Molecular DST in Real Practice

( Chang-ki Kim ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-

1. Background Drug resistance continues to threaten tuberculosis (TB) control efforts despite the availability of rapid diagnostics and standardized regimens for treatment of multidrug-resistant (MDR) TB, which is resistant to rifampicin (RIF) and isoniazid (INH). Rapid and accurate drug susceptibility testing is crucial to ensure early initiation of appropriate therapy and delay in diagnosis increases the risk of patient mismanagement, the amplification of drug resistance and ongoing transmission. In 2019, an estimated 10 million people fell ill with TB; of those 10 million people, 3 million were not reported to have been diagnosed and notified. The gap is proportionately wider for drug-resistant TB (DR-TB). Of the estimated 465 000 patients with multidrug-resistant TB or RIF-resistant TB (MDR/RR-TB), only 206 030 (44%) were diagnosed and notified. For the first time, the World Health Organization (WHO) has provided global estimates of the incidence of INH resistance: in 2019, there were 1.4 million incident cases of INH-resistant TB, of which 1.1 million were susceptible to RIF. Most of these people were not diagnosed with DR-TB and did not receive appropriate treatment. 2. Molecular WHO-recommended rapid diagnostic tests (mWRDs) Sputum-smear microscopy is a relatively insensitive test and cannot distinguish drug-susceptible strains from drug-resistant strains. The current gold standard method for the bacteriological confirmation of TB is culture using commercially available liquid media. However, culture is not used as a primary diagnostic test in many high-burden countries because of the cost, the infrastructure requirements and the long time required to generate results Moreover, phenotypic DST remains the reference standard for most anti-TB compounds; however, this test is slow and it requires specialized infrastructure and highly skilled staff. 1) Xpert MTB/RIF The Xpert MTB/RIF assay is a cartridge-based automated test that uses real-time polymerase chain reaction (PCR) on the GeneXpert platform to identify MTBC and mutations associated with RIF resistance directly from sputum specimens in less than 2 hours. WHO recommends using the Xpert MTB/RIF as an initial test for TB and detection of RIF resistance in adults and children with signs and symptoms of pulmonary TB. In addition, Xpert MTB/RIF can be used for TB meningitis and extrapulmonary TB. The Xpert MTB/RIF Ultra assay (hereafter called Xpert Ultra) uses the same GeneXpert platform as the Xpert MTB/RIF test and was developed to improve the sensitivity and reliability of detection of MTBC and RIF resistance. To address sensitivity, Xpert Ultra uses two multicopy amplification targets (IS6110 and IS1081) and a larger PCR chamber; thus, Xpert Ultra has a lower LoD than Xpert MTB/RIF (16 colony forming units [cfu]/mL and 131 cfu/mL, respectively). At very low bacterial loads, Xpert Ultra can give a “trace” result, which is not based on amplification of the rpoB target and therefore does not give results for RIF susceptibility or resistance. An additional improvement in the Xpert Ultra is that the analysis is based on melting temperature (Tm), which allows for better differentiation of resistance-conferring mutations. Planning the transition to the Xpert Ultra requires special consideration and a GLI document is available to assist in this process. 2) Moderate Complexity automated NAATs Rapid detection of TB and RIF resistance is increasingly available as new technologies are developed and adopted by countries. However, what has also emerged is the relatively high burden of INH-resistant, RIF-susceptible TB that is often undiagnosed. Globally, INH-resistant, RIF-susceptible TB is estimated to occur in 13.1% of new cases and 17.4% of previously treated cases. A new class of technologies has come to market with the potential to address this gap. Several manufacturers have developed moderate complexity automated NAATs for detection of TB and resistance to RIF and INH on high throughput platforms for use in laboratories. The tests belonging to this class are faster and less complex to perform than phenotypic culture based drug susceptibility testing (DST) and line-probe assays (LPA). They have the advantage of being largely automated following the sample preparation step. Moderate complexity automated NAATs may be used as an initial test for detection of TB and resistance to both first-line TB drugs simultaneously (RIF and INH). They offer the potential for the rapid provision of accurate results and for testing efficiency where high volumes of tests are required daily. Hence, these technologies are suited to areas with a high population density and rapid sample referral systems. WHO convened a Technical Expert Group to assess the results of an external laboratory validation of four novel centralized TB assays: the Abbott RealTime MTB and MTB RIF/INH assays, the Roche cobasR MTB and MTB-RIF/INH assays, the Hain FluoroTypeR MTBDR assay, and the BD MAXTM MDR-TB assay). The moderate complexity automated NAATs class of tests includes rapid and accurate tests for the detection of pulmonary TB from respiratory samples. Overall pooled sensitivity for TB detection was 93.0% and specificity 97.7%. Moderate complexity automated NAATs are also able to simultaneously detect resistance to both RIF and INH, and are less complex to perform than phenotypic DST and LPAs. After the sample preparation step, the tests are largely automated. Overall pooled sensitivity for detection of RIF resistance was 96.7% and specificity was 98.9%. Table 1. Mycobacterium genomic regions targeted by different assays for TB detection

A Comparison of Standard Methods for Evaluating the Water Resistance of Shell Fabrics

Kwon, Myoung Sook,Nam, Youn Ja The Costume Culture Association 2001 Fashion, industry and education Vol.4 No.3

