Donald E. Riley

Broad-Spectrum Bacterial rDNA Polymerase Chain Reaction Assay for Detecting Amniotic Fluid Infection Among Women in Premature Labor

We amplified bacterial 16S rRNA encoding DNA (rDNA) with the polymerase chain reaction (PCR) to detect amniotic fluid infection in 69 women in premature labor whose membranes were intact. Bacterial rDNA was detected by PCR in samples from 15 (94%) of 16 patients with positive amniotic fluid cultures. Bacteria were detected by PCR in samples from 5 (36%) of 14 patients with negative cultures and elevated interleukin (IL)-6 levels vs. 1 (3%) of 39 patients with negative cultures and IL-6 levels of < or = 2,000 pg/mL (P < .01). The median amniotic fluid cytokine levels and the pregnancy outcomes were similar for patients with positive amniotic fluid cultures and those with negative cultures and positive rDNA PCR assays. The association between amniotic fluid infection and premature labor may be underestimated on the basis of amniotic fluid culture results. The broad-spectrum bacterial 16S rDNA PCR assay may prove useful for diagnosing amniotic fluid infection.

Amniotic fluid interleukin-6 and preterm delivery: a review.

Objective To evaluate the potential role of amniotic fluid (AF) interleukin (IL)-6 as a predictor of preterm delivery and to consider possible explanations for the proportion of women with elevated AF IL-6 who deliver preterm yet lack microbiologically detectable intra-amniotic infection. Data Sources We searched the English language human literature in MEDLINE, 1966 through September 1999, using the keywords “labor/infant,” “premature,” “cytokines/interleukin-6,” and “AF.” We also examined abstracts from the 1999 meetings of the Society for Maternal-Fetal Medicine and the Society for Epidemiologic Research. We identified other studies by reviewing the reference lists of published articles. Methods of Study Selection The MEDLINE search yielded 55 citations. We focused on studies that reported on the association between AF IL-6 and preterm delivery. Tabulation, Integration, and Results There is consensus in the literature that elevated AF IL-6 is a stronger predictor of preterm delivery than intra-amniotic infection detected by either microbiologic culture or polymerase chain reaction (PCR). Among women with elevated AF IL-6, PCR could detect a higher proportion of intra-amniotic infection than culture. A number of women with elevated AF IL-6 (33–70%) deliver preterm and do not have evidence of intra-amniotic infection by either culture or PCR. Possible explanations for this observation are considered. Conclusion Elevated AF IL-6 is strongly associated with preterm delivery and merits future consideration in clinical settings to predict preterm delivery and guide patient care. Development of improved polymerase chain reaction-based clinical methods to detect intra-amniotic infection is necessary to better understand the relationship between elevated AF IL-6, intra-amniotic infection, and preterm delivery.

Epidemiology of prostatitis: new evidence for a world-wide problem

We review new data on the epidemiology of chronic prostatitis. These population-based studies used reasonable case-definitions to survey various populations from North America, Europe and Asia. Overall, 2–10% of adult men suffer from symptoms compatible with chronic prostatitis at any time and approximately 15% of men suffer from symptoms of prostatitis at some point in their lives. Other epidemiologic data suggest that chronic prostatitis may be associated with an increased risk for development of benign prostatic hyperplasia and prostate cancer. These data suggest that chronic prostatitis is an important international health care problem that merits increased priority from clinicians and researchers.

Chronic prostatitis: epidemiology and role of infection.

We review the epidemiology of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) and the role of infectious agents, emphasizing critical data necessary to define current research issues. The epidemiologic literature is limited, but the worldwide prevalence appears to be in the range of 2% to 10%, indicating that CP/CPPS represents an important international health problem. Recent molecular studies have documented bacterial DNA sequences in prostate tissue from CP/CPPS patients. These data suggest that colonization and/or infection occurs in the prostates of many patients with CP/CPPS. Further molecular research is needed to define the role of bacteria in the etiology of CP/CPPS.

