Broad-range Real-time Polymerase Chain Reaction Research Articles

Polymerase chain reaction test for diagnosis of infectious uveitis

BackgroundTo study the clinical utility of broad-range real-time Polymerase Chain Reaction (PCR) assay in patients suspected for infectious uveitis and to analyze the clinical relevance.MethodsMedical records of patients with uveitis were assessed in whom PCR analysis of intraocular fluids was performed between January 2018 and February 2021. Intraocular samples were investigated for cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV), herpes simplex viruses type 1 and 2 (HSV1,2), human T-lymphotropic virus type 1 (HTLV-1), Toxoplasma gondii and also for bacterial 16 S and fungal 18 S/28S ribosomal DNA (rDNA).ResultsAqueous paracentesis and vitreous sampling was done for 151 (81.2%) and 35 (18.8%) patients, respectively. Most of the patients had panuveitis (61.3%). PCR results were positive in 69 out of 186 patients (37%) according to the following order: CMV (18 cases), VZV (18 cases), fungal 18s/28s rDNA (17 cases), HSV (9 cases), bacterial 16s rDNA (3 cases), HTLV-1 (2 cases), and Toxoplasma gondii (2 cases). PCR positivity rate was 5.8% in patients with undifferentiated panuveitis. EBV was not detected at all. Initial treatment was changed in 38 patients (20%) based on PCR results. The overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of PCR test for aqueous samples was 82%, 91%, 96%, and 87%, respectively. No significant adverse effect related to sampling was reported.ConclusionPCR analysis of intraocular fluids in patients with suspected infectious uveitis plays an important role in confirming diagnosis or changing treatment with good predictive value. However, routine PCR test in patients with undifferentiated panuveitis in order to rule out possible underlying infectious etiology had low benefit.

A Novel Molecular Microbiologic Technique for the Rapid Diagnosis of Microbial Invasion of the Amniotic Cavity and Intra‐Amniotic Infection in Preterm Labor with Intact Membranes

The diagnosis of microbial invasion of the amniotic cavity (MIAC) has been traditionally performed using traditional cultivation techniques, which require growth of microorganisms in the laboratory. Shortcomings of culture methods include the time required (days) for identification of microorganisms, and that many microbes involved in the genesis of human diseases are difficult to culture. A novel technique combines broad-range real-time polymerase chain reaction with electrospray ionization time-of-flight mass spectrometry (PCR/ESI-MS) to identify and quantify genomic material from bacteria and viruses. AF samples obtained by transabdominal amniocentesis from 142 women with preterm labor and intact membranes (PTL) were analyzed using cultivation techniques (aerobic, anaerobic, and genital mycoplasmas) as well as PCR/ESI-MS. The prevalence and relative magnitude of intra-amniotic inflammation [AF interleukin 6 (IL-6) concentration ≥2.6ng/mL], acute histologic chorioamnionitis, spontaneous preterm delivery, and perinatal mortality were examined. (i) The prevalence of MIAC in patients with PTL was 7% using standard cultivation techniques and 12% using PCR/ESI-MS; (ii) seven of ten patients with positive AF culture also had positive PCR/ESI-MS [≥17 genome equivalents per PCR reaction well (GE/well)]; (iii) patients with positive PCR/ESI-MS (≥17 GE/well) and negative AF cultures had significantly higher rates of intra-amniotic inflammation and acute histologic chorioamnionitis, a shorter interval to delivery [median (interquartile range-IQR)], and offspring at higher risk of perinatal mortality, than women with both tests negative [90% (9/10) versus 32% (39/122) OR: 5.6; 95% CI: 1.4-22; (P<0.001); 70% (7/10) versus 35% (39/112); (P=0.04); 1 (IQR: <1-2)days versus 25 (IQR: 5-51)days; (P=0.002), respectively]; (iv) there were no significant differences in these outcomes between patients with positive PCR/ESI-MS (≥17 GE/well) who had negative AF cultures and those with positive AF cultures; and (v) PCR/ESI-MS detected genomic material from viruses in two patients (1.4%). (i) Rapid diagnosis of intra-amniotic infection is possible using PCR/ESI-MS; (ii) the combined use of biomarkers of inflammation and PCR/ESI-MS allows for the identification of specific bacteria and viruses in women with preterm labor and intra-amniotic infection; and (iii) this approach may allow for administration of timely and specific interventions to reduce morbidity attributed to infection-induced preterm birth.

