Comparative study of diagnosis of PD peritonitis by quantitative polymerase chain reaction for bacterial DNA vs culture methods

INTRODUCTION: Peritonitis remains one of the main complications that afflict peritoneal dialysis patients. We conducted a pilot study to determine the feasibility and potential advantages of quantitative PCR (qPCR) assays for the presence of bacterial DNA in this clinical scenario. METHODS: 14 patients attending with 'cloudy bags' had PD fluid analyzed in accordance with Renal Association Standards. In addition, quantitative bacterial DNA analysis was performed on 50 mL samples of PD fluid. DNA was extracted using a Qiagen kit. Quantitative PCR assays using primers and probes targeted at 16S rDNA were used to measure the levels of bacterial DNA. Samples from 13 patients attending the department for other reasons served as negative controls. Laboratory staff were blinded to clinical details at the time of analysis. RESULTS: We determined a threshold of bacterial DNA whereby 11 of 13 negative controls were 'negative'. Significant bacterial DNA was found in 6 of 9 culture positive' peritonitis cases (p < 0.05 by chi(2). The 3 cases of 'no growth' peritonitis had 'insignificant' bacterial DNA. Serial DNA analysis was performed in 8 patients. Of the 6 patients who were 'cured' with standard antibiotic therapy, only 1 showed a rise in bacterial DNA from Day 1 to 5. But the 2 patients who relapsed after antibiotics had marked rises in bacterial DNA (p < 0.05 by chi(2). DISCUSSION: We showed that results from quantitative bacterial DNA PCR assays correlate with current microbiological tests despite the small size of this study. We also suggest that this technology might be clinically useful as an adjunct for cases of 'no growth' peritonitis and to identify those patients likely to relapse despite apparent clinical improvement with standard antibiotic therapy.     

Comparison of polymerase chain reaction and bacteria culture in peritoneal dialysis associated peritonitis

Objective To evaluate clinical application value of polymerase chain reaction (PCR) detection for bacteria in peritoneal dialysis associated peritonitis (PDAP). Methods Peritoneal dialysis fluid specimens were collected from January 2014 to December 2014 in The First Affiliated Hospital of Anhui Medical University. Conventional bacterial culture and PCR detection were used respectively. According to the bacterial 16S rRNA gene, universal primers were devised and designed, based on reference, the specific primers of 17 kinds of experimental bacteria. Real-time fluorescent PCR (Real-time PCR, qPCR) amplification was implemented. The establishment of standard strain DNA extract was used as positive control; sterile double distilled water was used as negative control. Results (1) The traditional bacterial culture results showed that positive proportion was 26/40 in specimen of 40 cases, gram-positive strains accounting for 18/26. Main species were epidermis staphylococcus (5/26), hemolysis staphylococcus (4/26), escherichia coli (4/26), and streptococcus viridans (3/26). (2) The PCR detection results showed that total positive rate was 33/40 in 40 patients specimens, among which 2 cases of positive samples ended up with no specific strains being detected; the main bacteria strains in PCR were not different from ordinary culture results. (3) With bacterial culture as the gold standard, the detection sensitivity of PCR technology for PDAP pathogenic bacteria was 96.15% and specificity was 42.86%; the detection positive rate was significantly higher than ordinary culture method. (4) PCR technology for detecting pathogenic bacteria could produce results within 4-6 hours, while reported positive results in the traditional bacterial culture would take (77.88±15.53) hours, which was significantly longer than PCR. Conclusion Compared with traditional bacteria culture method, PCR method is more sensitive, simple, and quick. Bacteria detection using PCR technique is of clinic applied value in PDRP.     

Comparison of polymerase chain reaction for IS6110 and Amplicor in the diagnosis of tuberculosis.

BACKGROUND: Polymerase chain reaction (PCR) amplification of Mycobacterium tuberculosis DNA offers the potential of a sensitive and specific diagnostic test for tuberculosis. To evaluate this technique from the clinician's perspective, samples were collected from patients with chronic respiratory disease and the sensitivity and specificity of a newly introduced commercially available PCR kit (Amplicor) was compared with that of an established method to detect the target sequence IS6110. METHODS: Sputum or bronchial washings from patients with active tuberculosis, previously treated tuberculosis or other selected respiratory illnesses were analysed by both techniques and their sensitivity and specificity determined. RESULTS: Amplicor was more specific than IS6110 in the diagnosis of active infection (98% versus 79%). Both techniques were equally sensitive (92%). CONCLUSION: These results suggest that analysis of respiratory samples by Amplicor PCR in inner city populations of patients has greater specificity for a diagnosis of active tuberculosis than PCR for IS6110, and thus Amplicor PCR may aid the clinician in making a diagnosis of active tuberculosis.     

