Comparison of multiplex PCR assay with culture for detection of genital mycoplasmas

Ureaplasma, spp. Mycoplasma genitalium, and Mycoplasma hominis are associated with infection of the genitourinary tract, reproductive failure, and neonatal morbidity and mortality. We have developed a multiplex PCR for the detection of these Mycoplasmatales in a single amplification reaction. The analytical sensitivities of this assay were 10.8, 10.8, and 8.8 CFU for each organism, respectively. This multiplex PCR was compared to culture on 26 cervical swabs, 2 vaginal swabs, 4 female urine specimens, 49 semen samples, 2 male urine specimens, and 1 nonspecified sample. A total of 21 specimens were culture positive (25%); 17 of these were PCR positive. An additional 11 specimens were PCR positive but culture negative. Of the 21 culture-positive specimens, 17 (81%) grew Ureaplasma spp. and 4 (19%) grew Mycoplasma spp. Of the 28 PCR-positive specimens, Ureaplasma spp. was detected in 23 (82%), M. hominis was detected in 3 (11%), and both were detected in 2 (7%). In a confirmatory analysis, all samples were tested by amplification of a second target of the ureaplasma genome. True-positive cases were defined as a positive result by culture or by both amplification assays. The multiplex PCR detected organisms in 26 of the 30 true-positive specimens, as well as in 2 other specimens. Based on a 36% prevalence of infection, the sensitivity, specificity, and positive and negative predictive values of multiplex PCR analyses were 87, 96, 94, and 93%, respectively. Multiplex PCR offers a rapid, sensitive, and easy method to detect genital mycoplasmas.     

Detection of vancomycin-resistant enterococci in fecal samples by PCR.

Surveillance cultures for vancomycin-resistant enterococci (VRE) are time-consuming and expensive for the laboratory to perform. Therefore, we investigated the use of PCR as an alternative method of detecting and identifying VRE directly in fecal samples. PCR primers directed to vanA, vanB, vanC1, vanC2, and enterococcal ligase genes were used to detect and identify VRE in fecal material obtained by rectal or perirectal swabbing. Although PCR-inhibitory substances were present in DNA prepared directly from the swabs, the inhibitory substances could be reduced by processing the nucleic acid with two commercially available DNA preparation columns. Fecal material from 333 swabs was cultured on several selective agar media before and after broth enrichment. DNA was extracted from the fecal material and was analyzed by PCR. By using all four primer sets, only 59 (67.8%) of the samples were positive for vanA. However, after retesting the negative samples with only the vanA primer set, 77 (88.5%) of 87 specimens that were culture positive for Enterococcus faecium containing vanA were positive by PCR. One specimen was PCR positive for the vanA gene but culture negative for enterococci. The specificity of the vanA assay was 99.6%. PCR analysis of enrichment broth samples with all four primers sets after 15 to 18 h of incubation detected 74 (85.1%) of the 87 culture-positive specimens. The specificity of the vanA assay after the enrichment step was 100%. No vanB-containing enterococci were recovered by culture. Since 16 samples can be tested by PCR in 4 h (including electrophoresis), identification of VRE is possible within 8 h of specimen submission at a cost of approximately $10.12/assay. Thus, PCR may be a cost-effective alternative to culture for surveillance of VRE in some hospitals.     

Detection and typing of common human papillomaviruses among Jordanian patients

The epidemiology of human papillomaviruses (HPVs) genotype distribution of cutaneous warts in Jordanian patients were studied. A total of 200 samples were collected using skin swabs from patients with warts attending the dermatology clinic at the Jordan University Hospital over the period of June 2010 to October 2010. Another 100 control samples were taken from healthy Jordanian individuals with no current or previous history of warts. DNA extraction and sequencing was carried out using PCR with the FAP primer pair to detect HPV DNA, followed by multiple‐type‐specific (Multiplex) PCR combined with DNA sequencing. The prevalence of HPV among Jordanian patients tested with warts diagnosed clinically was 82% (157/192); of these 45% (87/192) were detected by FAP PCR method, and 37% (70/192) were detected by multiplex PCR method. Sequencing of the FAP positive samples shows that HPV 2 was associated with the highest prevalence (36%), followed by HPV 27 (28%) and HPV 57 (21%). A total of 6% of healthy persons were positive for HPV DNA. In conclusion, this study demonstrates that alpha HPV types (HPV 2, HPV 27, and HPV 57) are associated with the most prevalent cutaneous warts in Jordanian patients J. Med. Virol. 85: 1058–1062, 2013. © 2013 Wiley Periodicals, Inc.     

