Universal Polymerase Chain Reaction Research Articles

Evaluation of the Time Period for which Real-Time Polymerase Chain Reaction Detects Dead Bacteria

Real-time polymerase chain reaction (PCR) is currently widely used for the diagnosis of infections. We evaluated the time after treatment during which real-time PCR can detect dead bacteria. The presence of bacterial DNA was identified by real-time PCR through methicillin-resistant Staphylococcus (MRS)-PCR and universal PCR. Methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, and Escherichia coli were each killed with alcohol, antibiotics, or heat treatment in vitro. The detection periods of MRS-PCR for MRSA treated by alcohol, vancomycin, linezolid, and heat were found to be less than 16, 8, 12, and 8 weeks, respectively. The detection period of universal PCR for S. epidermidis treated by alcohol, cefazolin, and heat was less than 20, 20, and 4 weeks, whereas that for E. coli was 8, 20, and 4 weeks, respectively. The presence of detectable bacterial DNA in infected arthroplasty patients before and after successful treatment was also assessed by MRS- and universal PCR. MRS-PCR was positive in 6 patients before treatment and all became negative after a mean interval of 20.8 weeks (95% confidential interval, 13.2 to 33.7) after treatment. Universal PCR detected remnant bacterial DNA in 4 patients at a mean of 15.2 weeks (95% CI, 12.4 to 18.0) after treatment and was negative in 7 patients at a mean of 17.3 weeks (95% CI, 10.6 to 24.0) after treatment. Our studies revealed that real-time PCR detects dead bacteria for several weeks, but this capability decreases with time and is likely lost by 20 weeks after treatment.

Molecular diversity of the equine caecal microbiota and its correlation to postprandial fermentation metabolites: A preliminary approach

In this work we address the short-term postprandial dynamic changes in the equine caecal microbial composition and the microbial fermentation metabolites in the caecum. Samples were collected every half hour for 8.5 hours postfeeding from two horses on two consecutive days. The caecal content of volatile fatty acids (VFAs), lactate, and ammonia (NH3N), as well as microbiota composition and pH were analyzed. Mixed Sangers sequences generated from universal polymerase chain reaction (PCR)-amplified 16S rRNA gene were used to analyze the microbiota composition. Three main phylogroups were identified and classified as Clostridium cluster XIVa, Clostridium cluster III, and lactic acid bacteria. Furthermore, we found that the composition of the caecal microbiota was closely associated with the VFA profile. Our main findings were that short-term microbial fluctuations occur in healthy hindgut fermenting horses as a response to feed intake.

Cost-effectiveness of blood donor screening for Babesia microti in endemic regions of the United States.

Babesia microti is the leading reported cause of red blood cell (RBC) transfusion-transmitted infection in the United States. Donor screening assays are in development. A decision analytic model estimated the cost-effectiveness of screening strategies for preventing transfusion-transmitted babesiosis (TTB) in a hypothetical cohort of transfusion recipients in Babesia-endemic areas of the United States. Strategies included: 1) no screening; 2) Uniform Donor Health History Questionnaire (UDHQ), "status quo"; 3) recipient risk targeting using donor antibody and polymerase chain reaction (PCR) screening; 4) universal endemic donor antibody screening; and 5) universal endemic donor antibody and PCR screening. Outcome measures were TTB cases averted, costs, quality-adjusted life-years (QALYs), and incremental cost-effectiveness ratios (ICERs; $/QALY). We assumed a societal willingness to pay of $1 million/QALY based on screening for other transfusion-transmitted infections. Compared to no screening, the UDHQ avoids 0.02 TTB cases per 100,000 RBC transfusions at an ICER of $160,000/QALY whereas recipient risk-targeted strategy using antibody/PCR avoids 1.62 TTB cases per 100,000 RBC transfusions at an ICER of $713,000/QALY compared to the UDHQ. Universal endemic antibody screening avoids 3.39 cases at an ICER of $760,000/QALY compared to the recipient risk-targeted strategy. Universal endemic antibody/PCR screening avoids 3.60 cases and has an ICER of $8.8 million/QALY compared to universal endemic antibody screening. Results are sensitive to blood donor Babesia prevalence, TTB transmission probability, screening test costs, risk and severity of TTB complications, and impact of babesiosis diagnosis on donor quality of life. Antibody screening for Babesia in endemic regions is appropriate from an economic perspective based on the societal willingness to pay for preventing infectious threats to blood safety.

