P. Shawn Mitchell

Tropheryma whippelii DNA Is Rare in the Intestinal Mucosa of Patients without Other Evidence of Whipple Disease

Whipple disease is a systemic condition characterized by the presence of uniform rod-shaped bacteria in affected tissues and by macrophage inclusions that are positive on periodic acidSchiff (PAS) staining. The classic clinical features of the disease include diarrhea with malabsorptive findings, abdominal pain, weight loss, arthralgias, lymphadenopathy, and occasionally, neurologic abnormalities. Numerous attempts to cultivate the causative bacterium in the past have failed. Molecular methods have revealed this organism, Tropheryma whippelii, to be a novel actinomycete (1, 2). Recently, propagation in human fibroblasts has been reported (3), but this finding has not yet been reproduced by other investigators. Little is known about the pathogenesis of Whipple disease. Abnormalities of immune functions have been described (4) and are presumed to play a role as predisposing factors, but precisely defined preexisting immunologic defects have not been identified. Neither the natural habitats of the bacterium nor the route of infection are fully understood. Because of the frequency of intestinal manifestations, an oral route of acquisition is suspected (5, 6). The 16S ribosomal DNA (rRNA gene) sequence of the bacterium has been detected in a polymicrobial community of sewage effluent, suggesting a possible environmental habitat and source of infection (7). This possibility would be in accordance with the phylogeny of the bacterium (8) and with the epidemiologic features of the disease (5, 9). Two recent reports have described polymerase chain reaction (PCR)based detection of T. whippelii DNA in specimens of persons without the classic clinical or histologic features of Whipple disease. In one series, positive PCR results were obtained from saliva samples of 14 of 40 (35%) apparently healthy persons (10). In another series, positive PCR results were reported from intestinal biopsy or gastric juice samples of 14 of 105 (13%) patients undergoing elective endoscopy for reasons other than suspicion of Whipple disease (11). These investigators have speculated that T. whippelii is a commensal of the human gastrointestinal tract (10, 11). At the same time, several published series on diagnostic PCR have found no evidence of T. whippelii DNA in the intestinal biopsy specimens of controls (12-14). These conflicting and confusing data and the treatable nature of this disease emphasize the need for additional information on the prevalence of T. whippelii in the intestinal mucosa of persons without evidence of Whipple disease by classic diagnostic methods, such as PAS staining or electron microscopy. Such information would provide further insights into the epidemiology and pathogenesis of the disease and would be crucial information with which to judge the indications for and interpretations of diagnostic PCR results. Thus, we examined intestinal biopsy specimens from three groups of patients. Two of the groups were prospectively examined: Patients underwent endoscopy for reasons other than Whipple disease in the first group and for work-up of malabsorption in the second group. In addition, we evaluated a third groupcases without histologic evidence for Whipple disease among intestinal biopsy specimens submitted to Stanford University (Stanford, California), the Mayo Clinic (Rochester, Minnesota), or the University of Heidelberg (Heidelberg, Germany) for diagnostic PCR testing for T. whippelii. Methods Patients and Samples Endoscopic biopsy specimens of the small intestine in patients from three categories were examined by using routine histologic methods, including PAS staining, and PCR testing for T. whippelii. Group A consisted of 173 patients who underwent upper gastrointestinal endoscopy for evaluation of dyspepsia, abdominal pain, or possible gastroesophageal reflux or peptic ulcer disease, without signs of malabsorption or consideration of Whipple disease. Group B consisted of 37 patients who underwent endoscopy in the work-up for malabsorption (endoscopy was prompted by unexplained weight loss or chronic diarrhea, for example, or by the referring gastroenterologists concern about protein or fat malabsorption). Biopsy specimens from groups A and B were obtained prospectively at the Mayo Clinic. Patients gave informed consent to allow additional specimens to be obtained for PCR testing for T. whippelii. Group C consisted of 132 patients who underwent endoscopy for clinical suspicion of Whipple disease, based on intestinal or extraintestinal findings or both. The patients in group C were selected only if histologic studies (including PAS staining) of intestinal biopsy specimens gave no evidence of Whipple disease. The specimens in group C were submitted to the University of Heidelberg (70 specimens), Stanford University (30 specimens), and the Mayo Clinic (32 specimens). All biopsy specimens used in PCR analysis from groups A and B, as well as those from