Cobas Assay Research Articles

Evaluation of a novel domestic hepatitis B viral DNA quantitative fluorescence diagnostic kit

Objective Comparative performance characteristics of a novel domestic (Hunan Sansure) HBV DNA quantitative fluorescence diagnostic kit (PCR-fluorescence probing) and its counterpart Roche Cobas Ampliprep/COBAS Taqman HBV test kit. Methods Firstly,WHO international standard for HBV-DNA was diluted to 50,200,2000,20 000 IU/ml separately,and then all diluted samples were detected by both the novel domestic HBV DNA quantitative fluorescence diagnostic kit and Roche Cobas Ampliprep/COBAS Taqman HBV test kit to perform traceability analysis and to determine kits′reliability. Secondly,pre-diluted series of standard HBV B and C genotype plasma (25, 50, 200, 2000, 20 000, 200 000 IU/ml) were simultaneously performed 216 times for three-center detection by use of the domestic and Roche HBV DNA test kits separately.Accuracy and precision of those two types of HBV DNA kits were comparatively analyzed.Finally,a total of 42 clinical plasma samples including 5 negative HBV DNA and different concentration levels of 37 positive HBV DNA were detected by the above two types of kits to perform clinical quality evaluation. Results Traceability results showed that both HBV DNA test kits agreed with the HBV DNA WHO standard across all four titers tested and all absolute differences (observed mean HBV HBV-expected HBV DNA) were within 0.3 lg IU/ml.Accuracy results indicated that thedeviation of all observed values at 6 titers (both of HBV genotyping B and C) tested were within the acceptance criteria (0.005-0.280 lg IU/ml).Comparative performance of coefficient of variation of PCR assays to HBV genotypes B and C measured by both kits showed that there were no differences of precision were found (genotyping B: t=1.226,P=0.275; genotyping C: t=2.319,P=0.07).The clinical performance of the domestic assay compared to the COBAS assay had been assessed on a panel of 42 clinical specimens.The qualitative results indicated that the total concordant results between two tests were determined in 97.62% (41/42) of samples.Also,a significant correlation was observed between values tested by two HBV DNA kits in 37 HBV DNA positive samples (R2=0.934,P<0.0001). Conclusion No significant differences of the traceability,accuracy,precision and clinical detection are found between two kits.(Chin J Lab Med,2013,36:280-285) Key words: Hepatitis B virus; DNA; viral; Reagent kits; diagnostic; Eevaluation studies

Comparison of COBAS 4800 KRAS, PCR TaqMan, and PCR high-resolution melting assays for the detection of KRAS somatic mutations in formalin-fixed paraffin-embedded colorectal tumors.

103 Background: Colorectal cancer (CRC) is the 2nd most common cancer with more than one million new cases diagnosed every year. From 2006 to 2008, several studies showed the importance of the KRAS oncogene in the treatment of metastatic CRC (mCRC) and response to anti-EGFR therapies like cetuximab or panitumumab. KRAS is a small G protein which can bear activating mutations in 40% cases of mCRC. Numerous methods have been described for the detection of KRAS mutations in FFPE tissues. Direct sequencing is the gold standard but the most sensitive methods are real time PCR assays based. The new COBAS4800 KRAS developed by Roche Diagnostics is a CE IVD validated assay using real time PCR and TaqMelt technology. This assay is validated for the detection of 19 KRAS somatic mutations on exon 2 (codons 12/13) and exon 3 (codon 61) in specimen containing at least 5% of mutated tumoral cells. Methods: We compared COBAS with previously validated PCR TaqMan and PCR High Resolution Melting (HRM) assays. The PCR TaqMan and HRM are a real time PCR based assays. TaqMan can detect accurately 0.5 to 1% of mutated tumoral DNA on codon 12 (G12A, G12C, G12D, G12R, G12S and G12V) and on codon 13 (G13D). HRM can detect 5 to 10% of tumoral DNA on codon 12 and 13. 80 FFPE samples of colorectal tumors resections or biopsies have been collected and processed with the 3 assays. Results have been compared using kappa statistics. Results: On 80 samples, 69 were interpretable with COBAS and TaqMan and 62 with COBAS and HRM. No statistically significant difference between COBAS and TaqMan was found (k=0.942 ; p&lt;0.001). Only 2 discordant cases were found mainly regarding codon 61 mutations not detected with TaqMan. No statistically significant difference between COBAS and HRM was found (k=0.903 ; p&lt;0.001). Three discordant cases were found two regarding codon 61, not detected with HRM and one G12S mutation (detected by COBAS and TaqMan). Conclusions: The 3 assays can detect accurately KRAS mutations. Less ininterpretable results were found with COBAS and TaqMan assays. A pre screening with COBAS and mutation characterization with TaqMan seem to be a good scheme for routine diagnostic of KRAS mutations.