A Cross-Sectional Study of Helicobacter Infection Among Laboratory Animals and Animal Research Workers

A Cross-Sectional Study of Helicobacter Infection Among Laboratory Animals and Animal Research Workers

Identification and characterisation of oil sludge degrading bacteria isolated from compost

AbstractCompounds present in oil sludge such as polycyclic aromatic hydrocarbons (PAHs) are known to be cytotoxic, mutagenic and potentially carcinogenic. Microorganisms including bacteria and fungi have been reported to degrade oil sludge components to innocuous compounds such as carbon dioxide, water and salts. In the present study, we isolated different bacteria with PAH-degrading capabilities from compost prepared from oil sludge and animal manures. These bacteria were isolated on a mineral base medium and mineral salt agar plates. A total of 31 morphologically distinct isolates were carefully selected from 5 different compost treatments for identification using polymerase chain reaction (PCR) of the 16S rRNA gene with specific primers (universal forward 16S-P1 PCR and reverse 16S-P2 PCR). The amplicons were sequenced and sequences were compared with the known nucleotides from the GenBank. The phylogenetic analyses of the isolates showed that they belong to 3 different clades; Firmicutes, Proteobacteria and Actinobacteria. These bacteria identified were closely related to the generaBacillus, Arthrobacter, Staphylococcus, Brevibacterium, Variovorax, Paenibacillus, Ralstonia and Geobacillus.The results showed thatBacillus species were predominant in all composts. Based on the results of the degradation of the PAHs in the composts and results of previous studies on bacterial degradation of hydrocarbons in oil, the characteristics of these bacterial isolates suggests that they may be responsible for the breakdown of PAHs of different molecular weights in the composts. Thus, they may be potentially useful for bioremediation of oil sludge during compost bioremediation.

The Future Possibilities of Diagnostic Testing for the Evaluation of Febrile Infants

The Future Possibilities of Diagnostic Testing for the Evaluation of Febrile Infants

Microbial Adhesion on Membrane Oxygenators in Patients Requiring Extracorporeal Life Support Detected by a Universal rDNA PCR Test

Extracorporeal membrane oxygenation (ECMO) represents a temporary life-saving therapy for respiratory or circulatory failure, but infections during ECMO support are a life-threatening complication. Surface-related infections of ECMO are mentioned, but rarely described in the literature. A universal rDNA polymerase chain reaction (PCR) test was used to investigate the potential microbiological colonization of membrane oxygenators (MOs) in 20 patients undergoing ECMO. The overall patient-based positivity by PCR was 45%. Gram-positive bacteria (71%) represented the most abundant microorganisms on MO surfaces, followed by Gram-negative bacteria (22%) and fungi (7%). The most frequently detected causative pathogens were staphylococci (58%). Bacterial mixed infections represented 56% of all infections. In four PCR-positive cases, the pathogens detected on the MO surfaces were also found by blood culture or by culture of specimens obtained from the infectious focus. In conclusion, hollow fiber membranes of MOs can be colonized by microorganisms and appear to be a potential source of bacterial and fungal infections in ECMO patients. These infections may pose an increased risk for clinical worsening. As a consequence, persistent septic complications have to be discussed as an indication for MO exchange. The initial results suggest that the applied PCR assay is a valuable tool to investigate MOs.

Quantification of <italic>Saccharopolyspora rectivirgula</italic> in Composting Plants: Assessment of the Relevance of <italic>S. rectivirgula</italic>

