Rapid pyrazinamide drug susceptibility testing using a closed. We prepared a protocol for the literature search, article selection, data. Biological assay development, bioassay validation, biological assay analysis. 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. Line probe assay connecting our members with evidence and. We assessed the performance of the genotype mtbdr line probe assay that offers the simultaneous identi.
Tb online who recommends use of two alternative line. Multiple studies have shown poor treatment outcomes for isoniazid monoresistant cases when treated with the standardised tb treatment regimen 15. The contents in this lecture are based on the existing. 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. Evaluation of geno type mtbdrplus line probe assay for early detection of drug resistance in tuberculous meningitis patients in india. We developed a reverse hybridizationbased line probe assay with oligonucleotide probes designed to detect mutations in pnca. This platform uses a nitrocellulose strip for identification of mtbc and commonly isolated ntm by reverse hybridization. Mechanism of the reaction between uracil and carbodiimides. Jan 04, 2016 the line probe assay uses the reverse hybridization technology. Development and evaluation of a line probe assay for rapid. 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. Line probe assay for detection of mycobacterium tuberculosis. In the global dna methylation line1 assay, the genomic dna of interest is first enzymatically digested using the provided msei enzyme to generate the appropriate fragments to hybridize to a biotinylated.
Dec 08, 2015 this video showcases these different realtime pcr probe and primer formats. Lipa, innogenetics nv, ghent, belgium is a line probe assay lipa for in vitro use and allows the detection of mtb complex and its resistance to r after. The target dna, previously extracted by boiling, is pcramplified using. Assay design can probe signaling pathways relevant to drug mechanism at different stages. Taqman probes are hydrolysis probes that are designed to increase the specificity of quantitative pcr. Line probe assay lpa test detects tb infection presence and resistance to two drugs rifampicin and isoniazid.
Drug susceptibility testing line probe assay, molecular tests. The diagnostic utility of line probe assays for multidrugresistant. Pdf accurate prediction of macrolide resistance in. For the scientist new to dna hybridization assays, much is written about the obvious factors which influence the assay, such as probe composition and annealing temperatures, but many other details, some of which are subtle, can greatly influence the sensitivity and quality of data generated from hybridization assays. Rapid screening of mdrtb using molecular line probe assay is. Rapid screening of mdrtb using molecular line probe assay. Line probe assay for rapid detection of drugselected. The line probebased assay avoids the need for a sequencing instrument, but both assays require transfer of dna opening the tube either to load onto a sequencer or for hybridization onto a. Molecular lineprobe assay for the detection of resistance to secondline antitb drugs sllpa tuberculosis diagnostics background multidrug resistant tuberculosis mdr. We have the appropriate solutions for all your needs. Accuracy of line probe assays for the diagnosis of pulmonary and. Multicenter evaluation of the molecular line probe assay.
L stuyver, a wyseur, a rombout, j louwagie, t scarcez, c verhofstede, d rimland, r f schinazi, and r rossau. Comparison of xpert mtbrif with line probe assay for. Muhammad yahya noori assistant professor department of pathology dimc focal person for provincial tuberculosis reference lab sindh duhs 2. This assay became the gold standard for tb drug susceptibility testing. Line probe assay connecting our members with evidence. Rapid pyrazinamide drug susceptibility testing using a. Line probe assays for drug resistant tuberculosis detection. 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.
The role of line probe assays in the xpert mtbrif ultra era. Evaluation of a novel line probe assay to detect resistance to. Line probe assays lpas enable rapid drugsusceptibility testing for rifampicin rif. There are three commercially available line probe assays, two by hain and one by innogenetics. Gupta r, thakur r, gupta p, jalan n, kushwaha s, gupta m, gupta p, aggarwal a, manchanda v. Global dna methylation line1 kit to quantify changes in. Diagnostic utility of a line probe assay for multidrug resistant. This new powerful assay provides new data dimensions and unique insight into biological mechanisms. J global infect dis serial online 2015 cited 2020 may 3. Mechanism of voltage sensor probe technology and vsp. Due to their chemical properties, these reagents can permeated. In case the result is invalid, the lpa test needs to be repeated and gives result in 5 to 7 days. Disclaimer this lecture was developed during the period november 2017 to july 2018.
Sample dna is hybridized to probes on a membrane strip. The test was performed according to the manufacturers protocol hain. Muhammad yahya noori assistant professor department of pathology dimc focal person for provincial. In line probing is a functional assay that can be used to determine structural changes in rna in response to ligand binding. Costeffectiveness of a line probe assay test compared to.