Evaluation of the Cobas 4800 HPV Test for Detecting High-Risk Human Papilloma-Virus in Cervical Cytology Specimens.

As new platforms for high-risk strains of human papillomavirus (HR HPV) testing are introduced into the clinical laboratory, it is important to verify their performance and agreement. In this validation study, post-aliquot cervical cytopathology specimens (n = 226) were used to analyze agreement between the Invader HPV ASR assay (Hologic) and the recently FDA-approved Cobas 4800 high-risk HPV assay (Roche). Residual sample from 92 Invader positive and 134 Invader negative samples were analyzed with the Cobas 4800 test. Discordant results were further analyzed by Linear Array HPV genotype testing (Roche). To assess intra- and inter-run precision, 31 Invader positive samples were run in duplicate on the Cobas 4800 by different operators over multiple days and purchased HR HPV DNA control was run in ten replicates. Cross-contamination during cytology processing was evaluated by spiking 6 Invader negative samples with different volumes of Acrometrix HPV High Risk Positive Control and analyzed on the Cobas with 4 negative samples in between. There was significant discordance between the assays (p < 0.001; exact McNemar X2 test), with overall agreement of 82%. Of the 92 Invader positive samples, 58 (63%) were positive with the Cobas assay, while 34 (37%) were negative. Of the 134 Invader negative samples, 6 (4%) were positive with the Cobas while 128 (96%) were negative. The observed discordance may be attributed to the previously described false positive rate of the Invader ASR assay. The Cobas 4800 high-risk HPV assay is a viable new tool for use in the clinical setting to identify high-risk HPV.

Emergence of lamivudine resistance hepatitis B virus mutations in pregnant women infected with HBV and HIV receiving antiretroviral prophylaxis for the prevention of mother‐to‐infant transmission in Malawi

HIV/HBV co-infection is highly prevalent in sub-Saharan Africa. The aim of this study was to determine if the use of triple combination lamivudine-containing prophylaxis for the prevention of mother-to-infant HIV transmission was associated with the emergence of lamivudine HBV mutations. The study included 21 pregnant co-infected women in Malawi who received either zidovudine or stavudine plus lamivudine and nevirapine from week 25 of gestation until 6 months after delivery or indefinitely if they met the criteria for treatment (CD4+ <350/mm(3)). HBV-DNA was determined using the Roche COBAS assay. Resistance mutations were assessed by the Trugene assay (Siemens Diagnostics). At baseline 33% of the women were HBeAg positive and had HBV-DNA > 10(4) IU/ml. Median CD4 count was 237 cells/mm(3) and median HIV-RNA was 3.8 log(10) copies/ml. After a median of 259 days of treatment, HBV-DNA was detectable in 9 out of 21 patients (42.8%). In three cases the HBV-DNA level was >10(4) IU/ml. Resistance mutations (M204I in five cases and L180M + M204I/V in one case) were present in 6 (28.6%) patients. Women with a resistant virus had significantly higher baseline HBV-DNA levels than those not developing resistance (1.1 × 10(7) IU/ml vs. 20.8 IU/ml, P = 0.022). Levels of ALT and AST were higher in women with resistant viruses compared to those retaining a wild-type virus. A high rate of lamivudine resistance was seen in this cohort of pregnant women. Follow-up of these patients will clarify if the presence of resistance has a significant impact on liver disease.