Exposure to bioaerosols in composting plants can lead to negative health effects on compost workers. Health complaints vary between cough, irritation of the eyes and the skin, sinusitis, or dyspnea among others. It is fact that compost materials harbor high concentrations of microorganisms, which were aerosolized during handling compost. Within the present study, total cell numbers between 3.4 × 10(4) and 1.6 × 10(8) cell counts per m(3) air were determined after 4',6-Diamidin-2-phenylindol DAPI staining in 124 samples from German composting plants. Special attention should be paid to some specific microorganisms, which are able to cause health complaints. Saccharopolyspora rectivirgula, known to be one of the major causes of extrinsic allergic alveolitis (EAA, also called hypersensitivity pneumonitis, HP), was often found in environments of agricultural production, where the classical form of EAA ('farmer's lung disease') is common, but also in composting plants. In Germany, cases are known where workers had to terminate their work due to this disease. However, up to now, the relevance of S. rectivirgula at composting plants is unexplained. This study showed that high concentrations of airborne S. rectivirgula were found in composting plants similar to that found in agricultural production. Altogether, in 86.7% of the 124 analyzed samples, S. rectivirgula was detected using quantitative real-time polymerase chain reaction (PCR). Estimated concentrations ranged between 1.24 × 10(2) cell counts of S. rectivirgula per cubic meter air next to the rotted residues and 1.5 × 10(7) cell counts next to a converter. Furthermore, our methodical proceedings were verified. To analyze DNA extraction limits through the amount of cells within one sample, the DNA concentration was compared with total cell counts (TCCs). Altogether, when TCC was <1.4 × 10(5) cells per DNA extraction assay, no DNA was measurable; when TCC reached 3.5 × 10(6) cells, DNA was always detectable by fluorometric method. To overcome limitation of DNA measurement using fluorometric method, samples without measurable DNA were inserted in a PCR assay with universal primers. Results showed that a gain of 37% was possible, when samples were additionally analyzed by universal PCR. Hence, cell counts >2.0 × 10(6) cells were necessary to measure DNA concentrations in 90% of the analyzed samples, whereas cell counts <3.0 × 10(5) are sufficient to detect PCR products. Therefore, sampling of bioaerosols should be done in consideration of the expected cell count per cubic meter air. Note, to get measurable DNA using a fluorometer, >3.5 × 10(6) cells must be sampled for one DNA extraction assay. With this study, the real-time PCR approach for the detection of S. rectivirgula at workplaces in compost plants was revised, and the results revealed that this method is suitable for occupational exposure measurements.

Use of Real-Time Polymerase Chain Reaction for the Diagnosis of Infection and Differentiation Between Gram-Positive and Gram-Negative Septic Arthritis in Children

Diagnosis and identification of the etiological agent of septic arthritis (SA) in children is an important issue, as early treatment based on accurate diagnosis of joint infections can prevent potentially disabling complications. The purpose of this study was to evaluate the efficacy of real-time polymerase chain reaction (PCR) for the diagnosis of SA in children. Twenty children with suspected SA who had joint pain and underwent surgical treatment were enrolled in this study. Their preoperative clinical and laboratory findings were investigated. Tissues obtained during operation were subjected to microbiological culture and real-time PCR, including methicillin-resistant Staphylococcus (MRS)-specific PCR and broad range universal PCR. Infection was confirmed if the result of microbiological culture was positive. Furthermore, abnormal clinical and laboratory findings and improvement in the symptoms and posttreatment data were also defined as the final diagnosis of infection. Out of the 20 patients, 19 were diagnosed with the infection. The remaining patient was postoperatively diagnosed with juvenile idiopathic arthritis. Abnormal preoperative body temperatures, white blood cell counts, C-reactive protein levels, and erythrocyte sedimentation rates were observed in 6, 9, 15, and 12 cases, respectively. The results of microbiological culture, MRS-PCR, and universal PCR were positive in 9, 2, and 15 cases, respectively. Analysis of the melting peak in universal PCR revealed that of the 15 cases, 10 had gram-positive and 5 had gram-negative infections. The sensitivity and specificity for the diagnosis of SA were, respectively, 0.47 and 1.00 in microbiological culture and 0.79 and 1.00 in real-time PCR. Successful diagnosis of infection and differentiation between gram-positive and gram-negative bacteria were achieved using MRS-PCR and universal PCR. Hence, real-time PCR is useful and has greater sensitivity than microbial culture for diagnosing SA in children. Level II diagnostic study investigating a diagnostic test.