Comparison of broad-range bacterial PCR and culture of cerebrospinal fluid for diagnosis of community-acquired bacterial meningitis

Appropriate, rapid and reliable laboratory tests are essential for the diagnosis and optimal antibiotic therapy of acute bacterial meningitis. Broad-range bacterial PCR, combined with DNA sequencing, was compared with culture-based methods for examining cerebrospinal fluid (CSF) samples from patients with suspected meningitis. In total, 345 CSF specimens from 345 patients were analysed, with acute community-acquired bacterial meningitis being diagnosed in 74 patients. The CSF of 25 patients was positive by both PCR and culture; 26 patients had CSF specimens positive by PCR only, and 14 patients had specimens positive by culture only. The sensitivity of PCR and culture for clinically relevant meningitis was 59% (44/74) and 43% (32/74), respectively, while the specificity was 97% (264/271) and 97% (264/271), respectively. The commonest bacterial rRNA gene sequences detected by PCR only were those of Streptococcus pneumoniae and Neisseria meningitidis (n = 12). PCR failed to detect the bacterial rRNA gene in seven specimens from patients with symptoms compatible with acute bacterial meningitis. Overall, the results demonstrated that PCR in conjunction with sequencing may be a useful tool in the diagnosis of bacterial meningitis. PCR is particularly useful for analysing CSF from patients who have been treated with antibiotics before lumbar puncture.     

Quantitative real-time PCR for detection of Acinetobacter baumannii colonization in the hospital environment

A real-time PCR assay was developed for detecting the presence of Acinetobacter baumannii on hospital equipment and compared to conventional bacterial culture using 100 hospital environmental samples. The real-time PCR detected contaminated surfaces in 4 h with high sensitivity (100%) compared to conventional culture. Thirty-eight percent of samples were positive by real-time PCR and negative by bacterial culture (false positives), possibly indicating the widespread presence of bacterial DNA that is not associated with viable bacteria.     

Utility of the polymerase chain reaction in the diagnosis of tuberculous meningitis

Due to inconsistent clinical presentations and the lack of a rapid, sensitive and specific test, tuberculous meningitis (TBM) is particularly difficult to diagnose. The present study was carried out to determine the utility of the polymerase chain reaction (PCR) using INS primers in the diagnosis of TBM and to compare the efficacy of two different DNA extraction protocols. Fifty-seven cerebrospinal fluid (CSF) samples from suspected cases of meningitis -- 30 definitive/possible TBM and 27 non-TBM -- were processed for microscopy, culture and PCR. Results of computer tomographic (CT) scan findings were noted. The results of smear, culture and PCR were compared using culture and/or clinical response to treatment as the gold standard. The sensitivity of microscopy, culture, CT scan and PCR was 3.3%, 26.7%, 60.0% and 66.7%, respectively. PCR following QIAmp DNA extraction had a sensitivity of 66.7% compared to PCR following a DNA extraction protocol based on the use of cetyl trimethyl ammonium bromide (CTAB) (50%). PCR was positive in all culture-positive CSF samples using either extraction method. PCR is a rapid and sensitive technique; above all, it can diagnose tuberculous meningitis at a very early stage.     

Laboratory Diagnosis of Common Herpesvirus Infections of the Central Nervous System by a Multiplex PCR Assay

