, 53 had CMV disease, 37 of whom were first diagnosed at autopsy. CMV disease was diagnosed either by ophthalmoscopic findings typical of CMV retinitis, biopsy or autopsy. One or two plasma samples taken prior to the first diagnosis of CMV disease (alive or at autopsy) or death without CMV disease were analysed by CMV quantitative PCR. Sensitivity, specificity, positive and negative predictive values were calculated for different CMV viral load cut-offs and according to detection of viraemia in one versus two samples. RESULTS: Twenty-seven of 53 patients with CMV disease (51%) and 10 of 72 patients without CMV disease (14%) had detectable viraemia in at least one sample. Sensitivity and negative predictive value (NPV) of the test, maximised with a cut-off at the test's limit of detection of CMV viraemia (400 copies/mL), were 47% and 70%, respectively. With cut-off at 10 000 copies/mL, specificity and positive predictive value (PPV) were 100%. With a requirement for CMV viraemia in two samples, specificity and PPV were 100% in patients with CMV viraemia above the limit of detection. CONCLUSION: Our results indicate that quantitative CMV PCR is best used to rule in, rather than to rule out CMV disease in HIV-infected individuals at high risk.

A new automated method for isolation of Chlamydia trachomatis from urine eliminates inhibition and increases robustness for NAAT systems.

Chlamydia trachomatis is a leading cause of sexually transmitted infection. Diagnostic methods with easy non-invasive sample collection are important to increase testing and hence to reduce the spread of this infection. To enable more use of urine samples in C. trachomatis diagnostics, automation is an absolute requirement since obtaining high-quality DNA from urine specimens involves extensive processing. Here, we present a study in which a new automated sample preparation method, BUGS'n BEADS STI (BnB STI), was used up-front of the BDProbeTec ET end point analysis and compared with the full BDProbeTec ET method to analyze C. trachomatis in 1002 urine samples. The BnB STI system represents a new concept within magnetic sample preparation in which bacteria are first isolated from the sample material followed by purification of bacterial nucleic acid using the same magnetic particles. Similar sensitivity and specificity were obtained with both methods. None of the samples processed with BnB STI inhibited the BDProbeTec ET test whereas 1.8% showed inhibition when processed according to the manual BDProbeTect ET DNA preparation method. Moreover, the average MOTA scores obtained with the BnB STI system were 48% higher for all amplification controls and 57% higher for positive samples, compared to the manual sample preparation. Based on these results and the significant reduction in hands-on-time for urine sample processing, the automated BnB STI sample preparation method was implemented for routine analysis of C. trachomatis from urine samples.

Changes in circulating level of angiogenic factors from the first to second trimester as predictors of preeclampsia.

OBJECTIVE: This study was undertaken to assess changes in placenta growth factor and soluble fms-like tyrosine kinase-1 as predictors of preeclampsia. STUDY DESIGN: Nested case-control study of 154 preeclampsia cases delivered preterm and 190 delivered at term, and 392 controls. RESULTS: Comparing the lowest and highest quartile of placenta growth factor increase from first to second trimester, the odds for preterm preeclampsia was 13.8 (95% CI, 4.4-43.2) higher for women with the lowest increase. Compared with controls, women with preterm preeclampsia had lower soluble fms-like tyrosine kinase-1 in the first, but higher in second trimester. Comparing highest and lowest quartile of increase, the odds for preterm preeclampsia was 9.2 (95% CI 3.4-25.0) higher for women with highest increase. Low placenta growth factor and high soluble fms-like tyrosine kinase-1 increase combined yielded extremely high relative risk of preterm preeclampsia (odds ratio, 35.3, 95% CI, 7.6-164.2), compared with the combination of high (placenta growth factor) and low (soluble fms-like tyrosine kinase-1) increase. CONCLUSION: Low placenta growth factor and high soluble fms-like tyrosine kinase-1 increase from first to second trimester are strong predictors of preeclampsia.

HIV genetic diversity in Cameroon: possible public health importance.

To monitor the evolving molecular epidemiology and genetic diversity of HIV in a country where many distinct strains cocirculate, we performed genetic analyses on sequences from 75 HIV-1-infected Cameroonians: 74 were group M and 1 was group O. Of the group M sequences, 74 were classified into the following env gp41 subtypes or recombinant forms: CRF02 (n = 54), CRF09 (n = 2), CRF13 (n = 2), A (n = 5), CRF11 (n = 4), CRF06 (n = 1), G (n = 2), F2 (n = 2), and E (n = 1, CRF01), and 1 was a JG recombinant. Comparison of phylogenies for 70 matched gp41 and protease sequences showed inconsistent classifications for 18 (26%) strains. Our data show that recombination is rampant in Cameroon with recombinant viruses continuing to recombine, adding to the complexity of circulating HIV strains. This expanding genetic diversity raises public health concerns for the ability of diagnostic assays to detect these unique HIV mosaic variants and for the development of broadly effective HIV vaccines.

Comparison of nonviral transfection and adeno-associated viral transduction on cardiomyocytes.

