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.

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.

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.

Real-time PCR method for detection of Encephalitozoon intestinalis from stool specimens.

The prevalence of microsporidiosis is likely underestimated due to the labor-intensive, insensitive, and nonspecific clinical laboratory methods used for the diagnosis of this disease. A real-time PCR assay was designed to assess DNA extraction methods and to detect three Encephalitozoon species in feces. Modifications of the MagNA Pure LC DNA isolation kit protocol (Roche Applied Sciences, Indianapolis, Ind.) were compared by using the automated MagNA Pure LC instrument (Roche) and fecal specimens spiked with Encephalitozoon intestinalis spores. Extracted DNA was amplified by the LightCycler (Roche) PCR assay. Assay sensitivity, reproducibility, and efficiency were assessed by comparing threshold crossover values achieved with different extraction and storage conditions (fresh, refrigerated, frozen, and preserved specimens). Optimal extraction conditions were achieved by using a commercial buffer, tissue lysis buffer (Roche), as the specimen diluent. LightCycler PCR results were compared to those obtained from routine stool microscopy with trichrome blue stain. The lower limit of detection for the LightCycler PCR assay varied by storage conditions from 10(2) to 10(4) spores/ml of feces, a value which represented a significant improvement over that achieved by staining (> or =1.0 x 10(6) spores/ml). Melting temperature analysis of the amplicons allowed for the differentiation of three Encephalitozoon species (E. intestinalis, E. cuniculi, and E. hellem). The assay is readily adaptable to the clinical laboratory and represents the first real-time PCR assay designed to detect Encephalitozoon species.

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.

Immunoserologic evidence of Human Granulocytic Ehrlichiosis in Danish patients with Lyme neuroborreliosis.

Human Granulocytic Ehrlichiosis (HGE) is a recently described human illness in the US which manifests as fever, myalgia and headache combined with pancytopenia and elevated concentrations of hepatic transaminases. Genetic analyses indicate that the agent of HGE appears to be an Ehrlichia species that is closely related to E. equi and E. phagocytophila. Ixodes dammini and I. scapularis were identified as potential vectors of HGE. Ixodes ticks are also the vector of Borrelia burgdorferi, the agent of Lyme borreliosis. The presence of antibodies against Ehrlichia in 132 sera from Danish patients with definite Lyme neuroborreliosis were examined in order to provide immunoserologic evidence of this infection in Denmark. Patients with Lyme neuroborreliosis were chosen as a test cohort, as these patients had been infested by a tick sufficient for transmission of B. burgdorferi. All had cerebrospinal fluid lymphocytic pleocytosis. As controls, serum samples from 50 healthy Danish blood donors were included. Of the 132 patients with Lyme neuroborreliosis, 5 (3.8%) reacted with the E. equi antigen substrate at titres 1:128. None of the blood donors were found seropositive for E. equi. At least 2 of the patients found seropositive for HGE constituted probable cases of HGE with E. equi antibody titres of at least 80 combined with fever, headache and myalgias. However, in no cases were we able to detect the presence of the HGE agent in the serum by PCR. We conclude that human exposure to granulocytic Ehrlichiae species may also occur in Europe, although further studies will be necessary to document active infection with these potential pathogens.

Evaluation of an enzyme-linked immunoassay for the detection in serum of antibodies to Entamoeba histolytica.

A commercially available enzyme-linked immunoassay (ELISA) was compared to an indirect hemagglutination test (IHA) for the detection of antibodies to Entamoeba histolytica on 225 patients' serums. All of the 107 serums that had IHA titers of < 32 (interpreted as excluding the presence of invasive amebiasis) were ELISA negative. Sixty-three of 68 (93%) serums that had IHA titers of > or = 128 (interpreted as indicative of the presence of active or recent infection) were ELISA positive. Fifty serums had IHA titers of 32 or 64 that were considered "equivocal," and the ELISA results for these were: six positive, 35 negative, and nine "intermediate" (optical density values between the arbitrary positive cutoff and the low positive control). It was concluded that "intermediate" (and negative) ELISA results should be interpreted as excluding the presence of invasive amebiasis. Using these criteria, the results obtained with this ELISA appear to compare favorably with those of the "gold standard" IHA. Therefore, this ELISA provides a reliable alternative to the IHA for the serologic diagnosis of amebiasis, which may be advantageous for some laboratories in terms of lower cost, shorter test time, and improved efficiency.

Revictimization by polygraph: the practice of polygraphing survivors of sexual assault.

Sexual assault survivors are scrutinized in a manner unlike that meted out to any other victims of crime. Law enforcement officers or prosecutors may subject the survivor to a polygraph exam in an attempt to ascertain the truth or as a prerequisite to further investigation of the case. In a survey conducted with rape crisis centres across the United States, 63 centres in 17 states reported working with survivors of sexual assault who had been polygraphed. Rape crisis centres in 11 states reported that children had been polygraphed. This article examines the practice of polygraphing survivors of sexual assault.

Evaluation of an enzyme-linked immunosorbent assay for detection of Cryptosporidium spp. in stool specimens.

We evaluated a commercially produced enzyme-linked immunosorbent assay (ELISA; LMD Laboratories, Inc.) for the detection of Cryptosporidium spp. in 296 stool specimens submitted to the Mayo Clinic parasitology laboratory for routine examination. The specimens examined were fresh (4 specimens), were stored frozen at -65 degrees C (49 specimens), or were preserved in 10% formalin (243 specimens). Results were compared with those obtained by indirect immunofluorescent antibody detection (Merifluor Cryptosporidium/Giardia; Meridian Diagnostics, Inc.). One hundred of the specimens were positive by indirect immunofluorescent antibody and ELISA, while 187 were negative by both methods; 91 of these negative stool samples contained 121 parasites of 17 different species. Eight ELISA false negatives and one false positive were observed. The ELISA sensitivity was 93%, specificity was 99%, and the positive predictive value was 99%. Storage of specimens preserved in 10% formalin or frozen fresh at -65 degrees C for up to 18 months did not appear to affect the results. There was no cross-reactivity with Giardia lamblia (54 negative specimens) or with the 16 other parasites present in the ELISA-negative stool samples. The ELISA is a fast, easy-to-read, and accurate method for the detection of Cryptosporidium spp. in stool specimens.

Evaluation of an enzyme-linked immunosorbent assay for the detection of Giardia lamblia in stool specimens.

A commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of Giardia lamblia in stool specimens was evaluated on 342 specimens submitted to the Mayo Clinic Parasitology Laboratory for routine examination. The stools were either fresh or fresh/frozen at -65 degrees C (139 specimens) or were preserved in formalin (203 specimens). ELISA results were compared with those obtained by conventional microscopic examination: 143 stools were positive by both methods and 186 were negative. Sixty-six of the negative specimens contained 96 parasites belonging to 16 species other than Giardia, indicating a low rate of cross-reactivity. There were eight "false positive" ELISA results, which included specimens from one patient who had previously had a "true positive" and another patient with multiple family members infected with Giardia. Five stools that were "falsely negative" by ELISA contained only rare G. lamblia. The ELISA sensitivity was 97%, the specificity was 96%, and the positive predictive value was 95%. Results were evenly distributed between frozen and formalinized stools. The LMD/Seradyn ELISA appears to be a simple, rapid, and accurate method for the detection of G. lamblia in unprocessed stool samples.