Harmsen, William S.

ABSTRACT The COBAS AMPLICOR system has played a major role in the transition of molecular diagnostics from research to routine clinical laboratory use by automating the nucleic acid amplification and detection processes. However, sample preparation remains a labor-intensive portion of the procedure. In this study, we evaluated the performance of the COBAS AMPLICOR Hepatitis C Virus Test, version 2.0 (Roche Molecular Systems, Branchburg, N.J.) following manual hepatitis C virus (HCV) RNA extraction versus automated extraction with the MagNA Pure LC instrument (Roche Applied Science, Indianapolis, Ind.). Parallel replicate testing was performed with standard dilutions of 100, 75, 60, and 0 HCV IU/ml and 153 clinical specimens. An analytical sensitivity of 75 IU/ml was achieved with either the manual or the standard-volume (200 μl) automated extraction methodologies (25 of 26 [96.2%]; 95% confidence interval [95% CI], 80.4 to 99.9), whereas the clinical sensitivity and specificity were both 100% with either extraction method. A large-volume (1 ml) automated extraction method was also evaluated with standard dilutions of 40, 25, 10, and 0 IU/ml and the same 153 clinical specimens. The analytical sensitivity of the COBAS AMPLICOR assay with the large-volume extraction method was 25 HCV IU/ml (26 of 26 [100%]; 95% CI, 86.8 to 100), whereas the clinical sensitivity and specificity were both 100%. The MagNA Pure LC instrument is a versatile, labor-saving platform capable of integration with minimal modification of the existing assay procedure. The increased sensitivity of the COBAS AMPLICOR Hepatitis C Virus Test, version 2.0 performed in conjunction with large-volume HCV RNA extraction may be important in HCV diagnostic testing as new therapeutic strategies evolve.

ABSTRACT 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 (∼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).

AbstractIslet cell carcinoma (ICC) of the pancreas is a rare, indolent malignancy associated with higher resectability rate and better survival than ductal carcinoma. This retrospective study presents results of surgical treatment from a single institution. From 1985 through 1993 a total of 64 patients (36 men, 28 women) were surgically treated for ICC. Ages ranged from 22 to 80 years (median 55 years) with a median postoperative follow‐up of 39 months (range 10–97 months). Of the 64 patients, 30 (47%) had functioning and 34 (53%) nonfunctioning tumors. Gastrinoma (n= 11) followed by glucagonoma (n= 6) and insulinoma (n= 4) were the most common functioning tumors. In the patients undergoing a laboratory study, 67% of the nonfunctioning tumors had elevated peptide hormone levels. Potentially curative resections were performed in 17 patients (26%), palliative procedures in 35 (55%), and exploratory laparotomy alone in 12 (19%). One patient (2%) died within 30 days after operation. Symptomatic improvement was achieved in 96% of patients with a mean duration of 22 months. Three‐ and five‐year survivals were 66% and 49%, respectively. In patients with curative resection, the disease‐free survival at 3 years was 53% (95% CI: 32–86%). The presence of diffuse hepatic metastases was a predictor of poor survival at 3 years (74% versus 58%;p= 0.05); there was no statistically significant difference in survival between functioning and nonfunctioning groups (p> 0.1). Although curative resection for ICC is rare, meaningful palliation can be achieved in most patients with rare mortality and acceptable morbidity.

AbstractMultiple endocrine neoplasia type I (MEN‐I) is an autosomal dominant disorder characterized by endocrinopathies involving the anterior pituitary gland, parathyroid glands, and pancreas. The long‐term prognosis for patients affected with this disorder is uncertain. To better characterize this prognosis, we performed a retrospective review of all patients with MEN‐I treated at a single institution during the period 1951–1997. A group of 233 patients served as the study population. Their records were analyzed for confirmation of diagnosis, treatments received, long‐term survival, and cause of death. Altogether, 108 eight male patients (46%) and 125 female patients (54%) were identified. At the conclusion of the study, 164 (70%) were alive and 69 (30%) were deceased, with a median follow‐up for patients alive at last contact of 13.4 years (range < 1 month to 54.3 years). The cause of death was reliably obtained in 60 patients. Of these patients, 17 (28%) died of causes related to MEN‐I, most commonly metastatic islet cell tumors (10 patients). The remaining patients died of causes unrelated to MEN‐I, most commonly coronary artery disease and nonendocrine malignancies (14% each). The overall 20‐year survival of MEN‐I patients was 64% (95% CI was 56–72%), and that of an age‐ and gender‐matched upper Midwest population was 81% (p < 0.001). Patients with MEN‐I appear to be at increased risk of premature death. Earlier diagnosis and treatment of potentially malignant pancreatic islet cell neoplasms may result in a decrease of this premature mortality.