group C that were studied at the Mayo Clinic, were fresh-frozen samples; those from group C submitted to the University of Heidelberg and Stanford University were either fresh-frozen or formalin-fixed, paraffin-embedded specimens. PCR Testing The PCR assays performed at our institutions have been described previously in detail (2, 12, 13, 15). Briefly, assays were performed by using nonnested PCR protocols with an analytic sensitivity of 10 to 100 spiked copies of cloned T. whippelii 16S rDNA per reaction (12, 13, 15). The specificity of these assays has been verified by testing on a wide variety of other bacteria, including many closely related organisms and on control tissues from patients with other diseases (12, 13). Positive PCR results were confirmed by oligonucleotide hybridization or direct sequencing (12, 13, 15). In terms of diagnostic sensitivity, PCR testing gave positive results with histologically positive samples in 95 of 96 patients tested at our institutions. This series comprises 60 previously described patients (12, 13), as well as 36 previously unreported cases. Different rooms were used for pre- and post-PCR procedures at each of the three institutions. To assess the adequacy of DNA extraction and the presence of PCR inhibitors, all samples were tested by using PCR for human DNA (-globin or c- myc), as described elsewhere (12, 13). Results None of the intestinal biopsy specimens in groups A, B, and C showed histologic evidence of Whipple disease, as defined by previous histologic descriptions and criteria (5, 16). All specimens from the 173 patients in group A and the 37 patients in group B yielded negative PCR results for T. whippelii DNA (Table) and positive results for human DNA. In group C, all intestinal biopsy specimens from 70 patients tested at the University of Heidelberg, 30 patients tested at Stanford University, and 32 patients tested at the Mayo Clinic were negative for T. whippelii DNA (Table), and all were positive for human DNA. Symptoms in this group included diarrhea; arthritis; unexplained weight loss; and central nervous system signs, including cerebellar ataxia and features of hypothalamic disease. Included in this group was one patient with cerebellar ataxia who had PCR-positive cerebrospinal fluid. Table. Results of Polymerase Chain Reaction Analysis for Tropheryma whippelii in Small-Intestinal Biopsy Samples Discussion The distribution and abundance of the Whipple disease bacterium, T. whippelii, in nature remain poorly defined. Dependence on clinical recognition of this disease, and hence on its typical clinical features, may have led to biased and limited surveillance for this organism. Our data address this problem to some degree and allow an important comparison to be made with findings recently published by two groups that suggest a surprisingly high prevalence of T. whippelii DNA in the upper alimentary tract of persons without findings typical of Whipple disease (10, 11). We examined duodenal biopsy specimens from three different sets of patients. Group A represents patients with normal duodenal histologic findings who underwent endoscopy for reasons other than suspicion of Whipple disease and whose probability of harboring T. whippelii might be considered at most only slightly higher than that of the normal population. Groups B and C might be considered to have had a higher pretest probability of harboring T. whippelii because symptoms and clinical findings consisted of malabsorption, one of the cardinal symptoms of Whipple disease, or otherwise led to the inclusion of Whipple disease in the differential diagnosis. No evidence of T. whippelii DNA was found in any of the intestinal biopsy specimens of the three groups in this study. It should be mentioned that a previously tested intestinal biopsy sample from a patient with negative intestinal histologic findings examined at Stanford University yielded a positive PCR result for T. whippelii DNA, as reported elsewhere (15). This patient had uveitis, and positive results were also obtained at the extraintestinal site (ocular vitreous fluid) by PAS staining, electron microscopy, and PCR. In addition, two other previously examined patients with suspected Whipple disease and negative intestinal histologic findings had PCR results positive for T. whippelii (13). One of these patients had suggestive histologic findings in a lymph node. Thus, the results of this study and previous work performed at our institutions offer several insights. First, T. whippelii DNA was not detected in the histologically negative intestinal mucosa of patients for whom Whipple disease was not considered (group A). Second, Whipple disease was not a common diagnosis in patients with malabsorption (group B), despite the common occurrence of malabsorption in this disease and the importance of including Whipple disease in the differential diagnosis. Third, T. whippelii DNA may be detected in intestinal mucosa in the absence of histopathologic evidence, but the rate at which this was observed is very low,

