Evaluation of a novel internally controlled real-time PCR assay targeting the 16S rRNA gene for confirmation of Neisseria gonorrhoeae infections.

A novel HybProbe real-time LightCycler PCR assay was developed for confirmation of Neisseria gonorrhoeae in samples positive according to the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae PCR assay. The new assay amplifies 375 bp of the N. gonorrhoeae 16S rRNA gene and includes an internal amplification control introduced during DNA purification. The assay had 100% specificity because of the high specificity of the HybProbes and primers. Other Neisseria spp. failed to generate positive crossing-point values and melting peaks. The analytical sensitivity for N. gonorrhoeae DNA was 0.5 fg/PCR, corresponding to 0.3 CFU/PCR. Sensitivity was not impaired in the presence of higher DNA concentrations (>or=1000-fold) from Neisseria spp. other than N. gonorrhoeae. The sensitivity was similar to that reported for the COBAS AMPLICOR assay with cervical swab samples. To assess its clinical applicability as a confirmatory test, 38 (2.9%) of 1313 swabs that were positive according to the COBAS AMPLICOR assay were tested using the new in-house assay and the commercially available GenFlow Neisseria test. Twenty-one samples negative according to COBAS AMPLICOR also underwent confirmatory testing. Both confirmatory tests yielded identical results; the 21 negative samples remained negative, and only 11 (28.9%) of the samples positive according to COBAS AMPLICOR were positive after retesting, suggesting a low prevalence (0.84%) of N. gonorrhoeae infection in the study population. These data suggest that the novel real-time PCR assay is an excellent and easy to interpret confirmatory test for the existing COBAS AMPLICOR assay for N. gonorrhoeae.     

Evaluation of HbA1c on the Roche COBAS Integra 800 closed tube system

OBJECTIVES: Evaluate a new whole blood (WB) HbA1c immunoassay and system with closed tube sampling (CTS) capability. DESIGN AND METHODS: Compare the Tina-quant Haemoglobin A1c Gen.2 (A1C-2) application on the COBAS INTEGRA 800 (I800) and new I800 dedicated system with CTS capability to current Integra applications and a HbA1c method accurate with common haemoglobin (Hb) variants. RESULTS: CVs were < or =1.7%. Mean bias against National Glycohaemoglobin Standardization Program (NGSP) samples was 0.3 HbA1c %. Compared to the Hitachi Tina-quant(R) [a] HbA1c II (HbA1c II) assay (accurate with common Hb variants), mean bias was 0.04% and 0.21% HbA1c at 6% and 9%, respectively, with Hb AS variants; and -0.01% and 0.26% HbA1c at 6% and 9%, respectively, with Hb AC variants. CONCLUSIONS: The Integra A1C-2 application is precise, accurate against NGSP-assigned samples and the Hb variants tested; and, the I800 dedicated system with CTS capability offers increased throughput and reduced sample handling.     

Evaluation of COBAS AmpliPrep nucleic acid extraction in conjunction with COBAS AmpliScreen HBV DNA, HCV RNA and HIV-1 RNA amplification and detection