Detection of BRAF mutations in melanoma: Rate of mutation detection at codon 600 using Sanger sequencing as compared to the cobas 4800 method.

8596 Background: Detection of BRAF V600 mutations is currently a prerequisite for approved use of vemurafenib in patients with metastatic melanoma. The cobas 4800 BRAF V600 Mutation Test (Roche Molecular Diagnostics), a PCR-based assay approved to aid in selecting patients for vemurafenib therapy, primarily detects V600E. It is also reported to detect V600K, which has been associated with vemurafenib response as well. We compared the mutation detection rate of the cobas assay with that of Sanger sequencing. Methods: 125 de-identified FFPE tissues submitted for BRAF mutation analysis that all showed histologically-confirmed melanoma were tested. BRAF mutations were detected using both the cobas kit and bidirectional Sanger sequencing using BigDye kits (Applied Biosystems). DNA was extracted from 5-um sections without macrodissection using the cobas DNA extraction kit (for the cobas test) or from 5-10-um sections using Agencourt extraction kits (Beckman Coulter) following macrodissection. Results: The two methods showed agreement in 104/125 (83.2%) of cases (Table). Sanger sequencing detected V600 dinucleotide mutations in 9 samples that were negative by the cobas assay. Sanger sequencing produced no results in 10 cases owing to suboptimal PCR, including 2 that were positive by the cobas assay. The cobas assay produced 2 invalid results, including 1 that was positive for V600E by Sanger.The cobas assay detected 7/11 V600K mutations. Conclusions: Overall agreement between cobas and Sanger sequencing was 83.2%. The Sanger method had higher analytic sensitivity, resulting in nine additional V600 mutations not called by cobas compared to the two seen by cobas but not Sanger sequencing. Thus, 16% (9/57) more patients would be identified as candidates for vemurafenib therapy using the Sanger method. [Table: see text]

Challenges Ahead for Companion Diagnostics

T he U.S. Food and Drug Administration ’ s (FDA) approval of vemurafenib (Plexxikon/Roche) last August was greeted as a milestone not only for melanoma treatment but also for personalized medicine. The drug is indicated for patients who harbor a BRAF V600 → E mutation. Vemurafenib was developed along with a companion diagnostic, in a test case for how the FDA plans to handle personalized drugs from now on. In a draft guidance issued last July 14, the FDA defined companion diagnostics as devices that provide essential information for the safe and effective use of a corresponding therapeutic product. It stated that it would review personalized drugs only in tandem with the diagnostic that was used to select patients for clinical trials, adding that personalized drug labels should refer specifically to approved diagnostics for use in selecting patients for treatment. FDA offi cials emphasize that the approach is needed to ensure that personalized drugs are given only to likely responders. But as the experience with vemurafenib shows, the new method encroaches on entrenched diagnostic turf. Hospitals, drug companies, and professional medical societies have all reacted against the guidance, claiming that it locks researchers and clinicians into infl exible protocols that stifl e innovation and limit access to treatment. They say that laboratory-developed tests (LDTs) that individual institutions create and administer can adequately determine patient eligibility for personalized care. Vemurafenib ’ s creators at Plexxikon, a Berkeley, Calif. – based biotechnology company, rank among the guidance ’ s most outspoken critics; they claim it imposes excessive burdens on early-stage drug development. However, Plexxikon also anticipated diagnostic scrutiny from the FDA even before their BRAF inhibitor, then known as PLX-4032, had shown human proof-of-concept antitumor effects. So in 2005, to accelerate the drug ’ s development, Plexxikon teamed with Roche Molecular Diagnostics (RMD), in Pleasanton, Calif., on the development of an error-free system for detecting BRAF mutations. Producing the test took several years — partly because RMD had to devise a way to work with paraffi n-embedded, formalin-fi xed tumor samples, in which DNA is often damaged. From these efforts emerged the Cobas 4800 V600 Mutation Assay, a closed, PCRbased system that fl ags V600 → E mutations with a “yes” or “no” fl uorescence reaction. Plexxikon credits the test with propelling vemurafenib from discovery to approval in just 5 years, specifi cally by reducing the size of the trial population to V600 → E mutation carriers only. Vemurafenib ’ s label now indicates that because it was tested only in patients selected with the Cobas assay, doctors should avoid using it in BRAF-mutated patients selected using some other method. FDA spokesperson Erica Jefferson emphasized that the label doesn ’ t constitute regulation. “Product labels are intended to guide treatment decisions and provide clinical information,” she said. “The language is worded to allow for future approved tests for use in selecting patients for treatment.”