A sensitive multiplex PCR assay for single-tube amplification that detects simultaneous herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (CMV), and Epstein-Barr virus (EBV) is reported with particular emphasis on how the method was optimized and carried out and its sensitivity was compared to previously described assays. The assay has been used on a limited number of clinical samples and must be thoroughly evaluated in the clinical context. A total of 86 cerebrospinal fluid (CSF) specimens from patients which had the clinical symptoms of encephalitis, meningitis or meningoencephalitis were included in this study. The sensitivity of the multiplex PCR was determined to be 0.01 and 0.03 50% tissue culture infective doses/the reciprocal of the highest dilution positive by PCR for HSV-1 and HSV-2 respectively, whereas for VZV, CMV and EBV, 14, 18, and 160 ag of genomic DNA were detected corresponding to 48, 66, and 840 genome copies respectively. Overall, 9 (10.3%) of the CSF samples tested were positive in the multiplex PCR. HSV-1 was detected in three patients (3.5%) with encephalitis, VZV was detected in four patients (4.6%) with meningitis, HSV-2 was detected in one neonate (1.16%), and CMV was also detected in one neonate (1.16%). None of the samples tested was positive for the EBV genome. None of the nine positive CSF samples presented herpesvirus coinfection in the central nervous system. Failure of DNA extraction or failure to remove any inhibitors of DNA amplification from CSF samples was avoided by the inclusion in the present multiplex PCR assay of alpha-tubulin primers. The present multiplex PCR assay detects simultaneously five different herpesviruses and sample suitability for PCR in a single amplification round of 40 cycles with an excellent sensitivity and can, therefore, provide an early, rapid, reliable noninvasive diagnostic tool allowing the application of antiviral therapy on the basis of a specific viral diagnosis. The results of this preliminary study should prompt a more exhaustive analysis of the clinical value of the present multiplex PCR assay.     

An optimized PCR leads to rapid and highly sensitive detection of Borrelia burgdorferi in patients with Lyme borreliosis.

The present study aimed at developing an optimized PCR protocol fro the sensitive and specific detection of all three Borrelia burgdorferi genospecies pathogenic to humans in Lyme borreliosis patients. A rapid DNA extraction method using alkaline lysis was introduced and was found to be superior to other DNA extraction methods. Nested PCR was performed with primer sets targeting the plasmid-located ospA gene and a chromosomal gene segment encoding a 66-kDa protein (p66). In spiked synovial fluid (SF) fewer than three borreliae/sample were detected. The specificities of the amplicons were confirmed by Southern blot analysis with PCR-derived probes. Urine, cerebrospinal fluid (CSF), and SF specimens from 57 patients with Lyme borreliosis and from 58 controls were examined. In clinical samples the diagnostic sensitivity of PCR was 85% with SF samples, 79% with urine samples, and 91% with paired SF-urine samples from patients with Lyme arthritis and was 79% with CSF samples, 45% with urine samples, and 87% with paired CSF-urine specimens from neuroborreliosis patients. One patient each with neuroborreliosis and with Lyme arthritis had PCR-positive urine samples only. In 17% of all cases both primer sets yielded positive results, while the other patients were positive with only one primer set. Among these, more positive results were obtained with the p66 gene primer than with the ospA primer. The specificity exceeded 99%. We conclude that DNA from B. burgdorferi sensu lato species can sensitively and specifically be detected with the optimized PCR method described. At least two different primer sets should be used, and whenever possible, urine and CSF or SF should be analyzed in parallel to achieve maximum sensitivity of the test. This protocol, therefore, considerably enhances the diagnostic power of PCR in patients with B. burgdorferi infection.     

Acanthamoeba DNA can be directly amplified from corneal scrapings

This study evaluated the performance of direct amplification of Acanthamoeba-DNA bypassing DNA extraction in the diagnosis of Acanthamoeba keratitis in clinically suspected cases in comparison to direct microscopic examination and in vitro culture. Corneal scrapings were collected from 110 patients who were clinically suspected to have Acanthamoeba keratitis, 63 contact lens wearers (CLW), and 47 non-contact lens wearers (NCLW). Taken samples were subjected to direct microscopic examination, cultivation onto the non-nutrient agar plate surface seeded with Escherichia coli, and PCR amplification. The diagnostic performance of these methods was statistically compared. The results showed that Acanthamoeba infection was detected in 21 (19.1 %) of clinically suspected cases (110); 17 (81 %) of them were CLW and the remaining 4 (19 %) positive cases were NCLW. Regarding the used diagnostic methods, it was found that direct amplification of Acanthamoeba DNA bypassing nucleic acid extraction was superior to microscopy and culture in which 21 cases (19.1 %) were positive for Acanthamoeba by PCR compared to 19 positive cases by culture (17.3 %) and one case (0.9 %) by direct smear. The difference in detection rates between culture and direct smear was highly statistically significant (P?=?0.001). On the other hand, there was no significant difference in detection rates between culture and PCR (P?=?0.86). On using culture as the gold standard, PCR showed three false-positive samples that were negative by culture and one false-negative sample that was positive by culture. At the same time, direct smear showed 18 false-negative samples. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of PCR were 94.7, 96.7, 85.7, 98.9, and 96.4, respectively, while those of direct smear were 5.3, 100, 100, 83.5, and 83.6, respectively. In conclusion, direct amplification of Acanthamoeba-DNA bypassing DNA extraction is a reliable, specific, sensitive method in the diagnosis of Acanthamoeba keratitis in clinically suspected cases. It should set up in ophthalmological centers as an easy diagnostic tool.     

