16S rRNA Assays Research Articles

Comparison of generic bacterial molecular detection (16S rRNA) with C-reactive protein and blood culture in the diagnosis of late-onset sepsis preterm infants: Abstract G177 Table 1

Background Preterm infants are at great risk of bloodstream infection while receiving intensive care. The incidence of late-onset bloodstream infection varies between 10–60%. Diagnosis is currently made with a combination of clinical signs and laboratory investigations. The use of new molecular methods to improve diagnosis in a range of infections has been reported in other age groups. Objective To compare a molecular 16S rRNA assay to conventional laboratory methods (C-reactive protein (CRP) and blood culture) in the diagnosis of late-onset sepsis in preterm babies. Methods A single-centre, prospective observational study was conducted. All babies with suspicion of late-onset sepsis were included. Blood for the 16S rRNA assay was taken along with routine investigations for sepsis (blood culture, CRP). The decision to label a baby as being “septic” was based on clinical data and the assessment by a national nosocomial infection scoring system. Sepsis status was then compared to results of blood culture, CRP and the 16S rRNA assay. Diagnostic odds ratio, sensitivity, specificity, positive likelihood and negative likelihood ratios were calculated. Results Conclusion This assay, which enables generic detection of bacteria without reliance on culture, has superior sensitivity and specificity in the diagnosis of late-onset infection than conventional laboratory methods. These results indicate that molecular methods should have a place in the diagnosis of late-onset sepsis in preterm babies. Further study is required to determine the extent to which these methods could be utilised.

Quantification of “bacterial load” in late-onset neonatal sepsis using molecular techniques

Premature infants are at increased risk of bacterial sepsis. Few studies of neonatal infection have quantified the degree of “bacterial load”. Traditionally, bacterial numbers have been estimated by measuring colony-forming-units per ml (CFU/ml), using conventional culture methods. Modern molecular techniques allow direct estimation of bacterial numbers in preterm infants with late-onset sepsis. Levels of bacterial 16S ribosomal RNA (rRNA) can potentially be used to quantify metabolically active bacteria in clinical samples. Objective To estimate the “bacterial load” in infants presenting with late-onset bacterial sepsis (>48 h old) using quantitative molecular methods. Methods A single-centred, observational study was conducted in all infants greater than 48 h old with clinically suspected sepsis. Blood samples (0.5 ml) were taken for a calibrated TaqMan reverse transcriptase polymerase chain reaction (RT-PCR) in addition to normal infection investigations. This assay allows quantitative detection of bacterial 16S rRNA and rDNA. Clinical and laboratory data were recorded at the time of sampling. Copy numbers (rRNA and rDNA) were calculated and corrected for background genetic material to estimate bacterial load. Results 100 samples were taken from 125 babies at a median age of 14 days. Median (interquartile) birth weight was 1.09 (0.81–1.45) kg and gestation was 27.5 (23–40) weeks. The rate of clinically diagnosed late-onset infection was 33%. The mean calculated and corrected copy number estimate (bacterial rRNA+rDNA) was 1.51×107/ml of blood (range 8.91×104 to 9.77×1010). Estimated copy number correlated positively with white blood cell count, neutrophil number and C-reactive protein and negatively with platelet count, lymphocyte count, pH and base deficit. There was no correlation with birth weight or gestational age. Conclusion Bacterial load in late-onset sepsis can be estimated by quantitative RT-PCR using a 16S rRNA assay. Direct comparison to estimates of CFU/ml was not possible because of the variability of rRNA numbers per bacterial cell. However, the potential of this target as a quantitative biomarker of bacterial infection was demonstrated. Increasing numbers of bacteria appear to stimulate greater immune response. Further study is necessary to place these values in context with other age groups and disease severity.

Effect of oxidized leachate on degradation of lignin by sulfate-reducing bacteria

Municipal solid waste materials (MSWs) in landfills need a long period of stabilization because lignin compounds in MSWs and leachate are not readily biodegraded, but inhibit methanogenic metabolism. Recirculation of leachate into the landfill offers the potential advantage of increasing the rate of decomposition of organic matter. However, the degradation of lignin by leachate recirculation alone is quite difficult. Several recent studies have demonstrated that sulfate-reducing bacteria (SRB) were able to degrade lignin compounds. In this study, batch tests were conducted to investigate the impacts of SRB enrichment on lignin decomposition rates as well as the decomposition of other biodegradable organics. Further, the effects of nitrite and nitrate on lignin degradation rates were also studied. A 16S rRNA assay showed that the SRB used herein, which were obtained by enriching solid waste collected from a closed MSW landfill, were Thaurea sp. and Desulfovibrio sp. Lignin was found to be biodegraded by the SRB and the rate of lignin removal per unit of waste volatile suspended solid was 2.9 mg lignin g(-1) VSS day(- 1). It was found that the initial degradation rate increased under higher initial lignin concentrations. However, the degradation rate during days 6-19 became slower than that during the initial 9 days because lignin consisted of complexly bonded aromatic compounds that were not readily biodegradable. Adding other organics such as lactate seemed to improve the rate and amount of lignin degradation, probably due to the increase in SRB associated with consumption of the additional organics. The lignin removal percentage decreased with increases in oxidized nitrogen (nitrite or nitrate) concentrations, indicating that oxidized nitrogen could inhibit SRB activity. Conclusively, the study verified the existence of SRB in the landfill and showed that the SRB could be activated for the degradation of lignin by the recirculation of the leachate, which is consistent with other studies showing that leachate recirculation could shorten the stabilization period of the landfill.

