Inhibition controls for qualitative real-time PCR assays: are they necessary for all specimen matrices?

A retrospective analysis of 386,706 specimens representing a variety of matrix types used in qualitative real-time PCR assays determined the overall inhibition rate to be 0.87% when the inhibition control was added preextraction to 5,613 specimens and 0.01% when the inhibition control was added postextraction but preamplification in 381,093 specimens. Inhibition rates of ≤ 1% were found for all specimen matrix types except urine and formalin-fixed, paraffin-embedded tissue.

Tuberculosis attributed to household contacts in the Philippines.

Data on the burden of disease from tuberculosis (TB) in Filipino households are limited. To determine the magnitude of undiagnosed TB in TB households, and the demographic and socio-economic factors associated with TB in the Philippines, household contacts of adult smear-positive TB patients seen from July 2001 to June 2003 were assessed based on interview, chest X-ray, tuberculin skin test and sputum examination. History of TB and older age were independently associated with TB disease, and age and duration of cohabitation with TB infection. TB and TB infection are highly prevalent in TB households in the Philippines.

Real-time PCR in clinical microbiology: applications for routine laboratory testing.

Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.

Optimal testing parameters for blood cultures.

The effects of volume of blood, number of consecutive cultures, and incubation time on pathogen recovery were evaluated for 37,568 blood cultures tested with the automated BACTEC 9240 instrument (Becton Dickinson Diagnostic Instrument Systems) at a tertiary care center over the period of 12 June 1996 through 12 October 1997. When the results for this study were compared with previous data published for manual broth-based blood culture systems and patient samples obtained in the 1970s and 1980s, the following were found: (1) the percentage increase in pathogen recovery per milliliter of blood is less, (2) more consecutive blood culture sets over a 24-h period are required to detect bloodstream pathogens, and (3) a shorter duration of incubation is required to diagnose bloodstream infections. Guidelines developed in the 1970s and 1980s for processing and culturing blood may require revision.

Comparison of the Roche LightCycler vanA/vanB detection assay and culture for detection of vancomycin-resistant enterococci from perianal swabs.

We compared the performance characteristics of a real-time PCR method, the LightCycler vanA/vanB detection assay (Roche Diagnostics Corporation, Indianapolis, Ind.) to that of Enterococcosel agar (BBL, Sparks, Md.) for direct detection of vancomycin-resistant enterococci (VRE) from 894 perianal stool swabs. For 421 of 894 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing vancomycin at 6 microg/ml; for the remaining 473 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing 8 microg/ml vancomycin. The LightCycler method produced considerably more positive results than either the Enterococcosel plate containing vancomycin at 6 microg/ml (n = 25 versus n = 11; sensitivity, 100%; specificity, 97%; positive predictive value [PPV], 42%; negative predictive value [NPV], 100%) or the Enterococcosel plate containing vancomycin at 8 microg/ml (n = 31 versus n = 10; sensitivity, 100%; specificity, 95%; PPV, 32%; NPV, 100%). When possible, additional testing, including culture, LightCycler PCR, and/or a conventional PCR method (PCR-restriction fragment length polymorphism assay), were performed on either the original specimens or original cultures or subsequent specimens for cases in which the original specimen was positive by LightCycler PCR but the Enterococcosel plate was negative. This additional testing demonstrated positive results for 7 of 14 (50%) evaluable discordant specimens which initially tested as LightCycler PCR positive but culture negative using the Enterococcosel plate containing vancomycin at 6 microg/ml and 12 of 17 (71%) evaluable discordant specimens which initially tested as LightCycler positive but culture negative using the Enterococcosel plate containing vancomycin at (8 microg/ml). These results demonstrate that the LightCycler VRE detection assay is considerably more sensitive than the standard culture method for detecting VRE directly from perianal swab specimens. The LightCycler assay also provides results much faster than culture (approximately 3.5 versus > or =72 h). The use of this test could have important implications for the effective control and prevention of nosocomial outbreaks of VRE.

Comparison of LightCycler PCR, rapid antigen immunoassay, and culture for detection of group A streptococci from throat swabs.