BACTERIAL DNA SEQUENCES IN PROSTATE TISSUE FROM PATIENTS WITH PROSTATE CANCER AND CHRONIC PROSTATITIS

PURPOSE Although bacterial genetic material has been detected in prostate tissue from patients with various disorders, the prevalence of these organisms is unknown. We tested the hypothesis that bacterial detection rates differ between patients with prostate cancer and those with the chronic prostatitis/pelvic pain syndrome. MATERIALS AND METHODS Sterile prostate biopsies were obtained during radical retropubic prostatectomy from 107 patients with prostate cancer and using a perineal approach from 170 with the chronic prostatitis/pelvic pain syndrome. Numerous controls were also evaluated. Bacterial ribosomal encoding DNA (165 rDNA) sequences were detected using a polymerase chain reaction assay. Selected positives were cloned, sequenced and compared with DNA databases. RESULTS Bacterial DNA sequences were detected in 21 (19. 6%) of 107 patients with prostate cancer compared to 79 (46.4%) of 170 with chronic prostatitis (p <0.0001). These bacteria included urogenital pathogens, other described microorganisms and bacteria not reported previously. CONCLUSIONS Bacterial DNA sequences may be identified in prostate tissue from many patients. Bacterial detection rates in prostate tissue appear to differ among populations, with higher rates among patients with the chronic prostatitis/pelvic pain syndrome than among those with prostate cancer. Future studies of the role of various bacteria in the prostate may provide insight into the pathophysiology of prostate disease.

Bacteria in the chronic prostatitis-chronic pelvic pain syndrome: molecular approaches to critical research questions.

PURPOSE There is a pressing need to determine the causes and consequences of, and optimal therapy for the chronic prostatitis-chronic pelvic pain syndrome. MATERIALS AND METHODS New data suggest that bacterial infection may be critical in some patients. We examined the rationale for and technical approaches to hypothesis driven studies of bacteria in the chronic prostatitis-chronic pelvic pain syndrome. RESULTS The first hypothesis was that patients with the chronic prostatitis-chronic pelvic pain syndrome have prostatic bacteria that distinguish them from controls. In pilot studies patients with inflamed expressed prostatic secretions were more likely to have bacterial DNAs, that is 16S ribosomal DNAs. Current goals are to clone, sequence and compare ribosomal DNAs from patients and controls to determine which bacteria are most specific to the chronic prostatitis-chronic pelvic pain syndrome and which should be targeted in clinical trials. The second hypothesis was that bacterial viability correlates with the severity of the chronic prostatitis-chronic pelvic pain syndrome. Quantitative assays for bacterial elongation factor messenger RNA (tufA messenger RNA) provide tools to correlate bacterial viability with patient characteristics, will provide insights into the potential value of antimicrobial therapy and identify characteristics that distinguish patients most likely to respond. The third hypothesis was that patients with prostatic bacteria have similar bacteria in expressed prostatic secretions or on seminal fluid analysis and, furthermore, these bacteria differ from bacteria in controls. These studies would determine whether expressed prostatic secretions or seminal fluid analysis can be used to identify prostatic bacteria and may result in clinical methods for noninvasive diagnosis of prostatic infection. CONCLUSIONS These studies should provide important insights into the causes of the chronic prostatitis-chronic pelvic pain syndrome and may elucidate optimal clinical evaluation and treatment in patients.