Clinical Spectrum and Laboratory Characteristics Associated With Detection of Herpes Simplex Virus DNA in Cerebrospinal Fluid

OBJECTIVE To determine the clinical, neurologic, and laboratory characteristics of patients with herpes simplex virus (HSV) type 1 (HSV-1) or HSV type 2 (HSV-2) DNA detected in cerebrospinal fluid (CSF) with use of polymerase chain reaction. PATIENTS AND METHODS Clinical, laboratory, and demographic data were determined from 249 CSF specimens (collected from 247 patients >10 years of age) that tested positive for HSV-1 or HSV-2 DNA at the Mayo Clinic from January 1999 to August 2000. RESULTS The median age of the 200 patients whose age was available was 70 years vs 40 years for those with HSV-1 or HSV-2 DNA in CSF, respectively. Detailed data were available for 39 and 78 patients with positive polymerase chain reaction results for HSV-1 and HSV-2, respectively. Of those with HSV-1 DNA detected in CSF, 89% had encephalitis, whereas most patients with HSV-2 DNA detected in CSF had findings compatible with meningitis. Only 5 (7%) of 69 patients in whom HSV-2 was detected in CSF had genital lesions at presentation, and none of the assessable patients with HSV-2 who had recurrent meningitis had active genital lesions at presentation. CONCLUSION The vast majority (82%) of patients with HSV-2 detected in CSF had no history of genital herpes and no lesions at the time of presentation. Polymerase chain reaction assays designed to detect HSV in CSF should detect HSV-1 and HSV-2 and differentiate between HSV-1 and HSV-2.

Comparison of the Abbott RealTime Human Immunodeficiency Virus Type 1 (HIV-1) Assay to the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test: Workflow, Reliability, and Direct Costs

ABSTRACT The Abbott RealTime human immunodeficiency virus type 1 (HIV-1) assay (ART) and the Cobas AmpliPrep/Cobas TaqMan HIV-1 test (CTM) are commercially available assays for quantification of HIV-1 RNA in plasma. We evaluated performance characteristics, workflow, throughput, reliability, and direct costs of these assays. Both assays yielded good correlation of quantitative results (r = 0.95) among clinical specimens, with a mean difference of −0.34 log10 copies/ml. Testing of healthy donor plasma specimens yielded “target not detected” results by ART, with “HIV-1 RNA detected, <40 copies/ml” results for 3.3% (3 of 90 samples) of these specimens by CTM. Both the m2000sp/m2000rt (ART) and docked CAP/CTM96 (CTM) instrument systems were capable of operating with continuous, uninterrupted workflow. When daily maintenance and cleaning were included, ART and CTM run durations (5 h 52 min and 6 h 4 min, respectively) and hands-on times (53 min and 46 min, respectively) were similar for a run batch size of 24. While ART was more flexible in terms of run batch size, CTM required fewer user interventions and consistently produced higher specimen throughput rates at 8, 16, and 24 h. Assay run failure rates were 6.3% (1 of 16 runs) and 4.2% (1 of 24 runs) for ART and CTM, respectively (P = 1.000), with invalid specimen result rates of 1.0% (5 of 495 specimens) and 2.8% (11 of 399 specimens), respectively (P = 0.073). Direct reagent and consumable costs for each assay were comparable (difference of <10%). In selecting an assay for implementation, laboratories should consider how various assay and instrument features might impact laboratory operation and patient care.