Rapid diagnosis of Brucella epididymo-orchitis by real-time polymerase chain reaction assay in urine samples

PURPOSE: We studied the diagnostic yield of a real-time polymerase chain reaction assay in urine samples for the rapid diagnosis of brucella epididymo-orchitis compared to that of conventional microbiological techniques. MATERIALS AND METHODS: We used an SYBR Green I LightCycler based real-time polymerase chain reaction to retrospectively study 10 urine samples from patients with Brucella epididymo-orchitis. The assay amplifies a 223 bp sequence of a gene that codes for the synthesis of an immunogenetic membrane protein specific for Brucella genus (BCSP31). After amplifying this 223 bp sequence we performed melting curve analysis to verify the specificity of polymerase chain reaction products. RESULTS: Brucella melitensis was isolated from blood cultures in 9 cases (90%). Wright's seroagglutination was negative or inconclusive in 30% of cases. Brucella was isolated from urine in only 1 case, whereas real-time polymerase chain reaction assay in urine was positive in 9 (90%). Also, results were available in 4 hours, whereas mean time to availability of the final blood culture results was 5.8 days (range 4.5 to 7). CONCLUSIONS: SYBR Green I LightCycler based real-time polymerase chain reaction assay in urine samples is highly sensitive and specific, and easy to perform. It could provide the clinician with results in less than 5 hours. The technique could be a practical and useful tool for the rapid diagnosis of genitourinary complications of human brucellosis.     

聚合酶链反应与分离培养技术检测结核分支杆菌诊断关节结核的对照研究

目的：研究聚合酶链反应（polymerase chain reaction,PCR）技术在关节结核标本结核分支杆菌检测方面的作用,探讨PCR技术对关节结核诊断的临床价值。方法：自1993年6月至2001年8月,对95例（男55例,女40例;年龄2～75岁）关节结核标本分别应用PCR技术和分离培养法盲法检测结核分支杆菌,计算两者检测阳性率,通过统计学处理进行比较。结果：95例关节结核标本结核分支杆菌检测中,PCR技术检测阳性78例,阴性17例,阳性率82％;分离培养法检测阳性15例,阴性80例,P13性率16％。PCR技术与分离培养法比较,Х^2=67,P〈0．001,两种方法对于关节结核标本结核分支杆菌的检出率比较差异有统计学意义。PCR扩增整个过程自动化控制,可在数小时内完成。结论：PCR技术检测关节结核标本具有快速、简便、敏感与特异等优点,明显优于分离培养,对关节结核的早期快速诊断与鉴别诊断具有较重要的临床价值。     

聚合酶链技术在慢性前列腺炎诊治中的应用

目的：探索慢性前列腺炎的病因，提高其诊治水平。方法：应用聚合酶链技术(PCR)对门诊疑诊为慢性前列腺炎患者的前列腺液进行淋球菌(NG)、沙眼衣原体(CT)及解脲支原体(UU)检测。结果：检出单纯淋球菌感染33例，阳性率9．09％；沙眼衣原体感染32例，阳性率5．15％；解脲支原体感染250例，阳性率23．00％，沙眼衣原体与解脲支原体合并感染13例；淋球菌合并解脲支原体感染5例；淋球菌合并沙眼衣原体感染4例；并应用PCR监测治疗效果。结论：PCR检测慢性前列腺炎病原体快速、敏感性高、特异性强，是一种较为理想、值得推广的临床应用技术。     

Comparison between antigenemia and a quantitative-competitive polymerase chain reaction for the diagnosis of cytomegalovirus infection after heart transplantation