Cardiomyocytes are terminally differentiated cells that to date have been characterized as poor targets for nonviral gene transfer. This study was therefore designed to determine the optimal nonviral gene transfer parameters in cell cultures of neonatal rat cardiomyocytes and to compare them with the efficiency of gene transfer using adeno-associated viral vectors (AAV). Transfection efficiency was measured by quantitative chloramphenicol acetyltransferase type I (CAT)-enzyme-linked immunosorbent assay and beta-galactosidase staining, based on overexpression of reporter genes (CAT and LacZ). The efficiency of CAT/LacZ overexpression was assessed using the following techniques: (1) liposomal reagents, such as: FuGENE 6, LipofectAMINE 2000, LipofectAMINE PLUS, GenePORTER, Metafectene, and LipoGen; (2) electroporation and nucleofector techniques; and (3) an AAV2 vector harboring a lacZ reporter gene. Toxicity was monitored by total protein measurement and by analyzing cell metabolism. On average, Lipofectamine 2000 was the most effective nonviral method examined yielding consistently high transfection rates (8.1% beta-galactosidase-positive cells) combined with low toxicity. Electroporation also resulted in high transfection values (7.5%); however, cellular toxicity was higher than that of Lipofectamine 2000. Finally, transduction with AAV2 vectors provided the highest levels of transduction (88.1%) with no cellular toxicity. We conclude that although transduction with AAV is more efficient (88.1%), transfections with nonviral techniques, when optimized, may provide a useful alternative for overexpression of therapeutic genes in neonatal cardiomyocytes.

Lower respiratory tract infection caused by human metapneumovirus in two children: the first report of human metapneumovirus infection in Norway.

Human metapneumovirus (hMPV) is a newly described human pathogen associated with respiratory disease. A real-time reverse transcriptase-polymerase chain reaction method was developed to detect this virus. This reports present the first 2 cases of hMPV disease diagnosed in Norway. Both patients were children with serious lower airway disease.

Seroprevalence and correlates of herpes simplex virus type 2 among urban Tanzanian women.

BACKGROUND: Data on herpes simplex virus type 2 (HSV-2) among women in the general population of developing countries are limited. GOALS: The goal of the study was to determine the seroprevalence of HSV-2 and to identify clinical, demographic, and behavioral correlates among women attending primary health care clinics. STUDY DESIGN: This was a cross-sectional survey of 382 randomly chosen women aged 15 to 49 years. RESULTS: The seroprevalence of HSV-2 was 39%. Only 2% had a history of genital herpes. HSV-2 was associated with antibody to HIV-1 (OR=2.3 [CI, 1.1-4.7]), syphilis (OR=4.7 [CI, 1.4-4.7]), and genital ulcers (OR=9.7 [CI 2.5-36.9]). Age, sexual debut, number of sex partners, and history of spontaneous abortion were found to be significantly associated with HSV-2. Eighty-two percent of the women with genital ulcers were HSV-2-seropositive, while syphilis accounted for 6% of cases. HSV-2 may thus be the most common cause of genital ulcers in this population. CONCLUSION: In view of the high HSV-2 seroprevalence and its association with HIV-1 and genital ulcers, integration of HSV-2 therapeutic management in STD syndromic algorithms is recommended. Counseling on symptom recognition, asymptomatic shedding, and preventive measures is needed.

Herpes simplex virus type-2 infection in pregnancy: no risk of fetal death: results from a nested case-control study within 35,940 women.

OBJECTIVE: The aim of this study was to assess the association of fetal death with herpes simplex virus type-2 (HSV-2) antibody status during pregnancy: 1. presence of antibodies in first trimester; 2. appearance of antibodies (incident infection); 3. increase in antibody titre; and 4. loss of antibodies. DESIGN: Prospective study. POPULATION: The source population was a cohort of 35,940 pregnant women in Norway. METHODS: Nested case-control study within the cohort. Cases were all women in the study population who experienced a fetal death after the 16th weeks of gestation (n = 281), and controls were 961 randomly selected women with a live born child. MAIN OUTCOME MEASURES: HSV-2 antibody status. RESULTS: Twenty-nine percent (82/281) of women with a fetal death and 27% (256/961) of the controls had of HSV-2 antibodies present in the first trimester (odds ratio 1.1, 95% CI 0.8-1.5). HSV-2 antibodies appeared in 2% (3/136) of initially seronegative cases and 3% (16/623) of the controls during pregnancy (odds ratio 0.9, 95% CI 0.2-3.0). An increase in HSV-2 antibodies occurred in 4% (2/55) of initially seropositive cases and 7% (16/231) of the controls (odds ratio 0.5, 95% CI 0.1-2.3). Loss of HSV-2 antibodies in initially seropositive women was not associated with fetal death, 42% (23/55) of the cases and 45% (104/231) of the controls seroreverted (odds ratio 0.8, 95% CI 0.5-1.6). Differences in follow up time, age and parity were controlled and did not influence the comparisons between cases and controls. CONCLUSION: This study provides no evidence of an association between HSV-2 infection during pregnancy and fetal death.