Evaluation of the COBAS TaqMan HCV Test with Automated Sample Processing Using the MagNA Pure LC Instrument

ABSTRACT The COBAS TaqMan HCV Test (TaqMan HCV; Roche Molecular Systems Inc., Branchburg, N.J.) for hepatitis C virus (HCV) performed on the COBAS TaqMan 48 Analyzer (Roche Molecular Systems) currently relies on a manual sample processing method. Implementation of an automated sample processing method would facilitate the clinical use of this test. In this study, we evaluated the performance characteristics of TaqMan HCV following automated sample processing by the MagNA Pure LC instrument (MP; Roche Applied Science, Indianapolis, Ind.). The analytical sensitivity of TaqMan HCV following sample processing by MP was 8.1 IU/ml (95% confidence interval, 6.1 to 15.2). The assay showed good linearity (R2 = 0.99) across a wide range of HCV RNA levels (25 to 5 × 106 IU/ml), with coefficients of variation ranging from 10% to 46%. Among 83 clinical specimens, the sensitivity and specificity of TaqMan HCV were 100% and 95%, respectively, when compared to the COBAS AMPLICOR hepatitis C virus test, version 2.0 (COBAS AMPLICOR; Roche Molecular Systems), with TaqMan HCV detecting two more HCV RNA-positive specimens than COBAS AMPLICOR. Both specimens were confirmed to be HCV RNA positive by the VERSANT HCV RNA qualitative test (Bayer HealthCare LLC, Tarrytown, N.Y.). There was also strong correlation (R2 = 0.95) and good agreement between the results from TaqMan HCV and the VERSANT HCV RNA 3.0 assay (bDNA) (Bayer HealthCare LLC) among a group of 93 clinical specimens. The MP is a versatile, labor-saving sample processing platform suitable for reliable performance of TaqMan HCV.

Evaluation of the invader assay for genotyping hepatitis C virus.

ABSTRACT The Invader 1.0 assay (Invader HCV Genotyping Assay, version 1.0; Third Wave Technologies, Inc., Madison, WI) has been developed for the rapid differentiation of hepatitis C virus (HCV) genotypes 1 to 6 based on sequence variation within the HCV 5′ noncoding (NC) region. In the present study, we evaluated the compatibility of Invader 1.0 with the COBAS MONITOR (COBAS AMPLICOR HCV MONITOR Test, version 2.0; Roche Molecular Systems, Inc., Branchburg, NJ), COBAS AMPLICOR (COBAS AMPLICOR Hepatitis C Virus Test, version 2.0; Roche Molecular Systems, Inc.), and COBAS TaqMan (COBAS TaqMan HCV Test; Roche Molecular Systems, Inc.) assays. The minimum HCV RNA titers required for successful HCV genotyping (≥90% success rate) were 1,000 IU/ml for COBAS MONITOR, 100 IU/ml for COBAS AMPLICOR, and 10 IU/ml for COBAS TaqMan. Invader 1.0 results obtained from unpurified COBAS TaqMan amplification products of 111 retrospectively selected clinical serum specimens (genotypes 1 to 6, with virus titers ranging from 15.1 to 2.1 × 107 IU/ml) showed 98% concordance with results obtained from the TRUGENE HCV 5′ NC Genotyping Kit (Bayer HealthCare LLC, Tarrytown, NY), used in conjunction with COBAS AMPLICOR. Although the assay is sensitive, accurate, and easy to perform, additional optimization of the Invader 1.0 interpretive software (Invader Data Analysis Worksheet) may be necessary to reduce potential misidentification of HCV genotypes in low-titer specimens. In summary, Invader 1.0 is compatible with a variety of commercially available PCR-based HCV 5′ NC region amplification assays and is suitable for routine HCV genotyping in clinical laboratories.

Evaluation of the MagNA Pure LC Instrument for Extraction of Hepatitis C Virus RNA for the COBAS AMPLICOR Hepatitis C Virus Test, Version 2.0

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.