Unique Vaginal Microbiota That Includes an Unknown Mycoplasma-Like Organism Is Associated With Trichomonas vaginalis Infection

The prevalence of Trichomonas vaginalis infection is highest in women with intermediate Nugent scores. We hypothesized that the vaginal microbiota in T. vaginalis-infected women differs from that in T. vaginalis-uninfected women. Vaginal samples from 30 T. vaginalis-infected women were matched by Nugent score to those from 30 T. vaginalis-uninfected women. Equal numbers of women with Nugent scores categorized as normal, intermediate, and bacterial vaginosis were included. The vaginal microbiota was assessed using 454 pyrosequencing analysis of polymerase chain reaction-amplified 16S ribosomal RNA gene sequences. The 16S ribosomal RNA gene sequence of an unknown organism was obtained by universal bacterial polymerase chain reaction amplification, cloning, and sequencing. Principal coordinates analysis of the pyrosequencing data showed divergence of the vaginal microbiota in T. vaginalis-infected and T. vaginalis-uninfected patients among women with normal and those with intermediate Nugent scores but not among women with bacterial vaginosis. Cluster analysis revealed 2 unique groups of T. vaginalis-infected women. One had high abundance of Mycoplasma hominis and other had high abundance of an unknown Mycoplasma species. Women in the former group had clinical evidence of enhanced vaginal inflammation. T. vaginalis may alter the vaginal microbiota in a manner that is favorable to its survival and/or transmissibility. An unknown Mycoplasma species plays a role in some of these transformations. In other cases, these changes may result in a heightened host inflammatory response.

Development of an ethidium monoazide–enhanced internally controlled universal 16S rDNA real-time polymerase chain reaction assay for detection of bacterial contamination in platelet concentrates

Development of an ethidium monoazide–enhanced internally controlled universal 16S rDNA real-time polymerase chain reaction assay for detection of bacterial contamination in platelet concentrates

Non‐invasive prenatal testing for fetal aneuploidy: charting the course from clinical validity to clinical utility

Non‐invasive prenatal testing for fetal aneuploidy: charting the course from clinical validity to clinical utility

Study of the early molecular diagnosis methods in invasive fungal infection of clinical humoral specimens

Objective The aim of this study was to investigate the diagnostic impact of polymerase chain reaction (PCR) assays for fungal pathogens in fluid samples,and to evaluate the feasibility of fast PCR diagnostic method for the invasive fungal infection.Methods The sterility body fluid samples from 60 cases hospitalized immunocompromised patients with clinical underlying diseases and suspect of invasive infections with fungi between January 2008 and December 2011 were processed for microscopy and cultures.Applying fungal ITS4 and ITS86 universal primers to amplify pathogenic fungi genes from the sterility body fluid with method of rapid PCR.The results were compared between the standard and PCR methods.Results Humoral direct clinical specimens by PCR amplification of DNA fragments with the scan results were similar.Positive rate of PCR test with clinical body fluid samples and the traditional fungal cultivation was similar.There was no significant statistical difference [38.3％ (23/60) vs 33.3％ (20/60),P ＞ 0.05].Conclusions PCR test is feasibility with clinical fungal diagnosis from directly humoral specimens.To amplify the clinical sterility body fluid samples with ITS fungal universal primers and PCR method might provide an accurate and rapid approach to detect the pathogenic fungi.Its methods on early diagnosis and prognosis of invasive fungal infections are of guiding significance. Key words: SUPERFICIALITY & ORIGIN; Body fluids/microbiology; Mycoses/diagnosis; Polymerase chain reaction; Early diagnosis

16S rRNA Gene-targeted TTGE in Determining Diversity of Gut Microbiota during Acute Diarrhoea and Convalescence

The human gut microbiota play a vital role in health and nutrition but are greatly modified during severe diarrhoea due to purging and pathogenic colonization. To understand the extent of loss during and after diarrhoea, faecal samples collected from children (n=21) suffering from acute diarrhoea and from their healthy siblings (n=9) were analyzed by 16S rRNA gene-targeted universal primer polymerase chain reaction (PCR), followed by temporal temperature gradient gel electrophoresis (TTGE). The gut microbiota decreased significantly as indicated by the number of TTGE bands at day 0 of acute diarrhoea [patients vs healthy siblings: 11±0.9 vs 21.8±1.1 (mean ± standard error), p<0.01]. The number of bands showed a steady increase from day 1 to day 7; however, it remained significantly less than that in healthy siblings (15±0.9, p<0.01). These results suggest that appropriate therapeutic and post-diarrhoeal nutritional intervention might be beneficial for the early microbial restoration and recovery.