Comparison of Two Molecular Methods for Rapid Diagnosis of Extrapulmonary Tuberculosis

Application of real-time PCR for the detection of Mycobacterium tuberculosis enables results to be obtained in about 2 h. A total of 340 nonrespiratory samples were processed using two real-time PCR assay kits: Xpert MTB/RIF and Cobas TaqMan MTB. The sensitivity and specificity of the Xpert assay were 95% and 100%, respectively, compared to 78% and 98% for the Cobas assay.

Comparative RNA Quantification of HIV-1 Group M and Non-M With the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 v2.0 and Abbott Real-Time HIV-1 PCR Assays

A new version of the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 assay (CA/CTM v2.0) has been introduced to overcome the underquantification observed with the first version. We compared the Roche Cobas CA/CTM v2.0 and Abbott RealTime HIV-1 assays for HIV-1 group M and non-M viral load measurement. We found a good correlation (r = 0.96) between the 2 techniques for the 260 HIV-1 group M plasma samples tested. The Roche Cobas assay gave significantly higher values than the Abbott assay, and 51 samples (20%) yielded differences greater than 0.5 log10 copies per milliliter. Conversely, 2 samples were more than 0.5 log10 copies per milliliter higher with the Abbott assay than with the Roche Cobas assay. Among the 84 samples with undetectable viral load in the Abbott assay (detection limit 40 copies/mL), 17 (20%) were detectable with the CA/CTM v2.0 assay (detection limit 20 copies/mL), with values ranging from 41 to 897 copies per milliliter. Extrapolation of the Abbott curves led to 10/17 (59%) of these samples being quantifiable. HIV-1 groups O and P were similarly quantified by the two techniques. The results of the Roche Cobas CA/CTM v2.0 and Abbott RealTime HIV-1 assays correlate well. The new version of the CA/CTM assay shows improved sensitivity. Nevertheless, the 2 assays differ by more than 0.5 log₁₀ copies per milliliter for some samples.

Performance of the Automated COBAS AmpliPrep/COBAS TaqMan HIV-1 Test on a Genetically Diverse Panel of Specimens from China: Comparison to the COBAS Amplicor HIV-1 Monitor Test, v1.5

Objectives: To investigate the impact of genetically diverse HIV samples from China on the performance of the automated COBAS® AmpliPrep/COBAS TaqMan® HIV-1 Test (CAP/CTM). Methods: 185 samples from untreated HIV-1-infected patients were used to assess the performance of the CAP/CTM Test and COBAS Amplicor HIV-1 Monitor Test, v1.5 (Cobas). Results: A comparison of the qualitative results of both assays showed concordance for 1 negative and 184 positive samples (100%, ĸ = 1.000). A significant correlation (R = 0.862, p < 0.001) and high agreement (95.53%) was observed for 179 samples with viral loads within the dynamic ranges of both assays. For samples of clades predominant in China, the fitted regression line differed significantly from the line of equality, although significant correlations (R = 0.694–0.899, p < 0.05) and high agreements (91.30–95.35%) were found for the two assays. The mean differences for samples from clades B’ and BC were significant (p < 0.001) whilst no great difference (7.19–10.88%) between the quantitative values for both assays was found by plotting the regression line. Conclusion: The viral loads of different HIV genotypes in China measured by the CAP/CTM and Cobas assays were comparable.

