ABSTRACT
Latest major contributions in the field of sepsis diagnostics result from advances in PCR technologies permitting new standards in speed and quality, given the fact that a timely diagnosis is the decisive factor to the survival of patients with bloodstream infections.Multiplex real-time PCR is a quantitative method for simultaneous amplification and detection of different targeted DNA molecules within hours. Nevertheless, various studies have shown a number of technical shortcomings as well as a high heterogeneity in sensitivity.The present method allows the standardized and rapid detection and identification of 25 common bacteria and fungi responsible for bloodstream infections from whole blood samples by using LightCycler(®) SeptiFast (LC-SF) test, based on real-time PCR.
Subject(s)
Bacteremia/diagnosis , DNA, Bacterial/isolation & purification , DNA, Fungal/isolation & purification , Fungemia/diagnosis , Multiplex Polymerase Chain Reaction/standards , Real-Time Polymerase Chain Reaction/standards , Automation, Laboratory , Bacteremia/microbiology , Bacteremia/pathology , Bacteria/genetics , Bacteria/growth & development , Bacteria/isolation & purification , Blood Specimen Collection , Culture Media/chemistry , DNA, Bacterial/genetics , DNA, Fungal/genetics , Fungemia/microbiology , Fungemia/pathology , Fungi/genetics , Fungi/growth & development , Fungi/isolation & purification , Humans , Molecular Diagnostic Techniques , Multiplex Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/methods , Time FactorsABSTRACT
BACKGROUND: Standardization of quantitative HIV-1 tests to a global primary standard is required by regulatory authorities to ensure comparability of test results across different assays and platforms of different manufacturers. OBJECTIVES AND STUDY DESIGN: Three generations of quantitative HIV-1 tests, the COBAS(®) AMPLICOR(®) HIV-1 Monitor Test, v1.5 (HIV-1 Monitor test v1.5); the COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HIV-1 Test (HIV-1 TaqMan(®) test v1.0); and the dual-target-based COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HIV-1 Test, v2.0 (HIV-1 TaqMan(®) test v2.0), were assessed for accuracy to World Health Organization (WHO) 2nd International Standard for human immunodeficiency virus 1 (HIV-1) RNA (NIBSC code 97/650) at concentration levels below 1667 IU/mL including relevant medical decision points. RESULTS: With the 2nd WHO Standard the mean difference across all concentrations was -0.07 log(10) for the HIV-1 Monitor test v1.5; +0.12 log(10) for the HIV-1 TaqMan(®) test v1.0; and +0.09 log(10) for the HIV-1 TaqMan(®) test v2.0. Linearity, including concentrations below the claimed limit of quantitation, was demonstrated for HIV-1 TaqMan(®) test v2.0. The HIV-1 TaqMan(®) test v1.0 showed a trend towards higher quantitation at very low concentration levels and the HIV-1 Monitor test v1.5 had a tendency towards lower quantitation at low concentration levels. CONCLUSIONS: All three assays are closely traceable to the primary WHO HIV-1 RNA standard for in vitro diagnostic IVD assays. Compared to the other two assays, the HIV-1 TaqMan(®) test v2.0 showed better linearity around the limit of detection and below.
Subject(s)
HIV Infections/diagnosis , HIV-1/genetics , HIV-1/isolation & purification , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/standards , RNA, Viral/analysis , HIV Infections/virology , Humans , Limit of Detection , RNA, Viral/genetics , Reference Standards , Reverse Transcriptase Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/standards , Sensitivity and Specificity , Viral Load , World Health OrganizationABSTRACT
BACKGROUND: HIV-1 RNA viral load is a key parameter for reliable treatment monitoring of HIV-1 infection. Accurate HIV-1 RNA quantitation can be impaired by primer and probe sequence polymorphisms as a result of tremendous genetic diversity and ongoing evolution of HIV-1. A novel dual HIV-1 target amplification approach was realized in the quantitative COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 (HIV-1 TaqMan test v2.0) to cope with the high genetic diversity of the virus. OBJECTIVES AND STUDY DESIGN: The performance of the new assay was evaluated for sensitivity, dynamic range, precision, subtype inclusivity, diagnostic and analytical specificity, interfering substances, and correlation with the COBAS AmpliPrep/COBAS TaqMan HIV-1 (HIV-1 TaqMan test v1.0) predecessor test in patients specimens. RESULTS: The new assay demonstrated a sensitivity of 20 copies/mL, a linear measuring range of 20-10,000,000 copies/mL, with a lower limit of quantitation of 20 copies/mL. HIV-1 Group M subtypes and HIV-1 Group O were quantified within +/-0.3 log(10) of the assigned titers. Specificity was 100% in 660 tested specimens, no cross reactivity was found for 15 pathogens nor any interference for endogenous substances or 29 drugs. Good comparability with the predecessor assay was demonstrated in 82 positive patient samples. In selected clinical samples 35/66 specimens were found underquantitated in the predecessor assay; all were quantitated correctly in the new assay. CONCLUSIONS: The dual-target approach for the HIV-1 TaqMan test v2.0 enables superior HIV-1 Group M subtype coverage including HIV-1 Group O detection. Correct quantitation of specimens underquantitated in the HIV-1 TaqMan test v1.0 test was demonstrated.
Subject(s)
HIV Infections/diagnosis , HIV Infections/virology , HIV-1/genetics , Polymerase Chain Reaction/methods , RNA, Viral/analysis , Reagent Kits, Diagnostic , Viral Load , HIV-1/isolation & purification , Humans , Sensitivity and SpecificityABSTRACT
Early detection of bloodstream infections (BSI) is crucial in the clinical setting. Blood culture remains the gold standard for diagnosing BSI. Molecular diagnostic tools can contribute to a more rapid diagnosis in septic patients. Here, a multiplex real-time PCR-based assay for rapid detection of 25 clinically important pathogens directly from whole blood in <6 h is presented. Minimal analytical sensitivity was determined by hit rate analysis from 20 independent experiments. At a concentration of 3 CFU/ml a hit rate of 50% was obtained for E. aerogenes and 100% for S. marcescens, E. coli, P. mirabilis, P. aeruginosa, and A. fumigatus. The hit rate for C. glabrata was 75% at 30 CFU/ml. Comparing PCR identification results with conventional microbiology for 1,548 clinical isolates yielded an overall specificity of 98.8%. The analytical specificity in 102 healthy blood donors was 100%. Although further evaluation is warranted, our assay holds promise for more rapid pathogen identification in clinical sepsis.