BACKGROUND AND OBJECTIVES: This report describes the evaluation of the COBAS AmpliPrep instrument for fully automated generic nucleic acid extraction in conjunction with hepatitis B virus (HBV) DNA, hepatitis C virus (HCV) RNA, and human immunodeficiency virus (HIV)-1 RNA COBAS AmpliScreen amplification and detection using serial dilutions of the WHO international standards (IS) and the PeliCheck reference panels. MATERIALS AND METHODS: Serial diluted samples of the WHO IS and the PeliCheck reference panels were tested 24 times to determine the HBV DNA, HCV RNA and HIV-1 RNA detection limits by Probit analysis. The existence and extent of cross-contamination were assessed by testing alternating high titre HBV DNA-positive and -negative samples. The specificity of the AmpliPrep-AmpliScreen test for HBV was determined by testing 232 minipools consisting of six donations, all negative for HCV/HIV-1 nucleic acid testing (NAT) and HBsAg. In addition, a HBV genotypes A-G panel was tested. RESULTS: The respective 95% detection limits (and 95% CI) on the WHO IS and on the PeliCheck reference panels were 6.7 (4.3-13) IU/ml and 123 (68-301) gEq/ml for HBV DNA, 23 (11-106) IU/ml and 126 (84-233) gEq/ml for HCV RNA, and 187 (108-422) IU/ml and 183 (108-434) gEq/ml for HIV-1 RNA. Based on the WHO IS and the PeliCheck reference panels, no significant differences in sensitivity for HBV and HCV were found between AmpliPrep and the licensed MultiPrep extraction method. The sensitivity of AmpliPrep-AmpliScreen for HIV-1 was probably twofold lower as compared to the MultiPrep-AmpliScreen method. No cross contamination was observed. All 232 minipools were HBV NAT-negative. The AmpliPrep-AmpliScreen test for HBV detected HBV genotypes A-G with equal sensitivity. CONCLUSIONS: The AmpliPrep instrument combined with the AmpliScreen assays for HBV, HCV and HIV-1 is robust and suitable for NAT donor screening. The sensitivity criteria for HIV-1 and HCV as defined by the Paul Ehrlich Institute and the Food and Drug Administration for minipool NAT screening are met by this system. SINGLE SENTENCE SUMMARY: Generic COBAS AmpliPrep nucleic acid extraction in conjunction with COBAS AmpliScreen detection for HBV, HCV and HIV-1.     

Hepatitis C virus-polymerase chain reaction minipool testing: 3 years in the largest Swiss blood transfusion service

BACKGROUND AND OBJECTIVES: Hepatitis C virus-polymerase chain reaction (HCV-PCR) minipool testing can improve the safety of labile blood products owing to a reduction in the diagnostic preseroconversion window period. In Switzerland, HCV-PCR minipool testing for the release of labile blood components became mandatory in September 1999. In the largest Swiss blood transfusion centre, HCV-PCR minipool testing began in January 1999. This report analyses the performance of the test during a 3-year period: 1 January 1999 to 31 December 2001. MATERIALS AND METHODS: EDTA-blood was collected in either standard tubes or plasma preparation (PPT) tubes from 10 blood transfusion services in Switzerland and then sent to the Blood Transfusion Service SRC Berne. Up to 48 donor samples were pooled overnight using Tecan Genesis RSP 200/8 pipettors. Viral RNA was extracted by using the Qiagen QIAamp 96 viral RNA BioRobot kit on a BioRobot 9604. For PCR amplification and detection of HCV or internal control (IC) sequences, the Roche Cobas Amplicor v2.0 test kit was used. Data management, pool resolution and identification of positive samples were performed using the PMS Software from Tecan. RESULTS: In the 3-year period from 1 January 1999 to 31 December 2001, 839056 blood donor samples were tested in minipools of up to 48 samples. Thirty-five HCV-PCR-positive donations were identified. Thirty-four samples had antibodies against HCV and were therefore also detected by screening for antibody to HCV (anti-HCV). In October 2001, one seronegative (but PCR-positive) donor was detected. CONCLUSIONS: HCV-PCR minipool testing was successfully introduced in the largest Swiss blood transfusion service. It was shown that the release of HCV-PCR minipool results can be accomplished concurrently with the results of serological analysis. The challenge with a seronegative, but PCR-positive, donor demonstrates that the minipool testing strategy adds additional safety to blood products.     

A one-step dipstick assay for the on-site detection of nucleic acid

Objectives

We have developed a one-step nucleic acid dipstick assay (NADA) for visually detecting polymerase chain reaction (PCR) products within 3 min. “One-step” means that there were no additional procedures between amplification and detection.

Methods

This method was achieved through the use of asymmetric PCR and specially designed probes with appropriate melting temperature values. We initially combined one-step NADA with asymmetric capillary convective PCR (ACCPCR), an easy and rapid nucleic acid amplification technique, to construct an on-site nucleic acid diagnostic platform.