Comparison of HCMV loads using in-house quantitative CMV PCR, Artus CMV TM PCR and COBAS Amplicor CMV monitor test.

A real-time PCR assay based on highly conserved cytomegalovirus glycoprotein B (CMV-gB) gene was developed to quantify the CMV genome loads in human plasma obtained from immunocompromised patients. The sequence comparison from several strains resulted in the best possible set of primers and a probe that directed against the most highly conserved region. The detection limit of In-house TaqMan polymerase chain reaction assay for CMV-DNA was 2.3 log10 copies/ml and the linear measure interval was 2.3 to 8.3 log10 copies/ml (R > 0.99). The In-house TaqMan assay was compared to commercial quantitative PCR test, COBAS Amplicor CMV monitor test and Artus CMV TM PCR test, in the determination of CMV DNA load in plasma samples. The correlation between In-house TaqMan and COBAS assays was statistically significant, R = 0.915 (P < 0.05; n = 27). Also correlation between In-house TaqMan and Artus CMV assays was statistically significant and higher (R = 0.998; P < 0.05; n = 8). When the In-house TaqMan system was evaluated with the Nucleospin ® RNA virus kit and QIAamp blood minikit, no significant difference was found at the 98.2% confidence interval. Preliminary results indicate that the In-house TaqMan real-time PCR with the designed primer and probe set can be an alternative tool for quantification of CMV loads in clinical routine laboratories.

Performance characteristics of two real-time PCR assays for quantification of hepatitis B virus DNA

Detection and quantification for hepatitis B virus (HBV) DNA has been an essential tool in the clinical setting. We aimed to assess clinical performance of the RealArt HBV TM PCR (RealArt) assay and the COBAS TaqMan HBV (COBAS) assay. Serum levels of HBV DNA in 146 treatment-naïve chronic HBV (CHB) Taiwanese patients (118 males, 47 HBeAg + ; mean age, 34.7+/-13.0 y) were determined by both assays. The detection rate by the RealArt assay was 85.6% (125/146), which was not significantly different from the COBAS assay (89.7%, 131/146). The detection rate was also not significantly different between both assays irrespective of HBeAg seropositivity. The 2 assays were also comparable regarding quantification rate (92.8%, 116/125 vs 93.1%, 122/131). There was a positive correlation in the 109 specimens measurable by both assays (r=0.94,p<0.001). The mean HBV DNA level measured by the COBAS assay was significantly higher than the RealArt assay (5.24+/-1.83 vs 4.79+/-2.09 log IU/ml, p<0.001). This study demonstrated that both RealArt and COBAS assays were comparable regarding clinical performance in HBV DNA measurement.

A novel real-time PCR to detect Chlamydia trachomatis in first-void urine or genital swabs

Screening for Chlamydia trachomatis infections can be performed on urine samples and genital swabs using molecular techniques. A novel approach was developed that combined an automated extraction procedure, an automated liquid-handling system and real-time PCR to detect C. trachomatis from urine or swabs. This novel real-time PCR approach was compared to the commercial Cobas Amplicor system on 628 specimens. In a retrospective analysis, 51 samples that tested positive using the Cobas assay were also positive with the real-time PCR, whereas the 49 samples negative with Cobas were also negative with the real-time PCR, for an overall agreement of 100 %. Among 528 prospective samples consecutively received at the authors' laboratory with a request for C. trachomatis PCR, five PCR reactions were inhibited when tested with Cobas. These five inhibited samples were found negative with the real-time PCR. Among the remaining 523 samples, 45 (8.6 %) were positive with both methods, 476 (91 %) were negative with both methods, and 2 (0.4 %) were positive with Cobas but negative with the real-time PCR. Thus, when considering Cobas as the gold standard, the overall agreement was 99.6 %, the sensitivity of the real-time PCR was 95.7 % and the specificity was 100 %. The two discrepant samples were retested in parallel and were found negative with both methods. When testing a batch of 25 samples, both reagent costs and laboratory technician time were reduced with the new technique (7.30 euros per sample and 134 min) compared to Cobas (11.20 euros per sample and 232 min). Moreover, due to reduced organizational constraints, the median time from sample reception to result was only 24 h using the automated platform. Overall, this novel real-time PCR approach exhibited an excellent specificity and a sensitivity similar to that of Cobas Amplicor PCR for the detection of C. trachomatis. Given its high throughput potential and low costs/laboratory technician time requirement, it may be useful for future use in large C. trachomatis screening programs.