Effectiveness of two intracanal dressings in adult Portuguese patients: a qPCR and anaerobic culture assessment

To quantify bacterial equivalents before and after chemomechanical preparation using 3% sodium hypochlorite (NaOCl) and intracanal dressing with calcium hydroxide paste (Ca(OH)2 ) or 2% Chlorhexidine digluconate gel (CHX) in necrotic pulps associated or not with apical periodontitis and to further compare this quantification with counts of anaerobic microorganisms. Prospective clinical trial in 69 single-rooted adult teeth (strict inclusion criteria); CHX group: 34; Ca(OH)2 group: 35. Bacteria samples were taken at baseline (S1), after chemomechanical preparation (S2) and after 14 days of intracanal dressing (S3). Bacterial equivalents were assessed by broad-range real-time polymerase chain reaction (qPCR), and live viable bacteria measured with conventional anaerobic culture (CFU/mL). Descriptive/inferential analysis was performed with spss vs. 20.0 (α = 0.05) using the Kruskal-Wallis, Mann-Whitney and chi-squared tests and Spearman's correlation coefficients. Both groups showed a significant decrease between S1 and S2 (Mann-Whitney U-test; P < 0.001) both in qPCR and in culture. In the Ca(OH)2 -group, no variation was observed between S2 and S3 by qPCR and culture. In contrast, the CHX group showed a significant increase from S2 to S3 by both techniques. The two groups were only significantly different in S3 (Mann-Whitney U-test; P ≤ 0.001), with a worse performance in the CHX group. Again, these results were congruent by both approaches. Data from both approaches correlate reasonably (rS < 0.5). Infected root canals contained a high bacterial load, and the chemomechanical root canal preparation reduced bacterial equivalents by 99.1% and anaerobic counts by 98.5%. Intracanal dressings were not efficient at reducing bacterial load, but the 14-day intracanal dressing with Ca(OH)2 performed significantly better than CHX, particularly in cases with apical periodontitis.

Use of a Comprehensive Polymerase Chain Reaction System for Diagnosis of Ocular Infectious Diseases

To measure the genomic DNA of ocular infectious pathogens in ocular fluids and to analyze the clinical relevance of these pathogens in uveitis and endophthalmitis. Prospective clinical case series. A total of 500 patients with infectious uveitis and endophthalmitis were examined at Tokyo Medical and Dental University, Tokyo Medical University, Kyushu University, Osaka University, and Kyoto Prefectural University, all in Japan. Genomic DNA of bacteria, fungi, parasites, and viruses in collected intraocular samples were examined by comprehensive polymerase chain reaction (PCR). Samples were analyzed first by multiplex PCR and quantitative real-time PCR for human herpes viruses (HHVs) 1 through 8 and toxoplasma. Subsequently, samples were examined by broad-range real-time PCR for bacterial 16S and fungal 18S/28S ribosomal DNA (rDNA). Infectious uveitis and endophthalmitis diagnoses were obtained when using the PCR system. Calculations of the positivity and the diagnostic parameters such as sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) also were evaluated. In all of the tested infectious uveitis and endophthalmitis patients, either herpes simplex virus type 1 (n = 18), herpes simplex virus type 2 (n = 4), varicella-zoster virus (n = 55), Epstein-Barr virus (n = 17), cytomegalovirus (n = 68), HHV type 6 (n = 2), toxoplasma (n = 6), bacterial 16S (n = 33), or fungal 18S/28S (n = 11) genome was detected. Neither HHV type 7 nor HHV type 8 DNA was detected in any of the samples. Of the 21 false-negative results found during the PCR analyses, 12 cases were negative for patients clinically suspected of having bacterial endophthalmitis. Conversely, false-positive results for the comprehensive PCR examinations occurred in only 3 cases that subsequently were found to have bacterial 16S rDNA. Diagnostic parameters for the sensitivity, specificity, PPV, and NPV of our PCR examinations were 91.3%, 98.8%, 98.6%, and 92.4%, respectively. Use of our comprehensive PCR assay to examine ocular samples in patients with endophthalmitis and uveitis seems to be clinically useful for detecting infectious antigen DNA. Thus, this PCR method is a reliable tool for both diagnosing ocular disorders and further screening of patients for intraocular infections. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Broad-range real-time PCR assay for detection of bacterial DNA in ocular samples from infectious endophthalmitis

To evaluate a broad-range real-time polymerase chain reaction (PCR) targeting the bacterial 16S rRNA gene for detection of bacterial DNA in infectious endophthalmitis. The bacterial 16S rRNA gene was measured by quantitative real-time PCR. For the assay, bacterial DNA was prepared from 12 Gram-positive and 4 Gram-negative strains. To determine the optimum method for DNA extraction, four extraction procedures were selected by using DNA extraction program cards with and without the use of lysozyme. To evaluate PCR sensitivity, PCR fragments were amplified from Staphylococcus aureus and Escherichia coli DNA. DNA extraction using the Bacteria card(®) without enzymes resulted in detection of all the tested strains at concentrations ≥ 10(7) copies/mL. Extraction with the Bacteria card(®) with lysozyme resulted in detection of all the tested strains at concentrations ≥ 10(6) copies/mL, indicative of no significant difference between the two procedures. DNA extraction using the Virus card(®), both with and without enzymes, resulted in reduced efficiency of detection of all strains compared with use of the Bacteria card(®). The PCR could detect as few as 1-10 colony-forming units (CFU) in diluted vitreous samples per reaction, and all tested bacterial species known to cause endophthalmitis were detected. Bacterial 16S-specific PCR can comprehensively detect the main causative bacteria of clinically suspected endophthalmitis.