Results

We developed a diagnostic assay for the hepatitis B virus based on the ACCPCR-NADA platform to verify its feasibility. It exhibited an analytical sensitivity of three copies per test and a broad detection spectrum including genotype A–I. It also showed 97.9% sensitivity and 100% specificity based on the results observed using 67 serum samples with the Roche COBAS AmpliPrep/COBAS TaqMan (COBAS) system as the standard for comparison.

Conclusion

The results provide evidence for the feasibility of using an ACCPCR-NADA platform in practical applications, especially in on-site test.     

QIAGEN实时PCR与COBASTaqMan检测血清HBVDNA预测慢性乙型肝炎肝组织病理状态的性能比较

目的比较QIAGEN实时PCR与COBAS TaqMan检测血清HBV DNA水平预测慢性乙型肝炎肝组织病理状态的性能。方法慢性乙型肝炎患者278例,其中HBeAg阳性和阴性分别为162例和1 16例。采用QIAGEN实时定量PCR和COBAS TaqMan系统检测血清HBV DNA。肝组织病理学诊断采用Scheuer评分系统,其中病理学分级和分期包括G0~G4和S0~S4。结果血清HBV DNA(QIAGEN)和HBV DNA(C()BAS)在HBeAg阳性患者G2与G3,S1、S2、S3与S4之间的差异有统计学意义(P均0.05);在HBeAg阴性患者G1与G2、G3,S1与S2、S3、S4之间的差异有统计学意义(P均0.05)。血清HBV DNA(QIAGEN)与HBV DNA(COBAS)定量的不一致率,在HBeAg阳性患者G1-2、G3和S1-3、S4分别为4.24%(5/1 18)、9.09%(4/44)和3.68%(5/136)、7.69%(2/26),在HBeAg阴性患者G1、G2-3和S1、S2-4分别为6.02%(5/83)、3.03%(1/33)和3.29%(2/61)、3.64%(2/55)。血清HBV DNA(QIAGEN)和HBV DNA(COBAS)预测HBeAg阳性患者≥G3、≥S4的ROC曲线下面积分别为0.645和0.695、0.703和0.755,预测HBeAg阴性患者≥G2、≥S2的ROC曲线下面积分别为0.848和0.817、0.756和0.756。血清HBV DNA(QIAGEN)和HBV DNA(COBAS)预测HBeAg阳性患者≥S4的最佳截断值分别为≤3.784×10~6 IU/mL和≤6.668×10~7IU/mL,对应的灵敏度、特异度分别为0.654和1.000、0.735和0.581;预测HBeAg阴性患者≥G2的最佳截断值分别为≥5.821×10~3IU/mL和≥9.311×10~3 IU/mL,对应的灵敏度、特异度分别为0.970和0.909、0.614和0.651。结论血清HBV DNA预测肝组织病理状态的特点为预测HBeAg阴性患者≥G2的效能最大,并且血清HBV DNA(QIAGEN)与HBV DNA(COBAS)预测HBeAg阴性患者≥G2的性能有高度一致性。     

Considerar negativa una carga viral expresada como menor del límite inferior de cuantificación puede inducir a error en el diagnóstico y manejo terapéutico de la hepatitis C

Introduction

For years many clinical laboratories have routinely classified undetectable and unquantifiable levels of hepatitis C virus RNA (HCV-RNA) determined by RT-PCR as below limit of quantification (BLOQ). This practice might result in erroneous clinical decisions.

Aim

To assess the frequency and clinical relevance of assuming that samples that are BLOQ are negative.

Material and method

We performed a retrospective analysis of RNA determinations performed between 2009 and 2011 (Cobas/Taqman, lower LOQ: 15 IU/ml). We distinguished between samples classified as «undetectable» and those classified as «<1.50E + 01 IU/mL» (BLOQ).

