Pdf performance of line probe assay mtbdrplus in the. The lineprobe assays not only improved dst in our lab, but made it also extremely fast and efficient. The probabilistic sensitivity analysis showed there is a 70% chance that the additional cost of the line probe assay, compared with. In this context, we evaluated the performance characteristics of a combination of techniques, i.
Speciesspecific positive probes and negative control probes are needed to run with human or mouse control slides for a quality control check of the technique and with the sample for a sample or rna quality control check. The following steps are for the development of the assays manually. Technical document handbook on tb laboratory diagnostic methods for the eu 1 background and introduction francis drobniewski tuberculosis tb is a major cause of morbidity and mortality in europe. Mtbdrplus line probe assay lipa has been endorsed by the world health organization for pulmonary tuberculosis tb diagnosis. Tb online who recommends use of two alternative line probe. Rifampicin r and isoniazid h are key first line antituberculosis drugs. Handbook on tb laboratory diagnostic methods in the european. Development and evaluation of a pcrbased, line probe assay.
We have developed the research prototype cystic fibrosis assay 31, which uses multiplex pcr and colorimetric detection with sequencespecific oligonucleotide probes immobilized in a linear array on nylon membranes, to type 28 cf mutations and 3 cftr polymorphisms table 1. The line probe assay lpa, based on strip technology was used to diagnose tb and detect rif as well as isoniazid inh resistance due to mutations in rpo. The diagnostic performance of the genotype mtbdrplus version 2 line probe assay is equivalent to that of the xpert mtbrif assay. Tb lipa is a commercial line probe assay designed to rapidly detect rifampicin resistance, a marker of mdrtb. The world health organization validates the use of line probe assay lpa for rapid detection of mdrtb.
Line probe assay connecting our members with evidence and. All processing from this point on is fully automated. If the clinical picture is highly suggestive of tb, a repeat test can be ordered to increase sensitivity. The economic evaluation of lpa proved that it was a cost minimizing strategy for both healthcare system with cost minimization observed in costs of hospital days, laboratory costs, medications and for. We compared the line probe assay lipa to sequence analysis for the detection of mutations conferring resistance to nucleoside inhibitors of human immunodeficiency virus type 1 hiv1 reverse transcriptase rt. A total of 100 clinical samples were genotyped by tma. Line probe assays for drugresistant tuberculosis detection 2 laboratory staff and clinicians on the interpretation of the results for both, fl and sllpa, and to better understand and manage possible discrepancies between phenotypic and genotypic dst. Drug susceptibility testing line probe assay, molecular tests. Your competent partner in mycobacteria diagnostics and.
Pcr products are hybridized to probes on the strip. Limited performance data from line probe assays lpas, nucleic acid tests used for the rapid diagnosis of tuberculosis tb, nontuberculosis mycobacteria ntm, and mycobacterium tuberculosis drug resistance are available for hivinfected individuals, in whom paucibacillary tb is common. Accuracy of line probe assays for the diagnosis of pulmonary. Molecular lineprobe assay for the detection of resistance to secondline antitb drugs sllpa tuberculosis diagnostics background multidrug resistant tuberculosis mdr tb is a public health crisis and a global health security risk carrying grave consequences for those affected. In line probing is a functional assay that can be used to determine structural changes in rna in response to ligand binding. Disclaimer this lecture was developed during the period november 2017 to july 2018. Inline probing is a functional assay that can be used to determine structural changes in rna in response to ligand binding. Jan 04, 2016 the line probe assay uses the reverse hybridization technology.
This process requires an extensive assay design procedure aimed at generating an optimum primer probe amplicon combination to allow accurate quantification of nucleic acids with minimum need for postpcr analyses see figure 1. Line probe assays for drug resistant tuberculosis detection. There was a marked offset at the end of the bioreactor run, suggesting the ability of the canty analyzer to correlated to early and late stages of apoptosis. Rifampicin r and isoniazid h are key firstline antituberculosis drugs.
The test was performed according to the manufacturers protocol hain life science gmbh, nehren, germany. Comparison of genexpert and line probe assay for detection of. Molecular line probe assay for the detection of resistance to second line antitb drugs sllpa tuberculosis diagnostics background multidrug resistant tuberculosis mdr tb is a public health crisis and a global health security risk carrying grave consequences for those affected. A commercial line probe assay for the rapid detection of rifampicin. Line probe assays lpas are rapid molecular diagnostics that can detect m. These findings were line with other recent studies that line probe assay is a rapid tool for screening tb and dst 7, 8, 10, 15, 3441. The study purpose was to validate the use of the genotype mtbdrplus line probe assay lpa to detect resistance to r and h in mycobacterium tuberculosis strains directly from smearpositive sputum samples in. In r5, the six selected probes were used for types. Acd provides positive control probes that target speciespecific housekeeping genes and negative control probes that target the bacterial dapb gene. Genotype mtbdrsl line probe assay shortens time to diagnosis. Role of a genotype mtbdrplus line probe assay in early. Muhammad yahya noori assistant professor department of pathology dimc focal person for provincial tuberculosis reference lab sindh duhs 2. Failure to detect resistance to these two drugs early results in treatment failure and poor clinical outcomes. A commercial line probe assay for the rapid detection of.