A Reverse Transcriptase–Polymerase Chain Reaction Assay ofBorrelia burgdorferi16S rRNA for Highly Sensitive Quantification of Pathogen Load in a Vector

We developed a real-time quantitative detection assay for the pathogen Borrelia burgdorferi, a Lyme borreliosis (LB) agent, using reverse transcription-polymerase chain reaction (RT-PCR) with primers and probe for a Borrelia genus-specific region of 16S ribosomal RNA. The standard curve of the assay was linear by semi-log plot over more than five orders of magnitude, and the detection limit of the assay was one thousandth of a single cell of B. burgdorferi. The minimum target level for detection using the RT-PCR assay for 16S RNA was 40-fold lower than the RT-PCR assay for messenger RNA of ospA, a highly expressed, plasmid-borne gene, and 1600-fold lower than the RT-PCR assay for messenger RNA of p66, a chromosome-borne gene of B. burgdorferi. The 16S rRNA assay was then applied in an experimental setting for monitoring the spirochetal load in B. burgdorferi-infected Ixodes scapularis ticks before and after they fed on Peromyscus leucopus mice immunized with recombinant OspA. Unfed infected ticks had a mean of 2,240 spirochetes per tick, and after feeding on non-immunized mice and engorgement, the mean number of spirochetes increased to 223,900 per tick. In contrast, there were either no or <or=7 spirochetes in ticks that had fed on OspA-immunized mice.

Conjunctival Chlamydial 16S Ribosomal RNA Expression in Trachoma: Is Chlamydial Metabolic Activity Required for Disease to Develop?

Nucleic acid amplification testing frequently detects conjunctival Chlamydia trachomatis infection in subjects without clinical signs of trachoma. It is unclear whether such subjects are actually infected. We measured chlamydial 16S ribosomal RNA (rRNA) expression, a marker of chlamydial metabolic activity, in comparison with the quantitation of a chlamydial DNA target in subjects exposed to trachoma. Subjects from 2 Gambian villages where trachoma was endemic were examined. Conjunctival samples were tested for the presence of C. trachomatis DNA using a quantitative real-time polymerase chain reaction (PCR) assay for the omp1 gene and for the expression of C. trachomatis 16S rRNA using a 1-step, real-time reverse-transcriptase PCR assay. A total of 248 people were examined. The prevalence of clinically active trachoma was 16.9%. C. trachomatis was detected in 19.8% and 6.8% of subjects by the omp1 and 16S rRNA assays, respectively. For subjects with positive results for both tests, the number of copies of 16S rRNA was approximately 100-fold greater than the number of copies of the omp1 gene. In samples from subjects in whom the omp1 gene was detected in the absence of 16S rRNA, typically only a few copies of omp1 were present. The expression of 16S rRNA was strongly associated with the presence of clinical signs of active trachoma. The use of 16S rRNA expression for the detection of chlamydial metabolic activity appears to usefully discriminate established infections from the inoculation of the conjunctiva with dead or subviable organisms, which probably occurs frequently in settings in which trachoma is endemic. The data support conclusions from primate challenge studies that live Chlamydiae species or antigens derived from them are needed to provoke the clinical signs of disease.

Comparison of a New Quantitative ompA -Based Real-Time PCR TaqMan Assay for Detection of Chlamydia pneumoniae DNA in Respiratory Specimens with Four Conventional PCR Assays

Chlamydia pneumoniae, an important respiratory pathogen, is difficult to culture, and detection rates by conventional PCRs vary considerably. A new quantitative ompA-based real-time PCR assay based on TaqMan technology for detection of C. pneumoniae in respiratory samples is described, and its performance in terms of sensitivity and reproducibility is compared with those of four published conventional PCRs (one single-step PCR targeting a cloned PstI fragment; two nested PCRs, one targeting the 16S rRNA gene followed by hybridization and the other targeting the ompA gene; and a touchdown enzyme time-release [TETR] PCR also targeting the 16S rRNA gene). Both ompA-based PCRs showed the best analytical sensitivity. All five assays could detect even lower target levels from spiked sputum, with the 16S rRNA assays performing better than the ompA-based nested PCR (10(-6) inclusion-forming units [IFU] were detected in four of four and two of four replicates by the 16S rRNA TETR PCR and the 16S rRNA nested PCR, respectively). In general, the ompA-based real-time protocol produced the most consistent positive results for all replicates tested down to 10(-6) IFU. Eight of 45 patient sputum specimens (18%) were C. pneumoniae DNA positive in at least one of four replicates tested by at least one assay. Without taking into consideration the analytical sensitivity or the reproducibility of the test results, the numbers of C. pneumoniae DNA-positive sputum specimens (n = 8) were four, three, two, two, and one for the 16S rRNA TETR assay, the PstI-based single-step PCR, the ompA-based real-time PCR, the ompA-based nested touchdown PCR, and the 16S rRNA-based nested PCR, respectively. However, the overall rate of concordance of positive results was low. Only one cell culture-positive sputum specimen was positive by four of five assays (14 of 16 replicates; mean cycle threshold value, 25; 10(8) particles/ml of sputum). Thirty-seven specimens were C. pneumoniae negative by all five assays for all replicates tested, as were all negative controls (n = 65 to 100 per testing panel). No PCR inhibitors were detected by real-time PCR or by the 16S rRNA-based nested assay. We confirm that the analytical sensitivity of an assay for the detection of C. pneumoniae does not necessarily predict its ability to detect its target in sputum. A quantitative, fast, and easy-to-handle diagnostic approach such as the ompA-based real-time TaqMan PCR described here might improve the detection of C. pneumoniae in respiratory samples.