Novel diagnosis of fungal endophthalmitis by broad-range real-time PCR detection of fungal 28S ribosomal DNA

To detect the fungal genome in the ocular fluids of patients with fungal endophthalmitis by using a novel broad-range polymerase chain reaction (PCR) system. After informed consent was obtained, ocular fluid samples (aqueous humor or vitreous fluids) were collected from 497 patients (76 patients with infectious endophthalmitis including clinically suspected bacterial and fungal endophthalmitis and 421 patients with infectious or non-infectious uveitis). Forty ocular samples from non-infectious patients without ocular inflammation were collected as controls. Fungal ribosomal DNA (28S rDNA) was measured by a quantitative real-time PCR assay. Fungal 28S rDNA of the major fungal species, such as Candida, Aspergillus, and Cryptococcus, were detected by novel broad-range real-time PCR examination (>10(1) copies/ml). Fungal 28S rDNA was detected in the ocular fluids of 11 patients with endophthalmitis or uveitis (11/497, 2.2%). All 11 positive samples were detected in the infectious endophthalmitis patients (11/76, 14.5%). These PCR-positive ocular fluids had high copy numbers of fungal 28S rDNA (range, 1.7 × 10(3) to 7.9 × 10(6) copies/ml), which indicated the presence of fungal infection. Of the 11 patients who were PCR positive, further examinations led to a diagnosis of fungal endophthalmitis in ten patients. The fungal 28S rDNA was detected in one non-infectious case (a false-positive case). In addition, there were two PCR false-negative cases that were clinically suspected of having fungal endophthalmitis. This novel quantitative broad-range PCR of fungal 28S rDNA is a useful tool for diagnosing endophthalmitis related to fungal infections.

Detection of Candida and Aspergillus species DNA using broad-range real-time PCR for fungal endophthalmitis

The goal of this work is to establish a broad-range real-time polymerase chain reaction (PCR) diagnostic system for ocular fungal infection and to measure Candida and Aspergillus DNA in the ocular fluids obtained from unknown uveitis/endophthalmitis patients. After obtaining informed consent, intraocular fluids (aqueous humor and vitreous fluid samples) were collected from 54 patients with idiopathic uveitis or endophthalmitis. Samples were assayed for Candida or Aspergillus DNA using broad-range (18S rRNA sequences) quantitative real-time PCR. Candida or Aspergillus DNA was detected in seven out of 54 patient ocular samples (13%). These PCR-positive samples showed significantly high copy numbers of Candida or Aspergillus DNA. On the other hand, fungal DNA was not detected in any of the other 46 samples collected from these idiopathic uveitis or endophthalmitis patients. In the one PCR-negative case, PCR did not detect any fungal genome in the sample, even though this patient was clinically suspected of having Candida endophthalmitis. Real-time PCR results were negative for fungal DNA in the bacterial endophthalmitis patients and in various uveitis patients. In addition, fungal DNA was also not detected in patients without ocular inflammation (controls). Analysis of ocular samples by this broad-range real-time PCR method can be utilized for rapid diagnosis of patients suffering from unknown intraocular disorders such as idiopathic uveitis/endophthalmitis.

Evaluation of the LightCycler® SeptiFast test in the rapid etiologic diagnostic of infectious endocarditis

The SeptiFast test (Roche Diagnostics) is a new commercial molecular technique that has emerged for the detection of bacteria in blood. We compared in this study the sensitivity of blood culture to a commercially available broad-range real-time polymerase chain reaction (PCR) assay for the detection in blood of 19 bacterial species and six fungal species (SeptiFast test, Roche Diagnostics) in 63 patients with infectious endocarditis (IE). The SeptiFast test is not more sensitive for organisms such as Streptococci, Enterococci, and Staphylococcus aureus (11/29 versus 12/29 for blood culture). It has detected less commonly coagulase-negative Staphylococci (0/15 versus 3/15, P = 0.2) and significantly fewer other microorganisms (0/6 versus 4/6, P = 0.03). However, bacteria were detected from three IE treated by antibiotics, with blood culture negative on admission. The SeptiFast test may be useful in cases of IE in patients treated with antibiotics before admission.

