The analytical specificity of the assay was determined using Basic Local Alignment Search Tool (BLAST) analysis of the National Center for Biotechnology Information (NCBI) GenBank database and no sequences were detected that would interfere with the LightCycler PCR assay. Further, the assay was tested using a panel of 104 respiratory pathogens (bacteria and viruses) that were extracted and subjected to the LightCycler PCR assay. As predicted, only
Mycobacterium tuberculosis complex was detected from this panel. In addition, nearly 100 species of nontuberculous mycobacteria were evaluated using this PCR assay and there was no cross-reactivity detected. The analytical sensitivity of the assay was determined to be 10 target copies/microliter using a dilution series of M tuberculosis spiked into respiratory specimens in triplicate. The sensitivity and specificity of the assay for detection of M tuberculosis complex verses culture was found to be 100% and 100% respectively for 26 M tuberculosis -positive cultures and 266 M tuberculosis -negative cultures.
The PCR inhibition rate of the assay was determined to be 0% by spiking 100 negative extracted respiratory specimens with M tuberculosis at 100 targets/microliter. The PCR assay was able to detect 100% of specimens spiked at the limit of detection for bronchoalveolar lavage (BAL) fluid, muscle/skin tissue, organ tissues, bone, cerebrospinal fluid (CSF), and urine. One of 30 (3%) of spiked respiratory tissues was inhibited and 3 of 30 (10%) of spiked sterile body fluids other than CSF were inhibited. Not surprising increased inhibition was seen in stool (24 of 30 spiked specimens were positive) and formalin-fixed, paraffin-embedded tissue (19 of 30 spiked specimens were positive). Method comparison of the LightCycler PCR assay versus mycobacterial culture was done using 192 respiratory specimens. The results are shown in Table 1. Table 1. Results for the LightCycler PCR assay versus mycobacterial culture for 192 respiratory specimens.
Table 2 provides a comparison of the LightCycler PCR assay versus the GEN-PROBE Mycobacterium tuberculosis Direct (MTD) assay, performed using 542 respiratory specimens (226 BAL fluids, bronchial washings and lung washings plus 316 sputa, induced sputa, and tracheal secretions). The kappa coefficient of 0. 96 indicates excellent agreement between the 2 methods. Table 2. Clinical sensitivity of the LightCycler PCR versus MTD for respiratory specimens. Two melt peaks can be produced during this assay. A melt peak at a temperature of 64. 0 degrees C + or – 2. 5 degrees C can correspond to either isoniazid-susceptible or isonazid-resistant M tuberculosis and, therefore, no indication of isoniazid susceptibility is provided for these isolates. However, an isolate with melt peak occurring at a temperature of 58. 0 degrees C + or – 2. 5 degrees C correlated with isoniazid resistance determined using a broth reference method in 100% (26/26) of isolates tested.
Isolates with a peak at a temperature of 58. 0 degrees C + or – 2. 5 degrees C are reported as Positive, probable isoniazid resistance detected. The PCR result is available 7 to 14 days prior to the broth method and, therefore, may be helpful in selecting appropriate antibiotic therapy for these patients. Confirmation of isonaizid resistance must be done using a phenotypic method if the isolate grows in culture. Submit first morning Sputum on 3 consecutive days OR 510 mL Body fluids / Aspirates OR 2 mL CSF, OR 100 ml (50 ml min) first morning Urine collected on 3 consecutive days OR Fresh tissue biopsies including endometrial aspirate / curettage in sterile normal saline OR formalin fixed paraffin embedded Tissue block OR 3 mL (1 mL min. ) Whole blood/Bone Marrow in 1 Lavender Top (EDTA)/ Yellow Top (ACD) tube OR Menstrual blood in a sterile screw capped container OR culture isolates. All specimens to be collected aseptically and shipped refrigerated. DO NOT FREEZE.