Ostéoarthrite de hanche primitive et myosite multifocale à méningocoque B chez un enfant de 7 ans

Neisseria meningitidis is associated with severe invasive infections such as meningitis and fulminant septicemia. Septic arthritis due to N.meningitidis is rare and bone infections have been reported exceptionally. We report the case of a 7-year-old boy who presented with septic arthritis of the right hip associated with a septic location on the pelvis and pyomyositis of the adjacent muscle. Culture of the joint fluid was sterile but universal 16S polymerase chain reaction (PCR) of this fluid revealed group B N.meningitidis. Our patient had never presented any symptoms of meningitis or septicemia and blood cultures were all sterile. Despite appropriate antibiotic treatment, the course of the disease was unusually long and his status did not improve until surgical lavage of the hip was performed. Moreover, MRI imaging showed bilateral hypersignals of the adjacent muscles and revealed an abscess formation in the left gluteus maximus muscle. Presumptive diagnosis bacterial myositis was confirmed by an elevation of creatine phosphokinase in the sera up to 21-fold the normal value but the culture of the abscess, performed 10 days after initiation of antibiotics, was sterile. Despite an initially unfavorable course, this patient's status improved after surgical drainage and he fully recovered 1 month later. This observation illustrates an unusual presentation of invasive meningococcal infection. The respective roles of infection and an inflammatory phenomenon during the course of the disease are discussed. Moreover, this case emphasizes the value of PCR for bacteriological diagnosis of bone and joint infections.

Molecular typing of bacteria of the genus Asaia in malaria vector Anopheles arabiensis Patton, 1905

The acetic acid bacterium &lt;em&gt;Asaia&lt;/em&gt; spp. was successfully detected in &lt;em&gt;Anopheles arabiensis&lt;/em&gt; Patton, 1905, one of the major vector of human malaria in Sub-Saharan Africa. A collection of 45 &lt;em&gt;Asaia&lt;/em&gt; isolates in cellfree media was established from 20 individuals collected from the field in Burkina Faso. 16S rRNA universal polymerase chain reaction (PCR) and specific qPCR, for the detection of &lt;em&gt;Asaia&lt;/em&gt; spp. were performed in order to reveal the presence of different bacterial taxa associated with this insect. The isolates were typed by internal transcribed spacer-PCR, BOX-PCR, and randomly amplified polymorphic DNA-PCR, proved the presence of different &lt;em&gt;Asaia&lt;/em&gt; in &lt;em&gt;A. arabiensis&lt;/em&gt;.

Quantitative evaluation of periprosthetic infection by real-time polymerase chain reaction: a comparison with conventional methods

Several recent studies have demonstrated the limited accuracy of conventional culture methods for diagnosing periprosthetic infections. We have applied real-time polymerase chain reaction (PCR) assays for the rapid identification of bacteria around implants and reported its utility. However, the capability of quantification is also a useful feature of this type of assay. The aim of our study was to validate the usefulness of quantitative analyses using real-time PCR of cases with clinical periprosthetic infections in comparison with more established tests, such as C-reactive protein (CRP) levels, microbiologic cultures, and histopathology. Fifty-six joints with suspected infections were reviewed retrospectively. A universal PCR assay was used to perform the quantitative analyses. The differences in the threshold cycles between clinical samples and a negative control (∆Ct) in each case were calculated. The results of the quantitative PCR assay were compared with CRP levels, microbiologic cultures, and histopathology. There was a significant correlation found between the CRP and ∆Ct values. There were also significant differences found in the ∆Ct values according to CRP levels, with higher CRP levels showing higher ∆Ct values. Similarly, there were significant differences in the ∆Ct measurements in our culture results and among our pathologic evaluations. We confirmed that quantification by universal PCR based on the ∆Ct correlated with preoperative CRP levels and was associated with the microbiologic culture results and pathologic severity. This quantification method may be valuable for assessing infection severity.