DIAGNOSIS OF VENTRICULAR DRAINAGE-RELATED BACTERIAL MENINGITIS BY BROAD-RANGE REAL-TIME POLYMERASE CHAIN REACTION

To compare a broad-range real-time polymerase chain reaction (PCR) diagnostic strategy with culture to evaluate additional effects on the etiological diagnosis and the quantification of the bacterial load during the course of ventricular drainage-related bacterial meningitis (VR-BM). We applied a PCR that targeted conserved regions of the 16S ribosomal ribonucleic acid gene to cerebrospinal fluid (CSF) samples from patients with external ventricular drainage or a ventriculoperitoneal shunt during the course of VR-BM. We compared the PCR results with CSF cultures. A total of 350 routine CSF samples were consecutively collected from 86 patients. The CSF deoxyribonucleic acid was automatically purified and subjected to PCR. Amplicons from the PCR samples that were positive for VR-BM were subsequently deoxyribonucleic acid sequenced for final identification. Clinical data were extracted from patient files. Sixteen patients had at least one VR-BM-positive sample as diagnosed from culture or PCR. Nineteen episodes were diagnosed with signs of VR-BM (n = 16 patients) or were determined to be contaminated (n = 3 patients). Four episodes of VR-BM were diagnosed via PCR alone and were predominantly caused by gram-negative pathogens, five episodes were diagnosed via culture alone, and seven episodes were diagnosed via both culture and PCR. Five patients had mixed infections. Overall, 71 samples were positive for VR-BM as indicated by either one or both of the methods. Eighteen CSF samples were VR-BM positive as indicated by culture alone, and 21 CSF samples were positive as indicated via PCR alone. Culture supplemented with broad-range, real-time PCR may increase the number of etiologically diagnosed VR-BM episodes, particularly when these are caused by gram-negative bacteria.

Broad-range real time PCR and DNA sequencing for the diagnosis of bacterial meningitis

Rapid aetiological diagnosis of bacterial meningitis is crucial for the early targeting of antimicrobial and adjuvant therapy. Broad-range polymerase chain reaction (PCR) targeting the 16S rRNA gene allows aetiological diagnosis of bacterial meningitis when applied to cerebrospinal fluid (CSF). We assessed the additional diagnostic effect of applying a novel broad-range real time PCR and subsequent DNA sequencing to culture, microscopy, and broad-range conventional PCR on CSF in patients with suspected bacterial meningitis. Broad-range conventional PCR and broad-range real time PCR with subsequent DNA sequencing were applied to 206 CSF specimens collected consecutively from 203 patients aged 6 d to 86 y. Patients' charts were reviewed for clinical information. 17 pathogens were identified by PCR and DNA sequencing or culture. Three specimens were negative by culture but positive by broad-range real time PCR. Three specimens were positive by culture but negative by broad-range real time PCR. Compared with culture, the sensitivity of broad-range real time PCR was 86%, and the specificity 98%. Conventional PCR resulted in a sensitivity of 64% and specificity of 98%. Broad-range real time PCR was generally comparable to culture of CSF and may be a useful supplement, particularly when antimicrobial therapy has been administered. Broad-range real time PCR was more sensitive than broad-range conventional PCR and microscopy.

A Molecular Gram Stain Using Broad Range PCR and Pyrosequencing Technology

The bacteria associated with orthopaedic infections are usually common gram-positive and gram-negative bacteria. This fundamental grouping of bacteria is a necessary first step in the selection of appropriate antibiotics. Since polymerase chain reaction (PCR) is more rapid and may be more sensitive than culture, we developed a postamplification pyrosequencing method to subcategorize bacteria based on a few nucleotide polymorphisms in the 16S rRNA gene. We validated this method using well-characterized strains of bacteria and applied it to specimens from spinal surgery cases with suspected infections. Lysates of 114 bacteria including 75 species were created following standard cultivation to obtain DNA. The DNA was amplified by a broad-range real-time PCR. The amplicons were evaluated by pyrosequencing and were classified as gram-positive, gram-negative, or acid-fast bacilli based on the first three to five nucleotides sequenced. In addition, clinical cases of suspected infection were obtained from spinal surgery. The results of the "molecular Gram stain" were compared with the results of traditional Gram stain and culture. The lysates of 107 (93.9%) of the bacteria extracts tested were appropriately categorized as gram-positive and gram-negative or as acid-fast bacilli on the basis of this assay. The sensitivity and specificity of this assay were 100% and 97.4% for gram-positive and 88.3% and 100% for gram-negative isolates. All of the five clinical samples were appropriately categorized as containing gram-positive or gram-negative bacteria with this assay. This study demonstrates that high sensitivity and specificity of a molecular gram stain may be achieved using broad-range real-time PCR and pyrosequencing.