Line probe assays lpas enable rapid drugsusceptibility testing for rifampicin rif and isoniazid. The line probe based assay avoids the need for a sequencing instrument, but both assays require transfer of dna opening the tube either to load onto a sequencer or for hybridization onto a strip. Can use mgit liquid culture and solid media quick visual interpretation mostly automated using the auto. Line probe assays lpas have been recently approved for use in low income settings and can be used to screen smearpositive sputum specimens for. Evaluation of a novel line probe assay to detect resistance to pyrazinamide, a key drug used for tuberculosis treatment. Your competent partner in mycobacteria diagnostics and. This video showcases these different realtime pcr probe and primer formats. We developed a reverse hybridizationbased line probe assay with oligonucleotide probes. Tb online who recommends use of two alternative line probe.
Drug susceptibility testing drug resistance testing means finding out which tb drugs a person is sensitive to, and if they have drug resistant tb. Technical faqs on hain genotype mtbdrplus line probe assay 3 is 98. The line probe assays detect drugresistant tuberculosis in clinical samples. The genoscholar pzatb ii assay nipro corporation, japan. Another potential role of line probe assays is the detection of isoniazid resistance in patients diagnosed with rifampicin sensitive tb who respond poorly to standard first line treatment. Accuracy of line probe assays for the diagnosis of. The target dna, previously extracted by boiling, is pcramplified using biotinylated primers and finally incubated with the strip. Another potential role of line probe assays is the detection of isoniazid resistance in patients diagnosed with rifampicin sensitive tb who respond poorly to standard firstline treatment. A commercial line probe assay for the rapid detection of. Line probe assay for detection of mycobacterium tuberculosis complex. The main mechanism of pza resistance in mycobacterium. Original article evaluation of a novel line probe assay to detect resistance to pyrazinamide, a key drug used for tuberculosis treatment m. Molecular mechanisms of action, resistance, detection to the. Duallabeled bhq probes, bhqplus probes, molecular beacons, and scorpions primers.
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. Molecular mechanisms of action, resistance, detection to. We evaluated the performance of the novel genoscholar pzatb ii line probe assay pzalpa2. Evaluation of a novel line probe assay to detect resistance. If the clinical picture is highly suggestive of tb, a repeat test can be ordered to increase sensitivity. However, there is no clarity regarding the superiority of one over the other. Molecular detection of drugresistant tuberculosis by line.
Comparison of genexpert and line probe assay for detection of. Genexpert mtbrif assay is a nucleic acid amplification naa test which simultaneously detects dna of mycobacterium tuberculosis complex mtbc and resistance to rifampin rif i. Line probe assay has been shown to be highly sensitive and specific in the detection of rifampicinresistant tb when used on culture isolates. Resistance of mycobacterium tuberculosis to pyrazinamide pza derives mainly from mutations in the pnca gene. We assessed the performance of the genotype mtbdr line probe assay that offers the simultaneous identification of mycobacterium tuberculosis and its resistance to rifampin rif and isoniazid inh by detecting the most commonly found mutations in the rpob and katg genes. We assessed the performance of the genotype mtbdr line probe assay that offers the simultaneous identification of mycobacterium tuberculosis and its resistance to rifampin rif and. The line probe assay lpa, based on strip technology was used to. Recently, the nipro pza line probe assay lipa nipro corp. The resistance of human immunodeficiency virus type 1 hiv1 to drugs is a major cause of antiretroviral treatment failure. The dualprobe cpv2 qpcr assay consisted of a tandem simplemgb probes, amplifying a 340 bplong amplicon with the forward primer and first probe according to streck end colleagues 30.
The development of rapid molecular diagnostic assays for pyrazinamide pza resistance is considered technically challenging as mutations are highly diverse, scattered along the full length of. Another identification option the line probe assay. Role of molecular methods in tuberculosis diagnosis and. The development of rapid molecular diagnostic assays for pyrazinamide pza resistance is considered technically challenging as mutations are highly diverse, scattered along the full length of the pnca. Your competent partner in mycobacteria diagnostics and instrumentation no matter if you want to start with molecular genetic testing, expand your existing mycobacteria diagnostics or look for an easy way to automate the processing. Mechanism of voltage sensor probe technology and vsp applications voltage sensor probes vsps are a fluorescence resonance energy transfer fretbased voltagesensing assay technology, which. Evaluation of geno type mtbdrplus line probe assay for. 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. Prasad noted, could be categorized as the binding event, the molecular event.
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. In 2008 in kampala, mdrtb prevalence was reported as 1. Only 25% of multidrugresistant tuberculosis mdrtb cases are currently diagnosed. Line probe assay technology involves the following steps. Genotype mtbdrplusv1 was whoendorsed in 2008 but newer lpas have since been developed. A liquid culture will be the most definitive way of ruling out tb. Line probe assay for rapid detection of drugselected mutations in the human immunodeficiency virus type 1 reverse transcriptase gene. The detection of pza resistance was evaluated in 258 clinical isolates of m. We developed a reverse hybridizationbased line probe assay in which. A nestedpcr protocol was also evaluated for the assay to amplify pcr products from m. The following steps are for the development of the assays manually.