Development and implementation of real-time nucleic acid amplification for the detection of enterovirus infections in comparison to rapid culture of various clinical specimens

Several real-time PCR and nucleic acid sequence-based amplification (NASBA) primer pairs and a modified real-time PCR primer pair for the detection of enteroviruses were compared. The modified real-time PCR primer pair was evaluated on clinical samples in comparison with cell culture using the MagnaPure LC Isolation instrument for nucleic acid extraction. Six hundred forty samples could be examined both by cell culture and real-time PCR. Faecal specimens (n = 285), cerebrospinal fluid (n = 210), throat swabs (n = 113), biopsies (n = 1--, vesicular fluid (n = 11), and pleural fluid specimens (n = 9) were included. By culture, 26/640 (4%) samples were positive for enterovirus. By real-time PCR, the number of positive specimens was 50 (7.8%). Of the 210 cerebrospinal fluid samples, three were positive by culture and nine by real-time PCR. Seventeen and 33 of a total of 285 faecal specimens were positive by culture and real-time PCR, respectively. In case of discrepant results, the clinical symptoms were in accordance with an infection due to enteroviruses. Genotyping using the VP1 gene correlated with serotyping by neutralization. In contrast, six of the 19 specimens that could be typed both by neutralization and by sequencing using the VP4 domain yielded a different genotype, yet within the same species. Real-time PCR turned out to be suitable for the detection of enteroviruses in the daily routine setting. In comparison to rapid culture, it offers a rapid, more sensitive, and reliable assay; especially in cerebrospinal fluid, the yield of enteroviruses is much higher.     

Simultaneous detection and strain differentiation of Mycobacterium tuberculosis complex in paraffin wax embedded tissues and in stained microscopic preparations.

AIMS: To detect and differentiate Mycobacterium tuberculosis simultaneously by polymerase chain reaction (PCR) in clinical samples prepared for histopathological analysis and for microscopic detection of acid fast bacteria. METHODS: Paraffin wax embedded tissue samples and Ziehl-Neelsen (ZN) and auramine stained microscopic preparations from culture positive tuberculosis patients were subjected to DNA extraction and amplification by PCR. PCR was performed with primers specific for direct repeats and the product was detected by hybridisation to a set of 43 different oligonucleotides, a procedure designated as "spoligotyping". RESULTS: Mycobacterium tuberculosis complex DNA was detected in all of the 23 paraffin wax embedded tissues analysed. Strain differentiation was possible in 20 of the 23 paraffin wax embedded tissues. Mycobacterium tuberculosis complex DNA was also detected and typed in eight of 10 ZN stained microscopic preparations. The hybridisation patterns obtained from virtually all of these samples were identical to those obtained from DNA extracted from cultures. CONCLUSION: Simultaneous detection and strain differentiation of M. tuberculosis complex bacteria is possible in clinical samples prepared by current methods for microscopic and histopathological analysis, without the need to culture. The methodology described opens the way to rapid disclosure of outbreaks in high risk settings, such as hospitals and prisons, where dissemination of tuberculosis might be very fast as a result of a high prevalence of human immunodeficiency virus infected patients.     

Colorimetric one-tube nested PCR for detection of Trichomonas vaginalis in vaginal discharge.