Results

We analyzed 2.432 HCV-RNA measurements in 1.371 patients. RNA was BLOQ in 26 samples (1.07%) from 23 patients (1.68%). BLOQ results were highly prevalent among patients receiving Peg-Riba: 23 of 216 samples (10.6%) from 20 of 88 patients receiving treatment (22.7%). The clinical impact of BLOQ RNA samples was as follows: a) 2 patients initially considered to have negative results subsequently showed quantifiable RNA; b) 8 of 9 patients (88.9%) with BLOQ RNA at week 4 of treatment later showed sustained viral response; c) 3 patients with BLOQ RNA at weeks 12 and 48 of treatment relapsed; d) 4 patients with BLOQ RNA at week 24 and/or later had partial or breakthrough treatment responses, and e) in 5 patients the impact were null or could not be ascertained.

Conclusions

This study suggests that BLOQ HCV-RNA indicates viremia and that equating a BLOQ result with a negative result can lead to treatment errors. BLOQ results are highly prevalent in on-treatment patients. The results of HCV-RNA quantification should be classified clearly, distinguishing between undetectable levels and levels that are BLOQ.     

The diagnosis and management of MSUD in Saudi Arabia by using two different methods

Plasma amino acid concentrations were measured in Maple Syrup Urine Disease (MSUD) infants using reversed phase high performance liquid chromatography (HPLC). The technique involved an automated data acquisition system and phenylisothiocyanate (PITC) pre-column derivatization. During a period of three years more than 14 cases of MSUD have been confirmed in our hospital suggesting an alarmingly high rate of incidence of this disease in the Kingdom as compared to the West. We present here a simple and reliable method of quantitating the branched chain and other amino acid concentrations in plasma samples of children with metabolic disorders. In addition, we also present a fluorimetric COBAS based enzymatic method for the rapid semiquantitative measurement of branched chain amino acids for a disease in which a prompt initial diagnosis is essential.     

Consistency of quantitation of HCV genotype 4 from egypt across three HCV‐RNA amplification assays

Different quantitative assays for HCV‐RNA are available that report test results in International Units (IU)/ml based on the WHO International Standard. Thus, assays have been calibrated with standard material containing HCV genotype 1, so the consistency of quantitation might differ between assays for other HCV genotypes. Three commercial HCV nucleic acid amplification techniques (HCV–NAT) were compared for quantitation consistency of genotype 4 and 1 specimens from an Egyptian blood donor panel (n = 92). Consistency of quantitation between HCV–NATs was higher for genotype 1 than for genotype 4. Most quantitative results reported by the assays were in the same range, but some genotype 4 samples were missed by two of the assays. The Abbott assay showed higher concentrations for genotype 4 than the two Roche assays. Follow‐up investigations of individuals should use the same assay unless another assay has been validated properly. Standardization of HCV–NAT assays remains an issue. J. Med. Virol. 80:2086–2091, 2008. © 2008 Wiley‐Liss, Inc.     

Chlamydia/gonorrhea combo and HR HPV DNA testing in liquid-based pap

Residual material from liquid-based pap (LBP) test has been used for molecular testing of Human Papilloma Virus (HR HPV) DNA. LBP test specimens (1,025) were evaluated to determine how often residual material was insufficient to perform microbiological testing. The specimens were tested for Chlamydia trachomatis and Neisseria gonorrhea (CCGT) alone and in combination with HR HPV(CCGT + HR HPV), using PCR method and HYBRID CAPTURE 2, respectively. A minimum volume of 5 ml (CCGT: 0.5-1 ml, HPV: 4 ml) of residual material is required for both the tests. The residual material was measured at >5 ml, < or =5 ml, and 0.5 ml volumes and documented. Results were tabulated. The insufficient rate for cases submitted for CCGT was 0.2% and 7.6% for HR HPV, with a detection rate of 40.9% for HR HPV. Utilizing residual material from LBP test for microbiological testing, as well as DNA testing for HR HPV is a convenient, cost-effective means to enhance patient care.