BACKGROUND: Antigenemia and quantitative polymerase chain reaction (PCR) are widely used for cytomegalovirus (CMV) diagnosis after heart transplantation due to their enhanced predictive values for disease detection when specific cut-off values are used. The purpose of this study was to compare, in the same patient setting, the predictive values of quantitative PCR and antigenemia for CMV disease detection, using specific cut-off values. METHODS: Thirty heart transplant receptors were ch prospectively monitored for active CMV infection and disease detection, using quantitative PCR and anti- po genemia. Positive and negative predictive values for pr CMV disease detection were calculated using cut-off pr values for both antigenemia (5 and 10 positive cells/300,000 neutrophils) and quantitative-PCR (50,000 and 100,000 copies/10(6) leukocytes). RESULTS: Active CMV infection was diagnosed in 93.3% of patients and CMV disease in 23.3%. The positive and negative predictive (%) values for CMV disease detection were 35/100 and 46.7/100, respectively, for quantitative PCR and antigenemia. Using 5 and 10 positive cells/300,000 neutrophils as cut-off values for antigenemia, the positive and negative predictive values (%) for disease detection were respectively 63.6/100 and 70/100. For quantitative PCR, the positive and th negative predictive values (%) for cut-off values of to 50,000 and 100,000 copies/10(6) leukocytes were 53.8/100 and 60/94.1, respectively. CONCLUSION: In our series, antigenemia and quantitative-PCR had enhanced and similar predictive values for CMV disease detection when specific cut-off values were used. The choice between these two methods for disease detection may rely less on their efficiency and more on the experience and familiarity with them.     

PCR diagnosis on formalin-fixed, paraffin-embedded tissues with acid-fast stain and culture negativity in chronic dialysis patients of cervico-mediastinal tuberculous lymphadenitis

Background: Bacteriologic studies often provide negative results in tuberculous infection, and do not favour early diagnosis. Polymerase chain reaction (PCR) is known to diagnose tuberculosis quickly. With this in mind, we used PCR to detect mycobacterial DNA on formalin-fixed, paraffin-embedded tissues with acid-fast stain and culture negativity in two dialysis patients with cervico-mediastinal lymphadenopathy. Methods: Sections of neck lymph nodes were cut at two different levels. At each level, two semi-adjacent sections with a thickness of 5 &mgr;m each were cut using standard microtomes with disposable blades. The first section mounted on a glass slide was stained b Ziehl-Neelsen, and the second section was examined by PCR based on a 123 bp fragment of IS6110 that is specific for the Mycobacterium tuberculosis complex. Results: The histology of lymph nodes disclosed inflammatory necrotizing granulomas, but acid-fast stain for M. tuberculosis was negative in the two patients. DNA of M. tuberculosis was detected in lymph node samples from each patient by PCR on the IS6110 element and by dot-blot hybridization. Conclusions: PCR assay is a potentially useful approach for early and rapid diagnosis of tuberculous lymphadenitis in chronic dialysis patients, since mycobacterial staining and culture often provide negative results.     

Clinical usefulness of plasma quantitative polymerase chain reaction assay: diagnosis of cytomegalovirus infection in kidney transplant recipients

Background

Preemptive therapy is used to prevent cytomegalovirus (CMV) disease in transplant recipients. The CMV antigenemia assay, which has been commonly used as a predictive marker for preemptive therapy, requires intensive labor and immediate processing. We compared the cutoff value of plasma CMV polymerase chain reaction (PCR) with CMV antigenemia in kidney transplant recipients.

Methods

We compared two diagnostic methods for CMV infection in kidney transplant recipients: quantitative PCR (qPCR) versus antigenemia. We evaluated the optimal cutoff value of plasma CMV qPCR by using receiver-operating characteristic curves for specific antigenemia values. All kidney transplant recipients from January 2004 to January 2005 were enrolled and followed with CMV antigenemia and plasma CMV qPCR.

Results

The analyses were performed on 899 samples collected from 111 patients in the early posttransplant period, matching 84.1% of patients for the results of CMV antigenemia and plasma CMV qPCR. For patients with symptomatic CMV infection and disease, who showed ≥25 positive cells in the antigenemia assay, the cutoff value for qPCR was 17.8 copies/μL with a sensitivity of 97.1%, a specificity of 89.1%, and a positive predictive value of 26.6%.

Conclusions

Diagnostic assays for CMV such as CMV antigenemia and quantitative plasma PCR, showed similar diagnostic values. They are the methods of choice for the diagnosis and monitoring of active CMV infection after kidney transplantation. However, because of the relatively low positive predictive value of qPCR, this test may lead to unnecessary preemptive treatment in kidney transplant recipients.     

The use of the polymerase chain reaction test in the diagnosis of tuberculosis

Current techniques for laboratory diagnosis of tuberculosis have some serious limitations. These include the high cost and time required for the current assays. The development of a rapid, sensitive, specific and low-cost assay is therefore of considerable importance. We report here the development and laboratory testing of a polymerase chain reaction DNA-based diagnostic test for the presence of Mycobacterium tuberculosis in sputum. The assay shows a high level of sensitivity and specificity and requires considerably less capital, consumables and time inputs than existing laboratory tests. We believe this technology is ready for large-scale evaluation and use, particularly in hospital-based laboratories.     