High prevalence of occult hepatitis B in Baltimore injection drug users

Occult hepatitis B is defined by the presence of hepatitis B virus (HBV) DNA in a serum or liver in the absence of hepatitis B surface antigen (HBsAg). The prevalence and clinical correlates of occult hepatitis B remain incompletely defined. A cross-sectional study was performed to determine the prevalence of occult hepatitis B in a high-risk cohort composed of 188 injection drug users in Baltimore, Maryland. All individuals had chronic hepatitis C viral infections confirmed by RNA detection and liver biopsy. Serologic assays for HBsAg and core antibody (HBcAb) were performed. Serum HBV DNA was detected using the COBAS HBV AMPLICOR monitor assay (lower limit of detection, 200 HBV copies per milliliter) and a semi-nested polymerase chain reaction (PCR) assay (lower limit of detection, 15 HBV copies per milliliter). Although almost all individuals (96%) were anti-HBC positive, only 8 of 188 (4%) were HBsAg positive. Occult hepatitis B was not identified using the COBAS assay, but was found in 81 of 180 (45%) of individuals using semi-nested PCR. Of the 8 HBsAg positive individuals, HBV DNA was found in 1/8 using the COBAS assay and 6/8 using the nested PCR assay. Overall, liver disease was mild, with a median serum alanine aminotransferase (ALT) of 38 IU/L, median activity grade of 3/18, and median fibrosis stage of 1/6. No association was found between the serum AST (aspartate aminotransferase), activity grade, or stage of liver disease and the presence of occult hepatitis B. Serum ALT levels were slightly higher in patients without occult hepatitis B (46 vs. 35 IU/L), and the median years since first injection drug use was somewhat longer in those without occult hepatitis B (24 vs. 20 years). In conclusion, although further research is needed to assess its clinical significance, there is a high prevalence of occult HBV infection in this cohort of HCV-infected injection drug users.

Multicenter quality control for the detection of hepatitis C virus RNA in seminal plasma specimens.

The discrepant results available in the literature about the presence of hepatitis C virus (HCV) RNA in seminal plasma of men chronically infected by this agent are related, at least in part, to the molecular techniques used and particularly to the wide range of protocols dedicated to RNA extraction. In order to evaluate these protocols and to standardize the method of detection of HCV RNA in this fluid, a panel of coded specimens was tested blindly in 12 French laboratories; it included 14 seminal plasma specimens and four water controls spiked with HCV RNA ranging from 10 to 20000 IU/ml and two HCV-negative seminal plasma specimens. The extraction step was performed according to methods using either silica beads (NucliSens [Organon Teknika S.A., Fresnes, France]; RNA viral kit [Qiagen, Courtaboeuf, France]) or guanidinium thiocyanate (Amplicor HCV assay; Roche Diagnostics, Meylan, France), preceded or not by a centrifugation of the seminal plasma. For the amplification step, all the laboratories performed the same reverse transcription-PCR technique (Amplicor HCV Cobas assay). The percentage of correct results ranged from 53.3 to 100, the poorest results being obtained when no centrifugation step preceded the Amplicor extraction protocol. The rate of correct results was significantly higher in laboratories using a preliminary centrifugation of the specimen (P = 0.034 by chi-square test). By contrast, the overall number of correct results was not correlated to the initial volume of sample used for the test. These results allowed us to validate standardized techniques adapted to the performance of this test on a routine basis, especially in men infected with HCV and involved in programs of medically assisted reproduction.

