Risk-factors and predictors of mortality in patients colonised with vancomycin-resistant enterococci.

Vancomycin-resistant enterococci (VRE) have emerged as significant nosocomial pathogens. A hospital-wide prevalence study was performed to identify cases with VRE faecal colonisation. A case-control study using two randomly selected VRE-negative controls for each positive case was performed to assess risk-factors for VRE colonisation by univariate and multivariate analysis. VRE faecal colonisation was documented in 53 (14.3%) of 370 patients screened. Previous exposure to anti-anaerobic agents, as well as quinolones, was associated with VRE colonisation (p <0.05). The presence of an invasive device (OR 4.8, p 0.003) and the duration of any antimicrobial treatment before VRE isolation (OR 1.2, p <0.001) predicted VRE colonisation in multivariate models. The crude mortality rate for patients with VRE colonisation was 24.5%, but VRE colonisation was not an independent predictor of mortality in these patients. These results suggest that an active surveillance programme focusing on specific patient groups may help in the identification of VRE-colonised patients. Promptly implemented infection control strategies targeting these groups should help to combat the rising incidence of VRE.     

聚合酶链反应检测淋菌的结果分析

本文应用聚合酶链反应(PCR)技术对3853例疑为淋菌感染患者进行了检测。检出阳性者961例，阳性率24．9％，对照培养法576例．检出阳性者38例，阳性率6．6％。结果表明PCR法具有高度特异性、敏感性和选择性．且快速、简便．是临床处理大批标本的一种最理想的方法．适于临床推广。     

The polymerase chain reaction in histopathology

Thanks to the advent of the polymerase chain reaction (PCR) molecular genetic study of histological samples is now a relatively straightforward task and the vast histopathology archives are now open to molecular analysis. In this review we outline technical aspects of PCR analysis of histological material and evaluate its application to the diagnosis and study of genetic, infectious and neoplastic disease. In addition, we describe a number of newly developed methods for the correlation of PCR analysis with histology, which will aid the understanding of the molecular basis of pathological processes.     

Determination of hepatitis C virus genotypes by melting-curve analysis of quantitative polymerase chain reaction products

Hepatitis C virus (HCV) is the major causative agent of non-A and non-B viral hepatitis. Factors associated with disease progression following HCV infection include the viral genotype, the patient's alcohol consumption and viral load. In this study, the COBAS AMPLICOR HCV MONITOR test, a commercially available quantitative assay for HCV RNA, was used for HCV genotyping analysis. Amplification products obtained from 100 HCV-positive cases were subjected to real-time polymerase chain reaction (PCR) typing using a single pair of fluorescence resonance energy transfer (FRET) probes and melting-curve analysis. Of 100 samples tested, two inhibited the PCR, two samples yielded discrepancies between our results and the reference laboratory results and the remaining samples provided correct typing. The present report suggests that HCV genotypes can be determined rapidly with FRET probes directly from COBAS AMPLICOR MONITOR test PCR products.     

Increased detection of cutaneous leishmaniasis in Norway by use of polymerase chain reaction

Cutaneous leishmaniasis (CL) is a parasitic infection and occurs in tropical and subtropical regions worldwide and in the region of the Mediterranean Sea. The diagnosis is based on the clinical appearance and biopsy findings that may be supplemented with polymerase chain reaction (PCR). In this study 20 cases were selected if (i) a histopathological diagnosis of granulomatous dermatitis was made, (ii) CL was taken into consideration or (iii) the diagnosis was CL. PCR analysis with primers specific for leishmania was performed on archived histological specimens and was positive in 6 of the 20 cases. In two cases both the clinical and histopathological diagnosis concurred with CL. In the remaining four cases a clinical diagnosis other than CL was made. In two of these cases the histopathology showed granulomatous dermatitis, and detection of parasites led to consideration of CL. In the last two cases leishmaniasis was not taken into consideration by clinicians or pathologists. Our study shows that CL may occur more often than anticipated in Norway, but clinicians do not consider the diagnosis as often as they should. Pathologists may also fail to diagnose or suggest CL especially when parasites are not visualized in the histopathological specimen.     