Use of polymerase chain reaction in diagnosis of occult tuberculosis of the fibula

An unusual case of tuberculosis of the lower end of the fibula in a young patient is reported. The patient presented with symptoms of pain and swelling over the outer aspect of the right ankle with full range of painless ankle movements. The plain radiographs of the ankle were normal but MRI scan showed increased signals within the lower end of the fibula on T2-weighted images. The histology of the lesion showed only a few Langhans giant cells and culture failed to grow any organism. Polymerase chain reaction analysis of the biopsy specimen, however, showed growth of Mycobacterium tuberculosis. The patient responded to antitubercular treatment with complete resolution of symptoms. Polymerase chain reaction analysis should be considered in atypical presentations with bone pain to rule out an occult infectious pathology.     

Rapid diagnosis of donor infection and renal allograft contamination using a polymerase chain reaction technique and rapid shaking culture

Post transplant infection is one of the serious complications of the organ recipients. We detected the donor infections and allograft contaminations in a limited period of time by polymerase chain reaction (PCR) and rapid shaking culture (RSC). The pre-procurement blood from 86 possible renal donors as well as the preservation solution (PS) and renal pelvic urine (PU) from 158 grafts were examined in order to detect highly virulent organisms such as methicillin resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and fungi. The average age of donors was 48.8 years old. The average period between the admission and brain death was 4.9 days, and the average period between the brain death and cardiac arrest was 3.6 days. The inflammatory indices such as body temperature, white blood cell count and C-reactive protein increased to 37.9 +/- 1.1 degree C, 12,600 +/- 6300/microl, and 20.2 +/- 11.6 mg/dl, respectively. Following PCR and RSC, procurement operations of the three possible donors were cancelled because of systemic bacterial infections by MRSA or Bacteroides fragilis. Six out of 158 grafts were discarded due to the diagnoses of MRSA or Candida albicans in PS and/or PU. All the other 148 grafts were transplanted. Following transplant, no single infectious complication transmitted by the graft was noted. We conclude that PCR combined with RSC is very accurate and useful for detecting donor infections and allograft contaminations, which may cause severe complications in the recipients.     

Rapid sensitive molecular diagnosis of pyogenic spinal infections using methicillin-resistant Staphylococcus-specific polymerase chain reaction and 16S ribosomal RNA gene-based universal polymerase chain reaction

Background contextRapid diagnosis and accurate detection of etiological agents in pyogenic spinal infection (PSI) patients are important.PurposeThe purpose of this study was to evaluate the clinical usefulness of methicillin-resistant Staphylococcus-specific polymerase chain reaction (MRS-PCR) and broad-range universal PCR (U-PCR) for diagnosing PSI.Study designA prospective diagnostic study.PatientsThirty-two clinically suspect PSI patients and six control patients who underwent computerized tomography–guided biopsy and/or surgical treatment were enrolled.MethodsTissue samples were examined by microbiological culture, histopathology, and real-time PCR (MRS-PCR and U-PCR). The diagnostic accuracy of real-time PCR was analyzed based on the definitive diagnosis of infection, defined as a positive result from microbiological culture or histopathology.ResultsAll six control subjects were negative for PSI for all analyses. Twelve clinically suspect PSI subjects received definitive diagnoses (PSI group). The non-PSI group consisted of six control subjects plus the remaining 20 patients from the PSI clinically suspect group. MRS-PCR results were positive for all MRS-cultured PSI subjects. U-PCR was positive for all subjects in the PSI group with one discrepancy between real-time PCR and microbiological culture results in differentiation between gram-positive and gram-negative bacteria. In the non-PSI group, MRS-PCR and U-PCR were positive in three and seven cases, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value of MRS-PCR for diagnosing MRS infection were 1.00, 0.91, 0.57, and 1.00, respectively; those for the diagnosis of bacterial infection with U-PCR were 1.00, 0.73, 0.63, and 1.00, respectively.ConclusionIdentification of MRS infection and ability to differentiate between gram-positive and gram-negative bacteria is rapidly achieved using MRS-PCR and U-PCR. Real-time PCR provides a sensitive molecular diagnosis of PSI and may contribute to antibiotic selection.     

