Molecular bacterial load assay, a culture-free biomarker for rapid and accurate quantification of sputum Mycobacterium tuberculosis bacillary load during treatment.

A molecular assay to quantify Mycobacterium tuberculosis is described. In vitro, 98% (n = 96) of sputum samples with a known number of bacilli (10(7) to 10(2) bacilli) could be enumerated within 0.5 log(10). In comparison to culture, the molecular bacterial load (MBL) assay is unaffected by other microorganisms present in the sample, results are obtained more quickly (within 24 h) and are seldom inhibited (0.7% samples), and the MBL assay critically shows the same biphasic decline as observed longitudinally during treatment. As a biomarker of treatment response, the MBL assay responds rapidly, with a mean decline in bacterial load for 111 subjects of 0.99 log(10) (95% confidence interval [95% CI], 0.81 to 1.17) after 3 days of chemotherapy. There was a significant association between the rate of bacterial decline during the same 3 days and bacilli ml(-1) sputum at day 0 (linear regression, P = 0.0003) and a 3.62 increased odds ratio of relapse for every 1 log(10) increase in pretreatment bacterial load (95% CI, 1.53 to 8.59).

Validation of host-specific Bacteriodales 16S rRNA genes as markers to determine the origin of faecal pollution in Atlantic Rim countries of the European Union.

The recent implementation of the Revised Bathing Water Directive in the European Union has highlighted the need for development of effective methods to differentiate between sources of faecal contamination. It had previously been shown that amplification of 16S rRNA genes of host-specific Bacteriodales species using the HF183F and CF128F primers could be used as markers for human and bovine faecal contamination in the United States. This paper determined the sensitivity and specificity of these markers in four Atlantic Rim countries (France, Ireland, Portugal and the United Kingdom) to evaluate their usefulness in determining the origin of faecal contamination. It was shown that the HF183F marker displayed high sensitivity (80-100%) and specificity (91-100%), and is reliable as an indication of human faecal contamination. The CF128F marker displayed 100% sensitivity in all four countries. However, strong regional variations in specificity (41-96%) were observed, highlighting the need for local validation before this marker is employed in source tracking of faecal contamination.

Maximum sustainable work rate for five protective clothing ensembles with respect to moisture vapor transmission rate and air permeability.

The fabrics associated with protective clothing affect heat stress, which influences productivity and risks of heat-related disorders. This study compared the work limiting effects of five protective coveralls and a semiclothed condition (t-shirt and shorts). Two fabric characteristics determined from bench tests, moisture vapor transmission rate (MVTR), and air permeability were also examined as possible predictors of ensemble performance. A progressive metabolic rate protocol was used where environmentalconditions (T(db) = 32 degrees C; T(pwb) = 26 degrees C) were held constant while treadmill speed was slowly increased. The limiting metabolic rate to just maintain thermal equilibrium was the critical point. At this point, critical speed and critical metabolic rate were noted and total evaporative resistance was calculated for each ensemble. Five acclimatized subjects wore each of the six clothing conditions in a random order. Statistically significant differences were found among the five protective garments and a semiclothed ensemble for critical treadmill speed (S(crit)), critical metabolic rate (M(crit)), and total evaporative resistance (R(e-t)). The semiclothed condition (S(crit) = 1.77 m/sec; M(crit) = 580 W; R(e-t) = 0.0099 kPa m2/W) and ensembles made from spunbonded, melt blown, spunbonded polypropylene (SMS) (1.72 m/sec; 560 W; 0.0135 kPa m2/W) and spunbonded polypropylene (1.67 m/sec; 550 W; 0.0126 kPa m2/W) were able to support higher work rates than fabrics made from Tyvek 1422-A (a nonwoven spunbonded olefin) (1.48 m/sec; 470 W; 0.0183 kPa m2/W) and a microporous film supported by spunbonded polypropylene (1.34 m/sec; 420 W; 0.0231 kPa m2/W). A tightly woven polyester ensemble (1.59 m/sec; 510 W; 0.0130 kPa m2/W) had intermediate values and was not significantly different from either group. Air permeability was a better predictor of fabric work limiting performance than MVTR. An air permeability on the order of 10,000 L/min cm2 bar would have little effect on maximum sustainable work.