Proficiency assessment mechanism for lineprobe assay. Hainline probe assays advantages works on processed specimens rapid assay improved instrumentation for analysis and documentation of results used in laboratories now disadvantages. This follows the 2008 recommendation by who on the use of lpas for the rapid detection of rifampicin resistance. For rif resistance, both assays showed very good performances, with 98% of the test results concordant with the drug susceptibility testing results. Hain line probe assay hain lpa is a fast and reliable method to determine resistance to rifampicin and isoniazid by detecting rpob, katg, and inha genes mutation and was evaluated in a variety of settings34. The method was first reported in 1991 by researcher kary mullis at cetus corporation, and the. The genotype mtbdrplus test is a whoendorsed lineprobe. As per our laboratory standard operating protocol, an lj culture is included for. The lineprobe assay uses the reverse hybridization technology. Considerations in dna hybridization assays commentary. 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.
The development of rapid molecular diagnostic assays for pyrazinamide pza resistance is considered technically challenging as mutations are highly diverse, scattered along the full length of the pnca gene and not all are associated with pza resistance. These two dna line probe assays have been recently evaluated in a comparative study for their abilities to detect rif and inh resistance in m. Accurate prediction of macrolide resistance in helicobacter pylori by a pcr line probe assay for detection of mutations in the 23s rrna gene. Evaluation of geno type mtbdrplus line probe assay for early. Prewarm the twin incubator to 45 c and hyb green and str red solutions to 37 45 c in a. In 2008, hain lpa was endorsed by the who for mdr tb screening5. The nucleotide sequences of the katg and inha genes and the maba. Molecular line probe assay for the detection of resistance to second line antitb drugs sllpa diagnostics background multidrug resistant tuberculosis mdr tb is a public health crisis and a global health security risk carrying grave consequences for those affected. Comparison of dna sequencing and a line probe assay for. Mechanism of voltage sensor probe technology and vsp applications voltage sensor probes vsps are a fluorescence resonance energy transfer fretbased voltagesensing assay technology, which measures changes in cellular membrane electrical potential. In case of treatment failure, we also test for resistance to rifampicin and isoniazid. Pdf proficiency assessment mechanism for lineprobe assay.
The lineprobe assays of hain lifescience are valuable diagnostic tools. Evaluation of a new line probe assay for rapid identification of. Proficiency assessment mechanism for lineprobe assay testing for diagnosis of multidrugresistant tuberculosis in india article pdf available june 2015 with 318 reads how we measure reads. Line probe assay conclusion advantages easy to perform detection of 18 clinically relevant mycobacteriumsp. Evaluation of a novel line probe assay to detect resistance to pyrazinamide, a key. The method was first reported in 1991 by researcher kary mullis at cetus corporation, and the technology was subsequently developed by hoffmannla roche for diagnostic assays and by applied biosystems now part of thermo fisher scientific for research applications. At that time, the evidence assessed was for the lpa technology, hain version 1 assay. Technical faqs on hain genotype mtbdrplus line probe assay. Line probe assays lpas enable rapid drugsusceptibility testing for rifampicin rif and isoniazid inh resistance and mycobacterium tuberculosis detection. Line probe assays lpas enable rapid drugsusceptibility testing for. Jan 25, 2017 the dual probe cpv2 qpcr assay consisted of a tandem simplemgb probes, amplifying a 340 bplong amplicon with the forward primer and first probe according to streck end colleagues 30, and the. The mtbdrplus line probe assay lpa and xpert mtbrif have been endorsed by the world health organization for the rapid diagnosis of drugresistant tuberculosis.
The sensitivity and specificity of pza resistance obtained by this new. Can use mgit liquid culture and solid media quick visual interpretation mostly. About 500 new smearpositive multidrugresistant tuberculosis mdrtb cases are estimated to occur per year in uganda. Technical faqs on hain genotype mtbdrplus line probe assay 1 what is the hain genotype mtbdrplus and how does it work. 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. The innolipa rif tb assay innogeneticsbelgium, is a line probe assay that uses multiplex pcr amplification and reverse hybridization to identify mtb complex mtbc, and at the same time. The role of line probe assays in the xpert mtbrif ultra. In case the result is invalid, the lpa test needs to be repeated and gives result in 5 to 7. Performance of the genotype mtbdr line probe assay for. Tuberculosis most common infectious cause of mortality after hivaids 2 3.
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