Assay for Free and Total Carnitine in Human Plasma Using Tandem Mass Spectrometry

Measurements of free and total carnitine in plasma are important in the diagnosis and clinical management of patients with carnitine deficiency syndromes and certain inborn errors of metabolism (1). The most popular analytical methods are based on the enzymatic reaction in which an acetyl group is transferred from acetyl-CoA to carnitine with release of free CoA (2). A spectrophotometric assay based on this method, using a Cobas centrifugal analyzer (Roche Diagnostics), has been in use in this laboratory for the past several years (3). Limitations of the Cobas enzymatic method in our view include the initial filtration step, which requires a minimum sample volume of 300 μL, and the number and cost of the reagents required. Assay results are critically dependent on two labile reagents, the enzyme carnitine acetyltransferase and the color reagent 5,5′-dithiobis(2-nitrobenzoic acid). We have also observed that patients on valproic acid therapy sometimes give total carnitine values that are spuriously low for reasons that are not clear. Another limitation of the Cobas assay is that acylcarnitines with chain lengths >10 are lost in the filtration step (3), and the method will underestimate the total carnitine content of patients with defects in the oxidative degradation of long-chain or multiple acyl-CoAs, especially when metabolically decompensated. We have emphasized previously that the most valuable applications of the routine Cobas assay are the identification of patients with carnitine deficiency and the monitoring of patients on carnitine therapy (3). We have developed a new carnitine assay method based on isotope-dilution tandem mass spectrometry (TMS), which is inherently more straightforward and accurate than previously reported methods. This is true primarily because it has absolute molecular specificity, uses an isotope-labeled internal standard to compensate for any losses or variance resulting from the sample preparation, and has no known chemical interference. The new assay …

Evaluation of the COBAS AMPLICOR CMV MONITOR test for detection of viral DNA in specimens taken from patients after liver transplantation.

Detection of cytomegalovirus (CMV) DNA in blood by PCR is a sensitive method for the detection of infection in patients posttransplantation. The test, however, has low specificity for the identification of overt CMV disease. Quantitative CMV PCR has been shown to overcome this shortcoming. The COBAS AMPLICOR CMV MONITOR test was evaluated by using consecutive serum and peripheral blood mononuclear cell (PBMN) samples from liver transplant patients. Twenty-five patients had CMV viremia (by shell vial cell culture assay) and/or tissue-invasive disease (by biopsy); 20 had no active infection. A total of 262 serum and 62 PBMN specimens were tested. Of 159 serum specimens from patients with overt CMV infection, the COBAS assay detected CMV DNA in 21 patients (sensitivity, 84%). Only 1 of 103 samples from patients with no evidence of active infection had detectable CMV DNA (341 copies/ml). By comparison of 62 matching serum and PBMN samples by the same assay, 12 PBMN samples were exclusively positive, whereas only 2 serum samples were exclusively positive (P < 0.05). At the time of clinical CMV infection, viral copy numbers were higher in PBMNs than serum from four of five patients. The COBAS AMPLICOR CMV MONITOR test is a sensitive and specific test for the quantitative detection of CMV DNA in blood. Clinical applications of the assay will require further validation with samples from a larger population of transplant patients.

Automated Analysis for Free and Short-Chain Acylcarnitine in Plasma with a Centrifugal Analyzer

We describe a fully automated, spectrophotometric assay of free and total carnitine in plasma ultrafiltrates. The method, suitable for routine application in most hospital laboratories, incorporates the hydrolysis of acylcarnitines to free carnitine within the program of a Cobas Fara II centrifugal analyzer. The hydrolysis is monitored and calibrated with standard solutions containing octanoylcarnitine. Results correlated well with those from a reference isotope-dilution mass spectrometric assay. The ability to analyze a batch of samples for both free and total carnitine within 90 min enables analysis of > or = 100 samples per day. Used in conjunction with acylcarnitine species identification by mass spectrometry, the Cobas assay facilitates the diagnosis of carnitine-deficiency syndromes and specific metabolic disorders.