A colorimetric one-tube nested PCR was developed for the detection of Trichomonas vaginalis in clinical vaginal discharge specimens. A family of 650-bp specific DNA repeats from the T. vaginalis genome was targeted. There was no cross-reaction with human DNA or other infectious agents, including Pentatrichomonas hominis and Giardia lamblia. The colorimetric assay was applied as an adjunct to nested PCR for semiquantitative determination of T. vaginalis DNA at levels corresponding to 1 to 1,000 parasites. PCR of samples prepared by a rapid boiling method was as sensitive and specific as PCR of samples prepared by the standard DNA extraction method: the equivalent of one T. vaginalis organism in 20 microliters of vaginal discharge could be detected. The colorimetric nested PCR was compared with wet mount and culture for the detection of T. vaginalis. A total of 378 clinical vaginal discharge specimens from symptomatic patients were examined; 31 patients were positive for T. vaginalis both by culture and by nested PCR. However, only 17 of these 31 patients were positive by wet mount examination. In addition, of 113 asymptomatic patients, 9 were positive for T. vaginalis by nested PCR. Of these nine PCR-positive patients, only two were also positive both by wet mount and by culture, four patients were positive by culture but negative by wet mount, and three patients were negative both by wet mount and by culture. No specimens negative by nested PCR were positive by wet mount or by culture. The three asymptomatic patients with PCR-positive but wet mount- and culture-negative samples were subsequently found to have T. vaginalis infection after repeated and prolonged culture was performed. This colorimetric nested PCR was very sensitive compared with culture for the diagnosis of vaginal trichomoniasis, especially asymptomatic T. vaginalis infection. It is also simple, specific, rapid, and semiquantitative.     

Preparation of mycobacterial DNA from blood culture fluids by simple alkali wash and heat lysis method for PCR detection.

A sodium iodide-isopropanol (NI) method was compared with an alkali wash and heat lysis (AH) procedure for the preparation and extraction of DNA from BACTEC 13A blood culture fluid samples from AIDS patients for use in a PCR for the detection and identification of mycobacteria. The sensitivity and efficiency of the DNA extraction methods were assessed by a multiplex PCR which detected the members of the genus Mycobacterium and differentiated between M. intracellulare, M. tuberculosis, and M. avium isolates with a limit of detection of between 0.28 pg (67 cells) and 120 pg (28,571 cells) of standard mycobacterial DNA. The PCR amplified mycobacterial DNA prepared by the AH procedure from 40 acid-fast bacillus-positive blood cultures with growth index values of > 20 U but not from 48 blood cultures with growth index values of < 21 U. The AH method was about 10 times more sensitive than the NI method for extracting DNA from 13 acid-fast bacillus-positive BACTEC fluid samples for PCR analysis. The study shows that the AH procedure in combination with the multiplex PCR is a simple, specific, and sensitive method which can be used in the routine diagnostic laboratory to detect and identify different members of the genus Mycobacterium in blood culture fluid samples from AIDS patients.     

Detection of herpes simplex virus DNA in genital and dermal specimens by LightCycler PCR after extraction using the IsoQuick, MagNA Pure, and BioRobot 9604 methods

We evaluated two automated systems, MagNA Pure (Roche Molecular Biochemicals, Indianapolis, Ind.) and BioRobot 9604 (Qiagen, Inc., Chatsworth, Calif.) as effective replacements for the manual IsoQuick method (Orca Research, Inc., Bothell, Wash.) for extraction of herpes simplex virus (HSV) DNA from dermal and genital tract specimens prior to analysis by LightCycler PCR. Of 198 specimens (152 genital, 46 dermal), 92 (46.2%) were positive for HSV DNA by LightCycler PCR after automated extraction of specimens with either the MagNA Pure or BioRobot 9604 instrument. The manual IsoQuick method yielded HSV DNA (total n = 95) from three additional specimens that were negative by the automated method (P = 0.25, sign test). Although the mean numbers of LightCycler PCR cycles required to reach positivity differed statistically significantly among all three of the methods of extraction, the estimated means differed by no more than 1.5 cycles (P < 0.05). Seventy (76%) of the 92 specimens that were LightCycler PCR positive by all three extraction methods were also positive by shell vial cell culture assay. HSV DNA was detected by a lower LightCycler PCR cycle number (26.1 cycles) in specimens culture positive for the virus than in culture-negative samples (33.3 cycles) (P < 0.0001). The manual IsoQuick and automated MagNA Pure and BioRobot 9604 methods provide standardized, reproducible extraction of HSV DNA for LightCycler PCR. The decision to implement a manual versus an automated procedure depends on factors such as costs related to the number of specimens processed rather than on the minimal differences in the technical efficiency of extraction of nucleic acids among these methods.     