Quantification of Hepatitis B Virus (HBV) DNA with a TaqMan HBV Analyte-Specific Reagent following Sample Processing with the MagNA Pure LC Instrument

ABSTRACT TaqMan hepatitis B virus (HBV) analyte-specific reagent (ASR; Roche Molecular Systems, Inc., Branchburg, NJ) is designed for the quantification of HBV DNA in serum or plasma. The performance characteristics of TaqMan HBV ASR following automated sample processing with the MagNA Pure LC instrument (MP; Roche Applied Science, Indianapolis, IN) were evaluated in this study. Analytical sensitivity and precision were assessed with commercially available HBV standards, while clinical serum specimens from HBsAg-seropositive patients and healthy blood donors were used to determine clinical sensitivity, specificity, and correlation with other commercially available assays. Analytical studies yielded a limit of detection of 2.4 IU/ml, with good linearity and correlation (R2 = 0.9958) with expected HBV DNA titers over a wide range (6.0 × 100 to 2.1 × 108 IU/ml). Clinical sensitivity and specificity of the assay combined with automated sample processing were both 100%. Comparison of TaqMan HBV ASR and VERSANT HBV DNA 3.0 assay (bDNA; Bayer HealthCare LLC, Tarrytown, NY) results among clinical specimens yielded good correlation (R2 = 0.9237), with a mean difference in titer of −0.213 log10 IU/ml (95% confidence interval, −0.678 to 1.10 log10 IU/ml). The overall test failure rate was 2.0% among 204 clinical serum specimens tested. Total time required for MP sample processing and automated postelution handling of 24 samples was 224 min, with 57 min of actual hands-on time. MP is a reliable, labor-saving platform suitable for use with TaqMan HBV ASR, providing sensitive and accurate quantification of HBV DNA levels over a range of 8 log10 IU/ml.

Diagnostic detection of herpes simplex and hepatitis C viral amplicons by capillary electrophoresis: Comparison with southern blot detection

Background: As a result of the large number of DNA-based clinical assays, there is intense interest in making polymerase chain reaction (PCR)-amplified DNA product analysis faster, more cost-effective, and more automated. Methods and Results: In this study, an evaluation of the use of capillary gel electrophoresis with laser-induced fluorescence detection is described as a means of analyzing postamplification PCR products from clinical specimens. Sixty-six herpes simplex virus and 152 hepatitis C virus amplicons were analyzed after PCR and reverse transcription PCR, respectively. It is shown that the use of a physical gel buffer system in a short capillary in conjunction with laser-induced fluorescence detection allows for sensitive detection of herpes simplex virus- and hepatitis C virus-specific DNA fragments in an expedient manner. Interinstrument and intercapillary reproducibility of the migration time was evaluated and found to be excellent. The advantages and disadvantages over agarose gel electorphoresis-Southern blot analysis are summarized. Conclusions: The advantages offered by capillary gel electrophoresis with laser-induced fluorescent detection including rapid and sensitive analysis, ease of setup, reduced cost, and possibility for automation, make this procedure a viable alternative to more labor-intensive agarose gel electrophoresis-Southern blot analysis as molecular diagnostic methodology.

The cobas® 6800/8800 System: a new era of automation in molecular diagnostics.

ABSTRACT Introduction: Molecular diagnostics is a key component of laboratory medicine. Here, the authors review key triggers of ever-increasing automation in nucleic acid amplification testing (NAAT) with a focus on specific automated Polymerase Chain Reaction (PCR) testing and platforms such as the recently launched cobas® 6800 and cobas® 8800 Systems. The benefits of such automation for different stakeholders including patients, clinicians, laboratory personnel, hospital administrators, payers, and manufacturers are described. Areas Covered: The authors describe how molecular diagnostics has achieved total laboratory automation over time, rivaling clinical chemistry to significantly improve testing efficiency. Finally, the authors discuss how advances in automation decrease the development time for new tests enabling clinicians to more readily provide test results. Expert Commentary: The advancements described enable complete diagnostic solutions whereby specific test results can be combined with relevant patient data sets to allow healthcare providers to deliver comprehensive clinical recommendations in multiple fields ranging from infectious disease to outbreak management and blood safety solutions.

Impact of the COBAS AmpliPrep/COBAS AMPLICOR HIV-1 MONITOR Test, Version 1.5, on Clinical Laboratory Operations

ABSTRACT The COBAS AmpliPrep/COBAS AMPLICOR HIV-1 MONITOR Test, version 1.5 (CAP/CA), and the COBAS AMPLICOR HIV-1 MONITOR Test, version 1.5, were compared. CAP/CA reduced and consolidated labor while modestly increasing assay throughput without increased failure rates or direct costs, regardless of batch size and assay format.