Rapid sexing of selected Galliformes by polymerase chain reaction

Vent sexing of one-day-old chicks in commercial hatcheries has long been common practice and can be highly accurate. However, there are circumstances when this technique is not applicable such as smaller breeds, non-domestic birds, or where is the necessity of precise sexing. In this study we present a simple and reliable method for fast gender determination in selected Galliformes for which phenotypic determination of sex is difficult until maturity. Four species were tested: two commercial species &amp;ndash; chicken (Gallus gallus) and turkey (Meleagris gallopavo), and two game birds &amp;ndash; common pheasant (Phasianus colchicus) and wood grouse (Tetraro urogallus). DNA was tested with universal single-pair primers polymerase chain reaction (PCR) detecting W chromosome specific sequence yielding a single band of length specific for each species. The method was developed with regards to time consumption and cost-effectiveness giving results in less than two hours. The method may also be used for early sexing in commercial chicken and turkey flocks as well as sexing of smaller game birds flocks or for research laboratories when rapid sexing of selected Galliformes cells is required. &amp;nbsp;

Systematic Use of Universal 16S rRNA Gene Polymerase Chain Reaction (PCR) and Sequencing for Processing Pleural Effusions Improves Conventional Culture Techniques

Conventional culture of pleural fluid samples frequently provides false-negative results. Universal polymerase chain reaction (PCR) of the 16S ribosomal ribonucleic acid (rRNA) gene (16S PCR) has proven useful in the diagnosis of various bacterial infections. We conducted a prospective study to assess the value of 16S PCR in the etiologic diagnosis of pleural effusion. All pleural fluid samples received for culture were also studied using 16S PCR. Positive samples were sequenced for identification. Clinical records and conventional culture results were analyzed to classify pleural fluid samples as infected or not infected. We studied 723 samples. We excluded 188 samples because they were obtained from a long-term chest tube, there was a diagnosis of mycobacterial infection, or there were insufficient data to classify the episode. Finally, 535 pleural fluid samples were analyzed. According to our criteria, 82 (15.3%) were infected and 453 (84.7%) were not infected. In the infected samples, 16S PCR was positive in 67 samples (81.7%) while conventional culture was positive in 45 (54.9%). There were 4 false positives with 16S PCR (0.9%) and 12 with culture (2.6%). The values for the etiologic diagnosis of bacterial pleural effusion of conventional culture compared with 16S PCR were as follows: sensitivity, 54.9%/81.7%; specificity, 97.4%/99.1%; positive predictive value, 76.3%/94.4%; negative predictive value, 92.6%/96.8%; and accuracy, 90.8%/96.5%.When compared with conventional culture, 16S PCR plus sequencing substantially improves the etiologic diagnosis of infectious pleural effusion. In our opinion, this technique should be added to the routine diagnostic armamentarium of clinical microbiology laboratories.

NoteDetection of 16S rDNA of ‘Candidatus Phytoplasma mali’ in plum decline in Tunisia

‘Candidatus Phytoplasma mali’ was detected, for the first time, in plum (Prunus domestica L.) trees, with symptoms of plum decline in Tunisia. Infected trees showed leaf browning, leaf and fruit wilting followed by tree death in a few weeks. Phytoplasma was detected by polymerase chain reaction (PCR) using universal phytoplasma primer pairs P1/P7. A band with expected size was observed in samples collected from symptomatic, but not symptomless, plum trees. PCR using group-specific primers yielded a band of 1050 bp with 16SrX group specific primers fO1/rO1. Universal PCR products (1.8 kb) were used for restriction fragment length polymorphism analysis (RFLP) after digestion with endonuclease SspI. RFLP patterns obtained were similar to those previously reported for the Apple Proliferation phytoplasma (AP, 16SrX-A), the causal agent of apple proliferation disease. Blast analysis revealed that universal PCR sequences obtained from the infected Tunisian plums were identical and had the highest identity (99%) with the sequence of ‘Ca. Phytoplasma mali’. Phylogenetic analysis based on 16S rDNA gene sequences showed that the plum decline phytoplasma strain from Tunisia clustered with ‘Candidatus Phytoplasma mali’ strains. This is the first report on detection of 16S rDNA of ‘Ca. Phytoplasma mali’ in plum decline in Tunisia.