Large-scale use of polymerase chain reaction for detection of Mycobacterium tuberculosis in a routine mycobacteriology laboratory.

We investigated the use of DNA amplification by the polymerase chain reaction reaction (PCR) for detection of Mycobacterium tuberculosis from clinical specimens. Two-thirds of each sample was processed for smear and culture by standard methods, and one-third was submitted for DNA extraction, amplification of a 317-bp segment within the insertion element IS6110, and detection by agarose gel electrophoresis, hybridization, or both. DNA was prepared from over 5,000 samples, with 623 samples being culture positive for acid-fast bacilli. Of 218 specimens that were identified as M. tuberculosis, 181 (85%) were positive by PCR. In the M. tuberculosis culture-positive group, PCR was positive for 136 of 145 (94%) and 45 of 73 (62%) of the fluorochrome smear-positive and -negative specimens, respectively. Of 948 specimens that were either culture positive for mycobacteria other than M. tuberculosis or culture negative, 937 specimens were negative by PCR and 11 (1%) specimens initially appeared to be false positive for M. tuberculosis. The reason for discrepant results varied; some errors were traced to the presence of an inhibitor in the specimen (7.3% in unselected specimens), nucleic acid contamination, low numbers of organisms in the specimen antituberculosis therapy, and possible low-level nonspecific hybridization. In comparison with culture, the sensitivity, specificity, and positive predictive value were 83.5, 99.0, and 94.2%, respectively, for PCR. When PCR was corrected for DNA contamination, the presence of inhibitor, and culture-negative disease, the values became 86.1, 99.7, and 98.4%, respectively. If the results for multiple specimens submitted from the same patient are considered, no patient who had three of more sputum specimens tested would have been misdiagnosed.     

Fast and specific dermatophyte detection by automated DNA extraction and real-time PCR

The aim of this study was to develop and validate a rapid and sensitive real-time PCR method for detection of all known species of dermatophytes, including identification of Trichophyton rubrum and Trichophyton interdigitale. Fungal DNA was extracted directly from clinical samples by using a pre-lysis step, followed by automated DNA extraction on the MagNA Pure Compact. In total, 202 clinical samples were examined by both conventional culture and by the new PCR method. In 103 (51%) of the samples fungal nucleic acid was detected by PCR, while only 79 (39%) were found to be positive by culture. Out of 103 PCR-positive clinical samples, 94 (91%) were identified as T. rubrum and eight (8%) as T. interdigitale. This real-time PCR is far more sensitive and 2–4 weeks faster than conventional culture for detection of dermatophytes present in clinical samples.     

Improved monitoring of cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation by an ultrasensitive plasma DNA PCR assay

Cytomegalovirus (CMV) DNA amplification assays in plasma have shown limited sensitivity compared to the detection of pp65 antigen in leukocytes. Our goal was to increase the sensitivity of a commercial CMV DNA PCR quantitative assay. After modification, the new assay was able to reproducibly detect 20 CMV DNA copies/ml of plasma. We compared this new ultrasensitive PCR assay with the standard PCR and the pp65 test for CMV detection and quantification in 22 consecutive allogeneic hematopoietic stem cell recipients. CMV infection or reactivation was detected in 84 of 319 (26%) samples by the ultrasensitive PCR assay compared to 38 of 319 (12%) samples by the pp65 assay (P < 0.01). All samples positive by the pp65 assay were positive by the ultrasensitive PCR, and CMV episodes were detected on average 4 days earlier and 7 days later than the first and the last pp65-positive test, respectively. In addition, during CMV episodes, the ultrasensitive assay identified positive samples that were inconsistently detected by the pp65 assay. The ultrasensitive assay was also much more sensitive than the standard PCR, with 26 versus 12% of CMV DNA-positive samples (P < 0.01). This assay improved the monitoring of CMV infection or reactivation in hematopoietic allogeneic stem cell recipients.     

Rapid detection of cytomegalovirus in clinical specimens by using biotinylated DNA probes and analysis of cross-reactivity with herpes simplex virus.