Evaluation of the polymerase chain reaction for the detection of human papillomavirus from urine

The ability to detect the presence of human pap-illomavirus (HPV)-DNA sequences in urine was evaluated using polymerase chain reaction (PCR). DMA was purified and extracted from urine samples, and subjected to 40 cycles of amplification using the consensus primer pair MY11 and MY09. Coamplification using the β-globin primers, GH20 and PC04, was performed as an internal reaction control. Following assay optimization, urine samples from 22 women undergoing examination for cervical dysplasia were tested for the presence of HPV-DNA. PCR assay results were correlated with cytologic and histo-logic findings as well as Vira Type(tm) assay results. Overall, HPV was detected by PCR in 16 (76%) of the interpretable samples. HPV sequences were detected in 13 (87%) of the 15 specimens from women showing evidence of condylomata, dysplasia, or invasive carcinoma. HPV was detected in 3 (50%) of the women whose cytologic or his-tologic results were either negative or showed benign atypia. Although the sample size in this study is small, our results show that HPV can be detected by PCR in a majority of individuals showing evidence of HPV infection. The method described provides a means for the clinical laboratory to detect a broad range of HPV types from using a sample obtained by noninvasive techniques. The ability to easily obtain urine would allow for increased numbers of individuals to be tested, and thus, aid in our understanding of HPV. © 1995 Wiley-Liss, Inc.     

A nested polymerase chain reaction for detection of Legionella pneumophila in clinical specimens

Objective: Because presently used methods for diagnosis of Legionella pneumonia lack sufficient sensitivity and sometimes specificity and rapidity, the detection of Legionella spp. by amplification of nucleic acids might be valuable. However, performing polymerase chain reaction (PCR) on clinical samples such as sputum is difficult because of the presence of extraneous DNA and inhibitors of the reaction. An attempt to circumvent these problems was made.
Method: A nested PCR method was devised using primers from the mip gene of Legionella pneumophila. This PCR was tested on pure cultures of legionellae and clinical isolates of other bacteria. Clinical samples (bronchoalveolar lavage fluid, bronchial aspirate and sputum) from patients who suffered from legionellosis and samples from patients who suffered from other causes of pneumonia were also tested.
Results: The PCR was specific for L. pneumophila and no non- Legionella bacteria reacted. Ten to 50 colony forming units of Legionella in the sample could be detected. Twenty-two of 25 clinical samples were positive among patients suffering from pneumonia proven to be due to L. pneumophila serogroups 1, 3, 4, 5 and 6. Two of the three negative samples were from patients who had been treated with adequate therapy for at least 2 days and were culture negative. However, nine other culture-negative samples were PCR positive, of which seven came from patients who had been treated for 3–7 days. All pneumonia patients in the control group proved negative in PCR. A commercial kit for DNA preparation from clinical samples, based on absorption of nucleic acids to silica gel, was superior to the traditional phenol/chloroform extraction and increased the rapidity, simplicity and sensitivity of the procedure.
Conclusions: A nested, simplified and rapid PCR method using mip primers proved to be more sensitive than culture and as sensitive and specific as other PCR procedures previously reported.     

The utility of IS6110 sequence based polymerase chain reaction in comparison to conventional methods in the diagnosis of extra-pulmonary tuberculosis

IS6110 sequence based polymerase chain reaction (PCR) was compared with conventional bacteriological techniques in the laboratory diagnosis of extra-pulmonary tuberculosis (EPTB). One hundred and ninety one non-repeated clinical samples of EPTB and 17 samples from non-tuberculous cases as controls were included. All the samples were processed for Ziehl-Neelsen staining for acid fast bacilli (AFB) and 143 samples were processed by culture for M. tuberculosis . All the samples were processed for PCR amplification with primers targeting 123 bp fragment of insertion element IS6110 of M. tuberculosis complex. Of the total 191 samples processed, 34 (18%) were positive by smear for AFB. Culture for AFB was positive in 31(22%) samples among the 143 samples processed. Either smear or culture for AFB was found positive in 51(27%) samples. Of the total 191 samples processed 120 (63%) were positive by PCR. In 140 samples, wherein both the conventional techniques were found negative, 74 (53%) samples were positive by PCR alone. Among 51 samples positive by conventional techniques, 46 (90%) were found positive by PCR. PCR assay targeting IS6110 is useful in establishing the diagnosis of EPTB, where there is strong clinical suspicion, especially when the conventional techniques are negative.     

Vancomycin-resistant enterococci colonization in patients with hematological malignancies: screening and its cost-effectiveness

Background and objective

We evaluated the rates of vancomycin-resistant enterococci (VRE) colonization and VRE-related bacteremia in patients with hematological malignancies in terms of routine screening culture and its cost-effectiveness.