Line probe assay conclusion advantages easy to perform detection of 18 clinically relevant mycobacteriumsp. We have compared direct sequencing to a line probe assay lipa for the detection of drug resistancerelated mutations in 197 clinical samples, and we have investigated the sequential appearance of mutations under drug pressure. Technical faqs on hain genotype mtbdrplus line probe assay. The line probe assay is a commercially available line probe assay that rapidly detects mycobacterium tuberculosis mtb complex, as well as the most common mutations associated with rifampicin and. Technical faqs on hain genotype mtbdrplus line probe assay 2 conventional tests for tb involve afb smear, culture and dst. Accuracy of line probe assays for the diagnosis of. At that time, the evidence assessed was for the lpa technology, hain version 1 assay. Tuberculosis most common infectious cause of mortality after hivaids 2 3. Microscopy cannot detect drug resistance, while the mtbdrplus can rapidly detect resistance to rifampicin and isoniazid, the most important firstline tb drugs.
Plasma samples from 40 patients who had received zidovudine, dideoxyinosine, and dideoxycytosine, alone or in combination, and who were enrolled in the altis 2 clinical trial. The line probe assay is a commercially available lineprobe assay that rapidly detects mycobacterium tuberculosis mtb complex, as well as the most common mutations associated with rifampicin and. Comparison of xpert mtbrif with line probe assay for. Line probe assay connecting our members with evidence. However, there is no clarity regarding the superiority of one over the other. Collect sputum sample from the patient with suspected tb. Molecular lineprobe assay for the detection of resistance to isoniazid and rifampicin lpa tuberculosis diagnostics background multidrugresistant tuberculosis mdr tb is a public health crisis and a major threat to global tb control.
The line probe invalid rates were substantially lower, at 1. Role of molecular methods in tuberculosis diagnosis and. Line probe assay for detection of human immunodeficiency. This takes a long time, first to isolate a culture and then to.
This study determined the diagnostic performance of mtbdrplus in bal for mtb complex detection and isoniazidrifampicin resistance in. Another identification option the line probe assay. Hepatitis c genotyping by pcr and line probe assay. Hybrids are labeled and detected using a color metric assay. Hainline probe assays advantages works on processed specimens rapid assay improved instrumentation for analysis and documentation of results used in laboratories now disadvantages multiple beacons needed to cover overlapping regions silent mutations may result in false predication of resistance not customizable by user. This schematic shows the steps involved in the line probe assay. In a doubleblinded prospective study, we evaluated the efficacy of the xpert mtbrif on samples that were first tested by lpa under the revised national. As it is we are not able to ascertain what kind of results you have since lpa will also give inh and rif sensitivity patterns. Nov 18, 2015 06 november 2015 who has approved the use of two alternative line probe assays lpas, with the capability to detect tb and rifampicin resistance. Technical faqs on hain genotype mtbdrplus line probe assay 3 is 98. Accuracy of line probe assays for the diagnosis of pulmonary and multidrugresistant tuberculosis. Cost minimization analysis of line probe assay for.
However, when the treatment success rate for the line probe assay test was increased to 60% one of the targets set by who in tb treatment the icer was below the willingnesstopay level i. A multisite validation in india of the line probe assay. The mtbdrplus line probe assay lpa and xpert mtbrif have been endorsed by the world health organization for the rapid diagnosis of drugresistant tuberculosis. Accuracy of line probe assays for the diagnosis of pulmonary and. Molecular line probe assay for the detection of resistance to isoniazid and rifampicin lpa tuberculosis diagnostics background multidrugresistant tuberculosis mdr tb is a public health crisis and a major threat to global tb control. Molecular tests for detecting drug resistance such as lineprobe assay have shown promise for the diagnosis of drugresistant tb. Second, pcr amplifies dna, usually the 16 s or rpob genes. Comparison of dna sequencing and a line probe assay for. Plasma samples from 40 patients who had received zidovudine, dideoxyinosine, and dideoxycytosine, alone or in combination, and who were enrolled in the altis 2. In this study, the strategy of testing sputum with genotype mtbdrplus mtbdrplus and genotype direct lpa.
Testing a set of 424 clinical enterobacteriaceae strains showed 100% sensitivity and specificity for the detection and differentiation of tem, shv and ctxm esbl genes present in that group. Can use mgit liquid culture and solid media quick visual interpretation mostly automated using the auto. However, until this study there have been few data on direct testing of. Jul 28, 2005 line probe assay has been shown to be highly sensitive and specific in the detection of rifampicinresistant tb when used on culture isolates. Realtime pcr qpcr data are reliable only if they result from a robust qpcr assay that has been carefully designed, validated and optimised.