We compared the performance characteristics of a real-time PCR method, the LightCycler Strep-A assay (Roche Applied Science, Indianapolis, Ind.), to those of a rapid antigen immunoassay, the Directigen 1-2-3 Group A Strep Test kit (BD Diagnostic Systems, Sparks, Md.), and a standard culture method for detection of group A streptococci (GAS) from 384 throat swabs. The LightCycler PCR produced more positive results (n = 58) than either culture (n = 55) or the Directigen immunoassay (n = 31). The results of the LightCycler PCR and the Directigen method were independently compared to the results of the accepted "gold standard," bacterial culture. The sensitivities, specificities, and positive and negative predictive values for this comparison were as follows: for the Directigen method, 55, 99, 97, and 93%, respectively; for the LightCycler PCR, 93, 98, 88, and 99%, respectively. In no case was a throat swab positive by both the LightCycler PCR and the Directigen method but negative by culture. The medical histories of patients whose throat swabs were negative by culture but positive by either the LightCycler PCR (n = 7) or the Directigen method (n = 1) were reviewed. All of these patients had signs or symptoms compatible with GAS disease, and therefore, all of these discordant positive results (along with positive results by either the Directigen method or the LightCycler PCR that agreed with the culture results) were counted as true positives for statistical analysis. For this analysis, the LightCycler PCR detected more true-positive results than the culture method (58 versus 55 swabs); however, this difference was not statistically significant (P = 0.5465). In contrast, statistically significantly more true-positive results occurred by culture than by the Directigen method (55 versus 31 swabs; P < 0.0001) and by the LightCycler PCR than by the Directigen method (58 versus 31 swabs; P < 0.0001). The LightCycler PCR is a suitable stand-alone method for the detection of GAS from throat swabs. Additionally, this method requires less than half the personnel time and the procedure can be completed in considerably less time ( approximately 1 h) than our standard approach (up to 2 days) for detection of GAS in throat swabs (i.e., testing by the Directigen method with negative results verified by culture).

Frequency of isolation of Staphylococcus lugdunensis in consecutive urine cultures and relationship to urinary tract infection.

Recent reports associate Staphylococcus lugdunensis with severe infection in humans. The frequency of this microorganism in urine cultures is unknown. Five hundred isolates of coagulase-negative staphylococci (CoNS) were recovered from 4,652 consecutive urine specimens submitted for culture to the Mayo Clinic Microbiology Laboratory. Thirty-one (6%) of 500 isolates of CoNS were identified as S. lugdunensis. In no case was S. lugdunensis isolated in pure culture; 29 (94%) of 31 S. lugdunensis isolates were part of mixed nonpathogenic flora. Medical records were reviewed for 30 of the 31 patients from whom these 31 isolates were isolated. Twenty-one (70%) of the 30 evaluable patients were not treated with antibiotics; the remaining 9 (30%) of 30 patients were treated with antibiotics that may be effective against S. lugdunensis. S. lugdunensis may be an unrecognized yet infrequent cause of urinary tract infection.

Culture with BACTEC Peds Plus/F bottle compared with conventional methods for detection of bacteria in synovial fluid.

An evaluation was undertaken to determine the utility of the BACTEC Peds Plus/F bottle and the BACTEC 9240 instrument (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) for the detection of clinically significant microorganisms in synovial fluid specimens. The Peds Plus/F bottle was used because in our laboratory the quantity of synovial fluid available for culture is frequently in the range of 0.5 to 3.0 ml. The culture results obtained with the Peds Plus/F bottle were compared to those obtained by a conventional agar plate method for a total of 805 synovial fluid specimens. Microbial growth was produced by 74 cultures (9.2%) from 60 patients, yielding a total of 77 microorganisms. Organisms were classified as pathogens (n = 62), contaminants (n = 12), or indeterminate (n = 3) on the basis of a review of the patients' medical histories. Culture using BACTEC Peds Plus/F bottle detected statistically significantly more pathogens overall (62 versus 51 pathogens [P = 0.001]) and statistically fewer contaminants overall (1 versus 11 contaminants [P = 0.006]) than culture by the agar plate method. These results indicate the superior performance of the BACTEC Peds Plus/F bottle over the conventional agar plate method for the detection of clinically significant microorganisms from synovial fluid specimens.