Materials and Methods

All patients of the hematology department who were older than 14 years of age and who developed at least one febrile neutropenia episode during chemotherapy for hematological cancers between November 2010 and November 2012 were evaluated retrospectively.

Results

We retrospectively analyzed 282 febrile episodes in 126 neutropenic patients during a two-year study period. The study included 65 cases in the first study-year and 78 cases in the second study-year. The numbers of colonization days and colonized patient were748 days of colonization in 29 patients (44%) in the first study-year and 547 colonization days in 21 patients (26%) in the second study-year, respectively. Routine screening culture for VRE cost $4516,4 (427 cultures) in the first study-year, $5082,7 (504 cultures) in the second study-year depending on the number of patients and their length of stay.

Conclusion

In line with our study results, routine screening of hematological patients for VRE colonization is not costeffective. Routine surveillance culture for VRE should be considered with respect to the conditions of health care setting.     

解脲脲原体套式（Nested）PCR检测研究

本文报告解脲脲原体（ＵＵ）的套式（ＮＥＳＴＥＤ）ＰＣＲ检测方法。经方法学考核表明，本法的特异性、灵敏度以及试剂的稳定性和对临检标本的顺应性均较好。９３份各种生殖道炎症患者之宫颈拭子标本套式ＰＣＲ检出２１份阳性（阳性率２２．６％），而市售ＰＣＲ试剂盒仅检出１份阳性（阳性率１．１％）。前者阳性检出率明显高于后者（Ｐ＜０．０１）。     

Comparison of in-house polymerase chain reaction method with the Roche Amplicor technique for detection of Mycobacterium tuberculosis in cytological specimens

Two techniques have been approved by the United States FDA for diagnosis of tuberculosis in smear positive sputa: LCX M. tuberculosis, a ligase chain reaction procedure manufactured by Abbott Laboratories, and Amplicor, a polymerase chain reaction (PCR) procedure manufactured by Roche. However, these commercial methods are expensive and beyond the reach of laboratories in most developing countries. We compared the Roche Amplicor kit with an in-house PCR using a primer set for Mycobacterium tuberculosis/bovis directed at MPB 64 protein gene. It was able to distinguish between M. tuberculosis, M. avium, M. gordonii, M. intracellularae, and M. kansasii. Fifty-seven cytological samples were submitted to the laboratory for molecular diagnosis of M. tuberculosis. Both procedures were run on every sample submitted and the two methods agreed completely. The custom-made method is less expensive than the commercial technique.     

应用定量PCR方法快速基因诊断Down综合征

目的探讨用定量聚合酶链反应方法对Ｄｏｗｎ综合征进行基因诊断。方法以短串联重复序列（Ｄ２１Ｓ１１）作为遗传标记,合成特异引物,用同位素标记聚合酶链反应扩增后对１１名正常人（６例外周血,５例羊水）及２８例Ｄｏｗｎ综合征患者（外周血）进行定量检测。结果１１名正常人中１０人出现ＤＮＡ含量为１∶１关系的２条电泳带,１人为１条带。２８例患者中２４人出现ＤＮＡ含量２∶１的２条带,３人为ＤＮＡ含量为１∶１∶１的３条带,１人为１条带。结论Ｄ２１Ｓ１１位点短串联重复序列多态是对Ｄｏｗｎ综合征基因诊断很有应用价值的遗传标记,应用定量聚合酶链反应方法可在２４小时内对Ｄｏｗｎ综合征做出快速、准确的产前及临床基因诊断。     

Effectiveness of rapid multiplex polymerase chain reaction for early diagnosis and treatment of pertussis