Prostatitis: what is the role of infection

Although bacterial prostatitis is a common diagnosis, well documented infections of the prostate are uncommon. Culture studies of prostate tissue led our group to hypothesize that bacterial colonization/invasion of the prostate gland might occur more commonly than is appreciated by standard microbiological techniques. Specific polymerase chain reaction (PCR) assays were used for each of the pathogens previously implicated in chronic prostatitis as well as broad-spectrum PCR assays to identify tetracycline resistance genes and bacterial ribosomal-encoding genes (16S rDNAs), followed by cloning and sequencing of the PCR products. Only ten (8%) of the 135 patients with chronic prostatitis had positive specific PCR assays including: Mycoplasma genitalium in four men, Chlamydia trachomatis in three and Trichomonas vaginalis in two, as well as one man positive for both M. genitalium and C. trachomatis. In contrast to the specific probes, the broad-spectrum PCR assays had a substantial proportion of positives. We found evidence of tetracycline resistance in 25% of patients. 16S rDNA-encoding sequences in 77% of the subjects. The tetracycline resistance positives were a subset of the 16S rDNA positive patients. Patients with 16S rDNA-encoding sequences were significantly more likely to have expressed prostatic secretion leukocytes. Many patients with chronic prostatitis/chronic pelvic pain syndrome have a wide variety of bacterial DNA-encoding sequences despite extensive negative microbiological investigations. Understanding the precise role of infection in this syndrome may well lead to better methods to elucidate the microbiology of the prostate in health and disease.

Symptoms and inflammation in chronic prostatitis/chronic pelvic pain syndrome ☆

OBJECTIVES To evaluate the possibility that patients with inflammatory and noninflammatory chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) might present with different symptoms. Patients with CP/CPPS present with characteristic symptoms without bacteriuria. The new National Institutes of Health consensus suggests that CP/CPPS can be divided into inflammatory and noninflammatory categories. METHODS Standardized symptom surveys were completed by 130 subjects who met the criteria for CP/CPPS after clinical examination and urethral, urine, expressed prostatic secretion (EPS), and seminal fluid analysis evaluations. RESULTS When classified by either EPS or postprostatic massage urine (VB3) findings, subjects with and without inflammation had similar symptoms. However, when classified using the combination of EPS, VB3, and seminal fluid analysis, subjects with inflammatory CP/CPPS had more severe (P <0.02) and more frequent symptoms, in particular, difficulty reaching erection (P <0.01), weak urinary stream (P <0.01), urinary frequency (P = 0.03), and penile pain (P = 0.04). CONCLUSIONS The increased severity and frequency of symptoms among patients with inflammatory CP/CPPS provide empirical support for the new consensus classification on the basis of the combination of EPS, VB3, and seminal fluid analysis findings.

Analysis of inactive X chromosome structure by in situ nick translation

Nick translation assays of fixed interphase female fibroblasts with tritiated nucleotides demonstrated a characteristic absence of label over sex chromatin. The chromatin bodies were nearly always peripheral in location and a ribbon of nick translatable DNA was detected between the sex chromatin and the nuclear envelope. High voltage electron microscopy indicated the possibility of a special nuclear envelope attachment region. The apparent resistance of sex chromatin to nick translation did not appear to be due to resistance to DNase I attack.

X Chromosomal short tandem repeat polymorphisms near the phosphoglycerate kinase gene in men with chronic prostatitis

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) causes substantial morbidity afflicting approximately 10% of adult males. Treatment is often empirical and ineffective since the etiology is unknown. Other prostate and genitourinary diseases have genetic components suggesting that CP/CPPS may also be influenced by genetic predisposition. We recently reported a highly polymorphic short tandem repeat (STR) locus near the phosphoglycerate kinase gene within Xq11-13. Because this STR is in a region known to predispose towards other prostate diseases, we compared STR polymorphisms in 120 CP/CPPS patients and 300 control blood donors. Nine distinct allele sizes were detected, ranging from 8 to 15 repeats of the tetrameric STR plus a mutant allele (9.5) with a six base deletion in the flanking DNA sequence. The overall allele size distribution in the CP/CPPS patients differed from controls (Chi-square=19.252, df=8, P=0.0231). Frequencies of two specific alleles, 9.5 and 15, differed significantly in CP/CPPS vs. control subjects and allele 10 differed with marginal significance. Alleles 9.5 and 10 were both more common in CP/CPPS patients than controls while allele 15 was less common. These observations suggest that Xq11-13 may contain one or more genetic loci that predispose toward CP/CPPS. Further investigations involving family studies, larger patient populations, and other control groups may help elucidate this potential genetic predisposition in CP/CPPS.