Adaptation of red blood cell lysis represents a fundamental breakthrough that improves the sensitivity of Salmonella detection in blood.

AIMS: Isolation of Salmonella Typhi from blood culture is the standard diagnostic for confirming typhoid fever but it is unavailable in many developing countries. We previously described a Microwave Accelerated Metal Enhanced Fluorescence (MAMEF)-based assay to detect Salmonella in medium. Attempts to detect Salmonella in blood were unsuccessful, presumably due to the interference of erythrocytes. The objective of this study was to evaluate various blood treatment methods that could be used prior to PCR, real-time PCR or MAMEF to increase sensitivity of detection of Salmonella. METHODS AND RESULTS: We tested ammonium chloride and erythrocyte lysis buffer, water, Lymphocyte Separation Medium, BD Vacutainer(®) CPT(™) Tubes and dextran. Erythrocyte lysis buffer was the best isolation method as it is fast, inexpensive and works with either fresh or stored blood. The sensitivity of PCR- and real-time PCR detection of Salmonella in spiked blood was improved when whole blood was first lysed using erythrocyte lysis buffer prior to DNA extraction. Removal of erythrocytes and clotting factors also enabled reproducible lysis of Salmonella and fragmentation of DNA, which are necessary for MAMEF sensing. CONCLUSIONS: Use of the erythrocyte lysis procedure prior to DNA extraction has enabled improved sensitivity of Salmonella detection by PCR and real-time PCR and has allowed lysis and fragmentation of Salmonella using microwave radiation (for future detection by MAMEF). SIGNIFICANCE AND IMPACT OF THE STUDY: Adaptation of the blood lysis method represents a fundamental breakthrough that improves the sensitivity of DNA-based detection of Salmonella in blood.

Detection of two biological markers of intercourse: prostate-specific antigen and Y-chromosomal DNA.

BACKGROUND: Although biological markers of women's exposure to semen from vaginal intercourse have been developed as surrogates for risk of infection or probability of pregnancy, data on their persistence time and clearance are limited. STUDY DESIGN: During 2006-2008, 52 couples were enrolled for three 14-day cycles of abstinence from vaginal sex during which women were exposed in the clinic to a specific quantity (10, 100 or 1000 µL) of their partner's semen. Vaginal swabs were collected before and at 1, 6, 12, 24, 48, 72 and 144 h after exposure for testing for prostate-specific antigen (PSA) and Y-chromosome DNA (Yc DNA). RESULTS: Immediately after exposure to 1000 µL of semen, the predicted sensitivity of being PSA positive was 0.96; this decreased to 0.65, 0.44, 0.21 and 0.07 at 6, 12, 24 and 48 h, respectively. Corresponding predicted sensitivity of being Yc DNA positive was 0.72 immediately postexposure; this increased to 0.76 at 1 h postexposure and then decreased to 0.60 (at 6 h), 0.63 (at 12 h), 0.49 (at 24 h), 0.21 (at 48 h), 0.17 (at 72 h) and 0.12 (at 144 h). CONCLUSIONS: Overall findings suggest that PSA may be more consistent as a marker of very recent exposure and that Yc DNA is more likely to be detected in the vagina after 12 h postexposure compared to PSA.

A case control study of anovaginal distance and bacterial vaginosis.

We aimed to test the hypothesis that a short anovaginal distance may increase the risk of bacterial vaginosis (BV) due to faecal contamination and disruption of the vaginal microbiota. Women attending two sexually transmitted infection (STI) clinics in Baltimore, Maryland, USA, who complained of a vaginal discharge were asked to participate in a study to measure mucosal immune responses. In this pilot study of all enrolled women, a small plastic ruler was used to measure the anatomic distance from the posterior fourchette to the anus with the participant in the lithotomy position. Cases of BV, defined by Amsel's clinical criteria (n = 62), were compared with controls (n = 31) without BV. We used linear and logistic regression models to adjust for potential confounders. A total of 93 women were recruited (median age 28.6 years, 93% black, 4.4% gonorrhoea infection, 7.4% chlamydia infection, 8.6% trichomonas infection, 67% BV diagnosed). Mean anovaginal distance was 3.22 cm (SD: 0.74, range 1.8-5.2) for controls and 3.37 cm (SD: 0.76, range: 1.8-5.7) for cases (P = 0.38). There was no difference between cases and controls when comparing median values, quartiles and after adjusting for potential confounders. Among high-risk women with multiple co-infections, there was no association between anovaginal distance and clinical diagnosis of BV.

Real-time PCR assays compared to culture-based approaches for identification of aerobic bacteria in chronic wounds.

Chronic wounds cause substantial morbidity and disability. Infection in chronic wounds is clinically defined by routine culture methods that can take several days to obtain a final result, and may not fully describe the community of organisms or biome within these wounds. Molecular diagnostic approaches offer promise for a more rapid and complete assessment. We report the development of a suite of real-time PCR assays for rapid identification of bacteria directly from tissue samples. The panel of assays targets 14 common, clinically relevant, aerobic pathogens and demonstrates a high degree of sensitivity and specificity using a panel of organisms commonly associated with chronic wound infection. Thirty-nine tissue samples from 29 chronic wounds were evaluated and the results compared with those obtained by culture. As revealed by culture and PCR, the most common organisms were methicillin-resistant Staphylococcus aureus (MRSA) followed by Streptococcus agalactiae (Group B streptococcus) and Pseudomonas aeruginosa. The sensitivities of the PCR assays were 100% and 90% when quantitative and qualitative culture results were used as the reference standard, respectively. The assays allowed the identification of bacterial DNA from ten additional organisms that were not revealed by quantitative or qualitative cultures. Under optimal conditions, the turnaround time for PCR results is as short as 4-6 h. Real-time PCR is a rapid and inexpensive approach that can be easily introduced into clinical practice for detection of organisms directly from tissue samples. Characterization of the anaerobic microflora by real-time PCR of chronic wounds is warranted.