Comparison of polymerase chain reaction amplification of two mycobacterial DNA sequences, IS6110 and the 65kDa antigen gene, in the diagnosis of tuberculosis.

BACKGROUND: Knowledge of the sequences of mycobacterial genes and the availability of DNA amplification techniques have raised the possibility that identification of mycobacterial DNA may offer a rapid and specific diagnostic test for tuberculosis. The correlation between the presence of Mycobacterium tuberculosis DNA and clinical tuberculosis, however, is not known. This study compared the results of polymerase chain reaction amplification of two M tuberculosis DNA sequences, IS6110 and the gene encoding the 65kDa heat shock protein (65kDa Ag), from sputum, bronchoscopy washings, and bronchoalveolar lavage fluid and related these findings to the presence of active and past tuberculosis. METHODS: Highly specific primers were used for amplification of IS6110 and 65kDa Ag DNA. Analysis was performed on one or more samples from 87 patients. RESULTS: IS6110 DNA was identified in samples from all six patients with active tuberculosis, from 15 to 18 patients with past tuberculosis, from five of nine contacts of patients with tuberculosis, and from nine of 54 patients with lung disease unrelated to tuberculosis. The 65kDa Ag DNA was identified in samples from all patients with active and past tuberculosis, from contacts of patients with tuberculosis, and from 14 of 42 patients with non-tuberculous lung diseases. CONCLUSION: These data suggest that the presence of IS6110 DNA correlates more closely with a tuberculosis related diagnosis than that of 65kDa Ag DNA and that both DNAs are found in most subjects with past tuberculosis or contacts of patients with tuberculosis. This may limit the clinical usefulness of these tests.     

Early diagnosis of polyomavirus type BK infection in tailoring immunosuppression for kidney transplant patients: screening with urine qualitative polymerase chain reaction assay

Polyomavirus type BK (BKV) nephropathy is increasingly a significant cause of graft dysfunction and even failure. Early diagnosis followed by reduction of immunosuppression has been associated with an improved prognosis. We screened 250 patients with the urine qualitative polymerase chain reaction (PCR) for BKV DNA. We followed with blood BKV PCR if the urine screen was positive and then reduced immunosuppression in viremic patients. One hundred ninety-nine patients (80%) had no viuria; 43 (17%) viuria; and 8 (3%) both viuria and viremia. Graft biopsy performed in three patients (1%) with viremia and impaired graft function all revealed BKV nephropathy. After 6 months of follow-up, seven out of eight viremic patients (88%) had negative repeat blood PCR and stabilized graft function. An early diagnosis of BKV infection with reduction of immunosuppression may reverse viremia and retard progression of BKV nephropathy. BKV screening by PCR assays should be considered in kidney transplant recipients, especially those with impaired graft function.     

Rapid diagnosis of genitourinary tuberculosis by polymerase chain reaction and non-radioactive DNA hybridization

OBJECTIVE: To establish a polymerase chain reaction (PCR) assay for the rapid detection and identification of mycobacteria in urine, and to assess the value of such assay in routine laboratory diagnosis of genitourinary tuberculosis. MATERIALS AND METHODS: Urine specimens from 1000 patients with clinical suspicion of urinary tuberculosis were examined. Two assays for the detection and identification of Mycobacterium tuberculosis (M. tuberculosis) complex and mycobacteria other than tuberculosis (MOTT) by non-radioactive DNA hybridization of PCR-product were applied. The first assay used PCR primers and probe derived from M. tuberculosis species-specific DNA insertion sequence, IS6110. The second utilized mycobacterium genus-specific sequence encoding ribosomal ribonucleic acid (16S rRNA). The results obtained by PCR were compared with those obtained by standard microbiological methods, acid-fast bacilli (AFB) stain and culture. RESULTS: Compared with cultures, the sensitivity of AFB staining was 52.07% and the specificity was 96.7%. In comparison to the results of culture, the overall sensitivity and specificity of the IS6110-PCR assay was 95.59% and 98.12% respectively. While the corresponding results for the 16S rRNA gene-PCR were 87.05% and 98. 9%. CONCLUSION: The high sensitivity and specificity in addition to the potential for rapid detection of mycobacteria, makes this test a useful tool in the clinical management of mycobacterial infection in urine. Urine specimens may contain M. tuberculosis and/or other mycobacteria; therefore, there are advantages in using genus-specific primers in parallel with species-specific primers in PCR assay.