Two cases of non-O157:H7 Escherichia coli hemolytic uremic syndrome caused by urinary tract infection.

Escherichia coli serotype O157:H7 is a leading cause of diarrhea and hemolytic uremic syndrome (HUS). Because of the limitations of current diagnostic techniques, the prevalence of non-O157:H7 Shiga toxin-producing E coli strains is not known. We describe two patients with HUS in whom no E coli O157:H7 was demonstrable in stool cultures. On culture of the urine, the first patient was found to have E coli O113:H21 strain, and the second patient had E coli O6:H1 serotype. Shiga toxin production (stx2) by the O113:H21 isolate was confirmed. The first patient required 15 days of peritoneal dialysis and subsequently recovered renal function. At last follow-up, serum creatinine was 0.9 mg/dL. The second patient had preservation of renal function throughout the acute illness with serum creatinine of 0.5 mg/dL. The clinical presentation, bacteriology, course, and outcome as well as epidemiologic implications of the increasing number of patients with E coli urinary tract infections associated with HUS are discussed. These cases illustrate the need to investigate patients with nondiarrheal HUS for infection with Shiga toxin-producing E coli of the non-O157 strain variety.

Direct identification of bacteria from positive blood cultures by amplification and sequencing of the 16S rRNA gene: evaluation of BACTEC 9240 instrument true-positive and false-positive results.

In a previous study which evaluated the BACTEC 9240 automated blood culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.), we noted a 1.3% "instrument false-positive" rate. That is, the BACTEC system signaled that a bottle (BACTEC Plus Aerobic/F bottle or BACTEC Anaerobic Lytic/10 bottle) culture was positive but a Gram stain was negative and there was no growth of bacteria or yeasts on subculture to chocolate agar. Furthermore, from the same sample of blood, cultures for fungi using the Isolator blood culture system (Wampole Laboratories, Cranbury, N.J.) were negative for growth. For the present study, we evaluated 76 instrument false-positive samples for the presence of 16S ribosomal DNA using the MicroSeq 500 kit (PE Biosystems, Foster City, Calif.). These samples also were negative for fungi by the Isolator method. This kit has a PCR module and sequencing module for the amplification and sequencing of the 16S RNA gene and provides a database for sequence alignment and identification of bacteria. To optimize the assay, we evaluated the effect of adding 0.5% bovine serum albumin to the sample from blood culture bottles and found that it decreased the effects of inhibitors on the PCR. Two control groups of blood culture specimens were also evaluated. One group (n = 45) were "instrument true positives"; the instrument signaled positive, and subsequent Gram stains were positive and subcultures on chocolate agar grew bacteria. The other group (n = 20) were "instrument true negatives"; the instrument signaled negative, the Gram stain was negative, and subcultures on chocolate agar and from the Isolator tube on fungal media showed no growth. None of the 76 instrument false-positive samples had evidence for 16S rRNA gene sequences. All of the instrument true-positive samples and all of the instrument true-negative specimens were positive and negative, respectively, using the MicroSeq 500 kit. Total peripheral white blood cell counts were statistically significantly higher for patients who had instrument false-positive results than for patients who had instrument true-positive or true-negative results (P = 0.001). We conclude that instrument false positives signaled by the BACTEC 9240 system are not due to bacteria in the blood culture samples.

In vivo and in vitro studies of cytosolic phospholipase A2 expression in Helicobacter pylori infection.