ABSTRACT 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.

ABSTRACT A rapid real-time multiplex PCR assay for detecting and differentiating Bordetella pertussis and Bordetella parapertussis in nasopharyngeal swabs was developed. This assay (LC-PCR-IS) targets the insertion sequences IS 481 and IS 1001 of B. pertussis and B. parapertussis , respectively, and is performed using the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.). The analytical sensitivity is less than one organism per reaction. Results for Bordetella culture and/or direct fluorescent antibody testing and a second LightCycler PCR assay (target, pertussis toxin gene) were compared to results of the LC-PCR-IS assay for 111 nasopharyngeal swabs submitted for pertussis testing. Of the specimens, 12 were positive (9 B. pertussis and 3 B. parapertussis ) and 68 specimens were negative by all methods. Three other specimens were positive for B. pertussis by at least two of the methods (including the LC-PCR-IS assay), and another 28 specimens were positive for B. pertussis by the LC-PCR-IS assay only. No specimens were negative by the LC-PCR-IS assay and positive by the other methods. A conventional PCR method (target, IS 481 ) was also compared to the LC-PCR-IS assay for a different group of nasopharyngeal swab specimens ( n = 96): 44 specimens were positive and 41 specimens were negative for B. pertussis with both PCR methods. Nine specimens were positive for B. pertussis by the LC-PCR-IS assay and negative by the conventional PCR assay, and two specimens were positive for B. pertussis by the conventional PCR assay and negative by the LC-PCR-IS assay. Positivity of the two assays was not significantly different ( P = 0.0654). The insertion sequence IS 481 is also present in Bordetella holmesii ; specimens containing B . holmesii may yield false-positive results. The LC-PCR-IS assay takes approximately 45 min to complete post-nucleic acid extraction, compared to 24 h for the conventional PCR assay previously used in our laboratory. The LC-PCR-IS assay is easier to perform than the conventional PCR assay, and the closed system decreases the chance of contamination. All of these characteristics represent a significant improvement in the detection of B . pertussis and B . parapertussis in nasopharyngeal specimens.

ABSTRACT A comparison of quantitative results expressed in hepatitis C virus (HCV) international units per milliliter, obtained from the VERSANT HCV RNA 3.0 (bDNA-3.0) assay, the QUANTIPLEX HCV RNA 2.0 (bDNA-2.0) assay, and the COBAS AMPLICOR HCV MONITOR version 2.0 (HCM-2.0) test was performed. A total of 168 patient specimens submitted to the Mayo Clinic Molecular Microbiology Laboratory for HCV quantification or HCV genotyping were studied. Of the specimens tested, 97, 88, and 79% yielded quantitative results within the dynamic range of the bDNA-3.0, bDNA-2.0, and HCM-2.0 assays, respectively. Overall, there was substantial agreement between the results generated by all three assays. A total of 15 out of 29 (52%) of the specimens determined to contain viral loads of <31,746 IU/ml by the bDNA-3.0 assay were categorized as containing viral loads within the range of 31,746 to 500,000 IU/ml by the bDNA-2.0 assay. Although substantial agreement was noted between the results generated by the bDNA-2.0 and bDNA-3.0 assays, a bias toward higher viral titer by the bDNA-2.0 assay was noted ( P = 0.001). Likewise, although substantial agreement was noted between the results generated by the HCM-2.0 and bDNA-3.0 assays, a bias toward higher viral titer by the bDNA-3.0 assay was noted ( P ≤ 0.001). The discrepancy between the HCM-2.0 and bDNA-3.0 results was more pronounced when viral loads were >500,000 IU/ml and resulted in statistically significant differences ( P ≤ 0.001) in determining whether viral loads were above or below 800,000 IU/ml of HCV RNA, the proposed threshold value for tailoring the duration of combination therapy. The expression of quantitative values in HCV international units per milliliter was a strength of both the bDNA-3.0 and HCM-2.0 assays.