Use of PCR to diagnose Toxoplasma gondii chorioretinitis in eyes with severe vitritis.

Two cases are reported of intraocular inflammation in which severe vitritis hampered the fundal view, making an accurate clinical diagnosis impossible, and vitreous analysis using conventional techniques was unhelpful. PCR for Toxoplasma gondii was positive in both cases and provided the only way of confirming the diagnosis. Other ocular samples also underwent PCR for Toxoplasma DNA and the specificity of this approach is demonstrated.

Reduction of the rate of false-positive cultures of Mycobacterium tuberculosis in a laboratory with a high culture positivity rate.

Our laboratory, engaged in a prospective study of adult pulmonary tuberculosis, processed on average 1186 sputum samples per year for the detection of Mycobacterium tuberculosis (M. tuberculosis). Approximately 55% of all sputum samples were culture-positive. The study protocol required that all patients had their M. tuberculosis isolates DNA fingerprinted at diagnosis, and at subsequent time points if the patients either failed treatment or presented again with tuberculosis. Over a 22-month period, there were 14 apparent treatment failures from 109 patients who had completed 6 months of therapy. Only two of these were true treatment failures, while the other 12 had DNA fingerprints that were different from those obtained at diagnosis. It was concluded that these 12 cultures represented episodes of laboratory cross-contamination. Retrospective DNA fingerprinting of patient isolates was done so that each patient had at least two independent isolates fingerprinted. This survey revealed that 7.3% of DNA fingerprints were discordant. False-positive cultures with discordant DNA fingerprints generally arose late in chemotherapy and the isolates were usually co-processed with other strongly smear-positive sputum samples. Simple modifications of laboratory procedures were made, and over a following 10.5-month period the false-positive rate was reduced to 2.1%. These modifications did not increase the workload or the cost of processing samples and can thus be used successfully by any laboratory, and particularly by those in resource-poor settings.

Multiple Mycobacterium tuberculosis strains in early cultures from patients in a high-incidence community setting.

In an ongoing molecular epidemiology study, human immunodeficiency virus-negative patients with first-time pulmonary tuberculosis from a high-incidence community were enrolled. Mycobacterium tuberculosis strains were identified by restriction fragment length polymorphism analysis with two fingerprinting probes. Of 131 patients, 3 (2.3%) were shown to have a mixture of strains in one or two of their serial cultures. This study further investigated these cases with disease caused by multiple M. tuberculosis strains in the context of the molecular epidemiology of the study setting.

Localisation of mycobacterial DNA and mRNA in human tuberculous granulomas.

In situ hybridisation was used to detect the presence of Mycobacterium tuberculosis in paraffin-embedded lung tissue of nine patients diagnosed with tuberculosis (TB). Mycobacterial DNA was found in all nine patients and in 175 out of 191 granulomas examined. A combination of in situ hybridisation and immunohistochemistry techniques demonstrated that mycobacterial DNA was associated with CD68-positive cells with the morphology of macrophages and giant cells. Mycobacterial DNA was also found within the necrotic regions of some granulomas. mRNA for the mycobacterial RNA polymerase beta subunit (rpoB) was detected by RNA: RNA in situ hybridisation. The rpoB mRNA was also localised to CD68-positive cells with the morphology of macrophages and to giant cells of certain necrotic granulomas. No rpoB mRNA was found in the necrotic regions of granulomas. Mycobacterial DNA was detected in 92% of patient granulomas of which 8% were positive for rpoB mRNA. The ability to identify mycobacterial RNA transcripts within human tuberculous granulomas affords us the opportunity to analyse the interplay between pathogen gene expression and the human immune response and should provide valuable insight into the mechanisms used by M. tuberculosis to persist within the human host.