Modifications of mucosal phospholipids have been detected in samples from patients with Helicobacter pylori-positive gastritis. These alterations appear secondary to increased phospholipase A2 activity (PLA2). The cytosolic form of this enzyme (cPLA2), normally involved in cellular signaling and growth, has been implicated in cancer pathogenesis. The aim of this study was to investigate cPLA2 expression and PLA2 activity in the gastric mucosae of patients with and without H. pylori infection. In gastric biopsies from 10 H. pylori-positive patients, cPLA2 levels, levels of mRNA as determined by reverse transcriptase PCR, levels of protein as determined by immunohistochemistry, and total PLA2 activity were higher than in 10 H. pylori-negative gastritis patients. To clarify whether H. pylori had a direct effect on the cellular expression of cPLA2, we studied cPLA2 expression in vitro with different human epithelial cell lines, one from a patient with larynx carcinoma (i.e., HEp-2 cells) and two from patients with gastric adenocarcinoma (i.e., AGS and MKN 28 cells), incubated with different H. pylori strains. The levels of cPLA2, mRNA, and protein expression were unchanged in Hep-2 cells independently of cellular adhesion or invasion of the bacteria. Moreover, no change in cPLA2 protein expression was observed in AGS or MKN 28 cells treated with wild-type H. pylori. In conclusion, our study shows increased cPLA2 expression and PLA2 activity in the gastric mucosae of patients with H. pylori infection and no change in epithelial cell lines exposed to H. pylori.

Direct detection of Legionella species from bronchoalveolar lavage and open lung biopsy specimens: comparison of LightCycler PCR, in situ hybridization, direct fluorescence antigen detection, and culture.

We developed a rapid thermocycling, real-time detection (also known as real-time PCR) method for the detection of Legionella species directly from clinical specimens. This method uses the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and requires approximately 1 to 2 h to perform. Both a Legionella genus PCR assay and Legionella pneumophila species-specific PCR assay were designed. A total of 43 archived specimens from 35 patients were evaluated, including 19 bronchoalveolar lavage (BAL) specimens and 24 formalin-fixed, paraffin-embedded open lung biopsy specimens. Twenty-five of the specimens were culture-positive for Legionella (9 BAL specimens and 16 tissue specimens). BAL specimens were tested by LightCycler PCR (LC-PCR) methods and by a direct fluorescent antibody (DFA) assay, which detects L. pneumophila serogroups 1 to 6 and several other Legionella species. Tissue sections were tested by the two LC-PCR methods, by DFA, by an in situ hybridization (ISH) assay, specifically designed to detect L. pneumophila, and by Warthin-Starry (WS) staining. The results were compared to the "gold standard" method of bacterial culture. With BAL specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by Legionella genus LC-PCR, 100 and 100%; Legionella genus detection by DFA assay, 33 and 100%; and L. pneumophila detection by L. pneumophila species-specific LC-PCR, 100 and 100%. With open lung biopsy specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by LC-PCR 68.8 and 100%; Legionella genus detection by DFA assay, 44 and 100%; Legionella genus detection by WS staining, 63 and 100%; L. pneumophila species-specific detection by LC-PCR, 17 and 100%; and L. pneumophila species-specific detection by ISH, 100 and 100%. The analytical sensitivity of both LC-PCR assays was <10 CFU/reaction. LC-PCR is a reliable method for the direct detection of Legionella species from BAL specimens. The Legionella genus LC-PCR assay could be performed initially; if positive, L. pneumophila species-specific LC-PCR could then be performed (if species differentiation is desired). The speed with which the LC-PCR procedure can be performed offers significant advantages over both culture-based methods and conventional PCR techniques. In contrast, for the methods evaluated, culture was the best for detecting multiple Legionella species in lung tissue. WS staining, Legionella genus LC-PCR, and L. pneumophila species-specific ISH were useful as rapid tests with lung tissue.

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.

Natural history of vancomycin-resistant enterococcal colonization in liver and kidney transplant recipients.

At Mayo Medical Center (Rochester, MN), surveillance rectal (and other-site) cultures have been routinely collected from liver transplant recipients as part of a selective bowel decontamination program. Beginning in 1995, vancomycin-resistant enterococcus (VRE) colonization and infection were identified in Mayo Clinic liver and kidney transplant patients through our surveillance cultures. The purpose of this study is to describe the natural history of VRE colonization in this patient population. Fifty-two patients with VRE colonization (predominantly with a single vanB clone) were identified from September 1995 through December 1997. Five hundred ninety cultures were reviewed for this study (mean, 11.3 cultures/patient). The median time from initial VRE colonization to the last surveillance culture obtained was 306 days (range, 1 to 1,393 days). VRE infection was documented in 6 patients (11.3%). Eighteen patients (35%) met the criteria for clearance of VRE colonization, defined as VRE-negative rectal culture results on at least 3 consecutive occasions greater than 1 week apart. However, VRE was detected on subsequent surveillance cultures from 2 of these patients (11% relapse rate). Of the remaining 34 patients, 16 remained colonized with VRE and 18 did not meet the definition for clearance of VRE colonization because of incomplete follow-up. This study documents that VRE colonization usually persists for months to years in liver and kidney transplant patients.