BackgroundPertussis, is an infectious respiratory disease caused by Bordetella pertussis. The incidence of pertussis has been increasing in South Korea to due to waning vaccine-induced immunity. Culture has a low sensitivity and a long turnaround time (TAT). Recently, a rapid multi-polymerase chain reaction (mPCR) test with a TAT of about 1 h was developed for the detection of respiratory pathogens (17 viruses and three bacteria), including B. pertussis. This study aimed to investigate the effectiveness of mPCR for early diagnosis and treatment of pertussis.MethodsWe performed a retrospective study of patients with pertussis diagnosed from May 2017 to June 2019 at a university hospital in South Korea. Nasopharyngeal swab specimens were tested using mPCR. Data were extracted from medical records.ResultsA total of 27 patients with a median age of 48.9 years (range: 3.3–82.2 years) were diagnosed with pertussis, of whom 9 (33.3%) were male. Eleven (40.7%) had fever, 12 (44.4%) had dyspnea, three (11.1%) had paroxysmal cough, and nine (33.3%) had inspiratory whooping. The median interval from symptom onset to diagnosis was 9.0 days (range: 1–31 days). Twenty-four patients (81.5%) were diagnosed within 2 weeks from symptom onset. All but one patient was prescribed macrolide antibiotics. Twenty-two patients (81.5%) required hospitalization, including three (11.1%) who required intensive care unit care for ventilation.ConclusionTesting patients with respiratory symptoms using mPCR can improve early diagnosis of pertussis, ensure proper treatment, and may help with outbreak control.     

Comparison of the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide tube method with the conventional method and real-time polymerase chain reaction for the detection of rifampicin resistance in Mycobacterium tuberculosis

Colorimetric methods are cheap, reproducible, and rapid methods of detecting drug resistance in Mycobacterium tuberculosis. The MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) method is one such technique that has been established in our laboratory to detect rifampicin resistance. The present study compared the results of the MTT method with those of the proportion method and real-time polymerase chain reaction (RTPCR) in order to establish sensitivity and specificity of MTT. The mutations for rifampicin resistance occur in rpoB gene, and the commonest reported are in codons 526 and 531. Therefore, RTPCR was targeted at these two codons. The concordance of MTT with the proportion method and RTPCR was 94 and 72.77%, respectively, and that of RTPCR with the proportion method was 77.77%. While the study confirmed that the MTT method is a good method for detecting rifampicin resistance, it also brought out the fact that RTPCR when targeted for limited mutations is not a good tool. Either the genotypic method used should target the total 81-bp rpoB genome or methods such as DNA sequencing should be used. For resource-constraint laboratories, the MTT method can be considered as a better choice.     

Detection of HPV DNA in trichilemmomas by polymerase chain reaction

Paraffin sections of 11 formalin-fixed trichilemmomas were investigated for the presence of human papillomavirus (HPV) DNA by the polymerase chain reaction (PCR) with the degenerated consensus primer pairs. PCRs were conducted with different annealing temperatures. When the annealing temperature was reduced from 55°C to 50°C, amplification products of the expected size were obtained for all 11 cases investigated. Determination of the HPV type was performed by cloning and sequencing of the amplification products. The sequence analysis of the eleven cloned amplicons gave the following data: based on sequence comparison with published amino acid sequences, the best homology was found to epidermodysplasia verruciformis (EV)-associated HPVs (supergroup B). In four specimens an HPV type 23 related type was found; five specimens contained HPV sequences which did not match with one of the known HPV types, but had the closest homology to HPV types 15, 17, and 37. Three of the HPV variants which had not been characterised, displayed identical sequences. Two additional HPV amplification fragments displayed 100% homology to HPV-6b. These results demonstrate, for the first time, the presence of HPV DNA in trichilemmomas. The sequence data suggest that HPV variants or types in trichilemmoma are members of the EV-associated HPV supergroup B. J Med Virol 51:119–125, 1997. © 1997 Wiley-Liss, Inc.     

Evaluation of the Roche Amplicor polymerase chain reaction assay for detection of enteroviruses in cerebrospinal fluid and its potential impact on patient management

Objective: To evaluate the Roche Amplicor polymerase chain reaction assay (APCR) by comparing the detection of enteroviruses from cerebrospinal fluid (CSF) by the Roche assay with detection by viral culture and to determine whether routine use of enteroviral PCR will affect patient management.
Methods: One hundred and sixty-three CSF specimens were tested by APCR and viral culture. Some of the discrepant specimens were resolved by retesting with an in-house PCR assay. Other discrepant results were resolved by testing the patients' serum by APCR or by viral culture of throat and stool specimens.
Results: Thirty CSF specimens were positive by APCR, and 18 of these were positive by viral culture. There were no APCR-negative, viral-culture-positive CSF specimens. Six of the 12 discrepant specimens were resolved as true positives.
Conclusions: The APCR assay was more rapid and sensitive than viral culture for detection of enteroviruses from CSF. Routine use of this assay has the potential to reduce the amount of antibiotics used and the number of patient days spent in hospital.