A method for rapid identification of human cytomegalovirus (HCMV) was developed with biotinylated DNA probes. BamHI restriction fragments from HCMV strain AD169 were selected and tested for their ability to detect virus in patient urine samples. All probes detected 30 pg of HCMV AD169 DNA. The BamHI B fragment detected 15 of 29 cell-culture-positive samples (sensitivity, 52%). There were four samples which were probe positive and cell culture negative (specificity, 87%). The D and H fragments used as combined probes detected 17 of 21 cell-culture-positive samples (sensitivity, 81%). There were five probe-positive and cell-culture-negative samples (specificity, 68.8%). The H fragment, when used alone, detected 11 of 14 culture-positive samples, and 5 samples were culture negative and probe positive. Sensitivity (78.6%) and specificity (76.2%) for the H fragment were similar to those for the combined probes, but the color intensity of the positive reactions detected by the H fragment alone was lower. There was unexpected cross-reactivity with herpes simplex type 1 and 2 controls when the combined D and H probes were used. Specific hybridization was demonstrated between subfragments of the HCMV BamHI D fragment and the herpes simplex virus type 1 EcoRI M fragment.     

Evaluation of extraction methods from paraffin wax embedded tissues for PCR amplification of human and viral DNA

AIM: To evaluate the efficiency of phenol/chloroform, microwave, and Qiagen spin column based DNA extractions from paraffin wax embedded tissue for use in the polymerase chain reaction (PCR). In addition, to assess the reliability of amplifying a housekeeping gene to indicate successful viral DNA extraction. METHODS: DNA samples extracted from 20 blocks of cervical carcinoma tissues using the three methods were subjected to PCRs targeting 509 bp and 355 bp of the beta globin gene, and 450 bp and 150 bp of human papillomavirus (HPV) DNA. RESULTS: Microwave extraction showed the highest positive rate for beta globin PCR, whereas the spin column method was the most efficient for HPV DNA extraction. When the 509 bp beta globin and 450 bp HPV PCR results were correlated, two of 10, eight of 12, and nine of 10 beta globin positive extractions prepared by means of the phenol/chloroform, microwave, and spin column methods, respectively, yielded HPV DNA of the expected size. For the beta globin negative samples, HPV was detected in three of 10, two of eight, and four of 10 samples. CONCLUSIONS: HPV DNA extraction was most efficient using the Qiagen spin column and had the highest positive predictive value when a housekeeping gene was used as an indicator of successful viral DNA extraction; the phenol/chloroform method was the least efficient. The potential drawbacks of some extraction methods when using a human housekeeping gene to assess the quality of viral DNA extraction need to be considered.     

Controlled evaluation of the IDI-MRSA assay for detection of colonization by methicillin-resistant Staphylococcus aureus in diverse mucocutaneous specimens

Rapid and reliable detection of methicillin-resistant Staphylococcus aureus (MRSA) carriers is crucial for the effective control of MRSA transmission in healthcare facilities. The aim of this study was to verify the performance of the IDI-MRSA real-time PCR assay for direct MRSA detection in diverse mucocutaneous swabs from hospitalized patients. Swabs from nares (n = 522) and skin or other superficial sites (n = 478) were prospectively collected for MRSA screening from 466 patients admitted to an 858-bed teaching hospital. Swabs were inoculated onto selective chromogenic MRSA-ID agar, buffer extraction solution for IDI-MRSA assay, and enrichment broth. MRSA was detected by culture in 100 specimens from 47 patients. Compared to enrichment culture, the sensitivity and specificity of the PCR assay were 81.0 and 97.0%, respectively, and its positive and negative predictive values were 75.0 and 97.9%, respectively. The IDI-MRSA assay was more sensitive on swabs from nares (90.6%) than from other body sites (76.5%, P < 0.01). The PCR assay detected MRSA in 42 of 47 patients with culture positive study samples. Of 26 patients with culture-negative but PCR-positive study samples, 11 were probable true MRSA carriers based on patient history and/or positive culture on a new sample. The median turnaround time for PCR results was 19 h versus 3 days for agar culture results and 6 days for enrichment culture results. These data confirm the value of IDI-MRSA assay for rapid screening of MRSA mucocutaneous carriage among hospitalized patients. Cost-effectiveness studies are warranted to evaluate the impact of this assay on infection control procedures in healthcare settings.