Susceptibility testing. Phenotypic and genotypic tests for bacteria and mycobacteria.

Genotypic-based methods hold promise for the rapid and accurate detection or confirmation of antimicrobial resistance; however, phenotypic methods will continue to have an advantage when resistance to the same antimicrobial agent may be caused by several different mechanisms. The diversity of genetic mechanisms may exceed the capabilities of current molecular technology. Genotypic assays have the ability to detect resistance but not susceptibility. Although resutls can be obtained rapidly, many molecular methods are labor-intensive, expensive, and lack standardization. Clinical studies will be required to validate the genotypic approach to detection of antimicrobial resistance. Molecular assays are also at risk for false-positive results because of contamination of specimens by other specimens that carry the DNA targeted for the assay, or carryover of amplified target DNA (amplicons) from a previous PCR assay during sample preparation. Detection of certain genetic resistance loci in clinical specimens must be interpreted with caution, because organisms in normal flora may also harbor the same loci. All these factors must be taken into consideration when introducing a genotypic method in the clinical laboratory. Other considerations include cost, turnaround time, and assay performance. It must be emphasized that the bedside assessment of the patient should always be considered in addition to the results of antimicrobial susceptibility tests (whether phenotypic or genotypic) so that the best outcome is assured for the patient.

Multiplex Polymerase Chain Reaction Detection of vanA, vanB, vanC-1, and vanC-2/3 Genes in Enterococci.

Resistance to the glycopeptide antibiotic vancomycin in enterococci, is phenotypically and genotypically heterogeneous. Three glycopeptide resistance phenotypes, VanA, VanB, and VanC, account for most glycopeptide resistance in enterococci; they can be distinguished on the basis of the level and inducibility of resistance to vancomycin and teicoplanin.

Frequency of isolation of Staphylococcus lugdunensis among staphylococcal isolates causing endocarditis: a 20-year experience.

Eighty-nine staphylococcal isolates recovered from patients with bacterial endocarditis at the Mayo Clinic from 1980 to 1999 were studied to determine the prevalence of Staphylococcus lugdunensis among clinical isolates of staphylococci causing endocarditis. Four isolates, all from patients with native mitral valve endocarditis, were identified as S. lugdunensis.

Performance of five agar media for recovery of fungi from isolator blood cultures.

We studied the recovery of 1,270 fungal isolates from 176,144 Isolator blood cultures (0.72% positive) on bacterial and fungal media, under routine and differing incubation conditions. Except with Histoplasma capsulatum, chocolate agar incubated for only 3 days proved to be an excellent medium for the recovery of fungi from the Isolator system.

Evaluation of the Alexon-trend ProSpecT Campylobacter microplate assay.

We evaluated stool specimens known to contain or be free of Campylobacter by traditional culture, using the ProSpecT Campylobacter microplate assay (Alexon-Trend, Ramsey, Minn.). This rapid enzyme immunoassay for the detection of Campylobacter-specific antigens demonstrated 96% sensitivity and 99% specificity and is an acceptable alternative method of Campylobacter detection.

The initial outpatient-physician encounter in group A streptococcal necrotizing fasciitis.

Of 15 patients in whom the diagnosis of streptococcal necrotizing fasciitis was missed at initial outpatient evaluation, 8 died. Although influenza-like and gastrointestinal symptoms were common, the most consistent clinical clue was unrelenting pain out of proportion to the physical findings. Necrotizing fasciitis should be considered in patients presenting with the latter complaint, even if there is only mild or no fever or erythema.