Although lpas are more technically complex designed for reference or regional laboratory settings and take longer to perform than the xpert mtbrif assay cepheid, sunnyvale, ca, usa, they. A multisite validation in india of the line probe assay for. Line probe assay for identification of the genus mycobacterium and 16 different mycobacterial species including m. Lipa, which links highly sensitive tma of hcv rna to the versant hcv genotype assay version 1.
Resistance to mycobacterium tuberculosis is a reality worldwide, and its diagnosis continues to be difficult and time consuming. Line probe assay has been shown to be highly sensitive and specific in the detection of rifampicinresistant tb when used on culture isolates. We evaluated the routine use once a week of a line probe assay genotype mtbdrplus for early diagnosis of resistance and for assessment of the main. We have developed the research prototype cystic fibrosis assay31, which uses multiplex pcr and colorimetric detection with sequencespecific oligonucleotide probes immobilized in a linear array on nylon membranes, to type 28 cf mutations and 3 cftr polymorphisms table 1. The sputum is mixed with the reagent that is provided with the assay, and a cartridge containing this mixture is placed in the genexpert machine. Line probe assay for detection of mycobacterium tuberculosis. The line probe assay was shown to detect with 100% accuracy esbl genes for which oligonucleotide probes are present in the assay. The resistance of human immunodeficiency virus type 1 hiv1 to drugs is a major cause of antiretroviral treatment failure. A liquid culture will be the most definitive way of ruling out tb.
Published studies have shown that the assay performs well when using culture isolates. Appropriate laboratory staff also need to be trained to conduct lpa procedures. The study evaluates the performance of lpa mtbdrplus compared to proportional method pm. Line probe assay for detection of mycobacterium tuberculosis complex. However, no single genetic alteration has yet been found that results in the mdr phenotype resistance to at least. The line probe assay is used for the diagnosis of mdrtb. There is a paucity of data on application of this test directly to clinical specimens, although based on a small number of studies, the test appears to be less promising. Drug susceptibility testing molecular tests, line probe assay. Globally in 2015, there were 4 80 000 new cases of. Displaying line probe assay principle powerpoint presentations there was a marked offset at the end of the bioreactor ppt presentation summary. Tb online who recommends use of two alternative line. Development and evaluation of a pcrbased, line probe. Because of the high rate of mdrtb cases, we need a fast and efficient method to detect resistance against firstline drugs. This process requires an extensive assay design procedure aimed at generating an optimum primerprobeamplicon combination to allow accurate quantification of nucleic acids with minimum need for postpcr analyses see figure 1.
The use of molecular line probe assays for the detection of. The target dna, previously extracted by boiling, is pcramplified using biotinylated primers and finally incubated with the strip. We prepared a protocol for the literature search, article selection, data. Molecular line probe assays lpa permit rapid diagnosis of tb, isoniazid and rifampin resistance, and. Handbook on tb laboratory diagnostic methods in the. Evaluation of the aid esbl line probe assay for rapid. The standard dst test is a twostep procedure involving culture and sensitivity testing. Kinases are a group of enzymes that modifies a target substrate by phosphorylation. Cost minimization analysis of line probe assay for detection. Evaluation of two line probe assays for rapid detection of. The contents in this lecture are based on the existing. Line probe assays lpas enable rapid drugsusceptibility testing for rifampicin rif.
The lineprobe assay uses the reverse hybridization technology. Test strips were interpreted in a twostep procedure. When considering line probe assay performance in comparison to culturebased methods, liquidbased mycobacterial culture is limited by contamination rates that can be as high as 14. High throughput laboratory in a high mdrxdr burden country approx. To face this challenge, the world health organization has recommended the use of rapid molecular tests.
Comparison of genexpert and line probe assay for detection. This follows the 2008 recommendation by who on the use of lpas for the rapid detection of rifampicin resistance. It can directly show the change in flexibility and binding of regions of rna in response to a ligand, as well as compare that response to analogous ligands. Evaluation of the line probe assay for the rapid detection. Purpose kinase assays are used to detect the activity of specific kinases from cells. Secondgeneration line probe assay for hepatitis c virus genotyping. What sample was used to diagnose the tb and what was the reading on the bands. Genotype mtbdrsl line probe assay shortens time to. Line probe assay technology involves the following steps. However, its value for mycobacterium tuberculosis mtb detection in bronchoalveolar lavage bal needs exploration. The mtbdrsl test is a molecular line probe assay for the rapid detection of mutations associated with resistance to fluoroquinolone and aminoglycoside antibiotics and cycloserine, drugs used to treat multidrugresistant tuberculosis.
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