title: Rapid diagnosis of mycobacterial infections and quantitation of Mycobacterium tuberculosis load by two real-time calibrated PCR assays.
abstract: Sensitive and specific techniques to detect and identify Mycobacterium tuberculosis directly in clinical specimens are important for the diagnosis and management of patients with tuberculosis (TB). We developed two real-time PCR assays, based on the IS6110 multicopy element and on the senX3-regX3 intergenic region, which provide a rapid method for the diagnosis of mycobacterial infections. The sensitivity and specificity of both assays were established by using purified DNA from 71 clinical isolates and 121 clinical samples collected from 83 patients, 20 of whom were affected by TB. Both assays are accurate, sensitive, and specific, showing a complementary pattern of Mycobacterium recognition: broader for the IS6110-based assay and restricted to the M. tuberculosis complex for the senX3-regX3-based assay. Moreover, the addition of a synthetic DNA calibrator prior to DNA extraction allowed us to measure the efficiency of DNA recovery and to control for the presence of PCR inhibitors. The mycobacterial burden of the clinical samples, as assessed by direct microscopy, correlates with the M. tuberculosis DNA load measured by the senX3-regX3-based assay. In addition, reduced levels of M. tuberculosis DNA load are present in those patients subjected to successful therapy, suggesting a potential use of this assay for monitoring treatment efficacy. Therefore, these assays represent a fully controlled high-throughput system for the evaluation of mycobacterial burden in clinical specimens.
authors.affiliation: Unit of Human Virology, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Broccolo
authors.forename: Paolo
authors.initials: P
authors.lastname: Scarpellini
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Locatelli
authors.forename: Anna
authors.initials: A
authors.lastname: Zingale
authors.forename: Anna M
authors.initials: AM
authors.lastname: Brambilla
authors.forename: Paola
authors.initials: P
authors.lastname: Cichero
authors.forename: Leonardo A
authors.initials: LA
authors.lastname: Sechi
authors.forename: Adriano
authors.initials: A
authors.lastname: Lazzarin
authors.forename: Paolo
authors.initials: P
authors.lastname: Lusso
authors.forename: Mauro S
authors.initials: MS
authors.lastname: Malnati
mesh_terms: D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis; D021901:DNA, Intergenic / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics / Q000302:isolation & purification*; D009164:Mycobacterium Infections / Q000175:diagnosis* / Q000382:microbiology; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D021901:DNA, Intergenic