The water resistance of shell fabrics intended for use in outdoor apparel was measured using three different standard test method, ASTM D 751, hydrostatic resistance, procedure A (Mullen test - with and without a fabric support) and Procedure B (Hydrostatic head test). A database of information on their water resistance performance was created. The data collected with different methods were correlated and the advantages and disadvantages of each method were compared. The Mullen test with a support appears to give higher and more favorable water resistance value on shell fabrics preventing fabrice rupture during the test. The hydrostatic head test gave lower hydrostatic pressure values than those measured on the two Mullen tests. The Mullen test is recommended for testing the water resistance of fabrics that have a relatively high water resistance because the Mullen tester applies a wide range of pressure. The hydrostatic head test is recommended for testing the fabrics that have relatively low water resistance. The area of the fabric sample that is in contact with the water is smaller in the Mullen test, so higher pressure levels can be reached and more samples should probably be tested to get a representative value for each fabric type. Futhermore, the hydrostatic head test was deemed more repeatable than the Mullen tests in this study.

Glycopeptide 내성 장구균의 항균제 감수성 검사법 비교 -E-test, Vitek, 디스크 확산법 및 한천 희석법-

이수연,박진희,박향숙,이미애,강은숙,홍기숙 대한임상병리학회 2000 대한임상병리학회지 Vol.20 No.3

Rifampin 내성 결핵균의 조기진단을 위한 rpoB 유전자의 염기서열 분열

전희선,박애자 중앙대학교 의과대학 의과학연구소 1996 中央醫大誌 Vol.21 No.1

The recent increase in new cases of Mycobacterium tuberculosis infection and rapid emergence of rifampin resistant and multidrug resistant strains have prompted new interest in tuberculosis worldwide. It has been reported that rifampin resistance can also be used as the surrogate marker for multidrug resistance. Therefore, rapid detection of rifampin resistant strain of M. tuberculosis is the key to control M. tuberculosis infection. Molecular biological methods based on rpoB gene sequencing and reverse hybridization line probe assay after polymerase chain reaction have a greater potential for early detection of rifampin resistance. This study was conducted to evaluate the clinical effectiveness of new rapid assays to detect rifampin resistance on the basis of molecular biological methods. The mutation pattern of rpoB gene in rifampin resistant strains isolated from sputum of patients in Korea was also studied. The results of rpoB gene sequencing and INNO-LiPA(INNO-LiPA Rif. TB Kit: Line Probe Assay, Innogenetics, Belgium) tests after polymerase chain reaction form clinical specimens were compared with that of conventional antimicrobial susceptibility tests. From February, 1995 through February, 1996 thirty rifampin resistant and thirty susceptible strains of M. tuberculosis were collected; and those were then subjected to rpoB gene sequencing and INNO-LiPA testing. The findings were as summarized below. Twenty eight patients(93.3%) out of thirty who had resistant strain showed multidrug resistance while two(6.7%) showed single drug resistance to rifampin. The rpoB gene sequencing revealed twenty eight(93.3%) sensitive strains agreed with results of conventional culture and susceptibility testing, but two(6.7%) strains disagreed with the conventional test results. Those two showed substitution of 526 histidine(CAC) by tyrosine(TAC) and asparagine(AAC). Twenty five(83.3%) of thirty resistant strains showed amino acid substitution within rpoB gene region. The patterns of this substitution by sequence were as following: ten(33.3%) strains of 531 serine(TCG) to leucine(TTG); seven(23.3%) of 516 aspartic acid(GAC) to valine(GTC), tyrosine and histidine ; seven(23.3%) of 526 histidine to arginine(CGC), tyrosine, aspartic acid, cystidine and serine ; and one(3.3%) of 533 leucine to proline(CCG). Of those the substitution of 526 histidine by serine has not been reported previously. INNO-LiPA test showed twenty eight(93.3%) of thirty sensitive strains agreed with conventional test results. Twenty seven(90.0%) of thirty resistant strains were agreeable with the INNO-LiPA results. The profiles of INNO-LiPA of resistant strains revealed absence of S5 band(43.4%), S4 band(23.3%), S2 band(13.3%), S1 band(10.0%) and S3 band(0%) in sequence. Of twenty seven strains showing absence of S bands, seventeen(63.3%) had confirmatory mutant R-probes. The retrospective review of patients' record showed 73.3% of patients who had resistant strains also had cavitary lesions of lungs(p<0.01). Twenty two(73.3%) of thirty resistant strains were isolated from patients having cavitary lesions of lungs. The most frequently observed mutation pattern of the resistant strains from the cavitary lesions was 531 substitution of serine by leucine(90.0% of serine -> leucine). Twenty seven(90.0%) of thirty sensitive strains and twenty four(80.0%) of thirty resistant strains were agreeable with both methods. Therefore, it is concluded that rpoB gene sequencing and INO-LiPA test after polymerase chain reaction are effective methods for rapid detection of rifampin resistant M. tuberculosis which is the surrogate marker for multidrug resistance. In particular, rpoB gene sequencing is the choice of method to discover a new mutant strain.

Evaluation of Three in-season Tests for Rapidly Detecting Resistance to Acetolactate Synthase Inhibitors in Schoenoplectus juncoides

WeiQiang Jia,Sug-Won Roh,Jae Eun Song,Aung Bo Bo,Kwang Min Cho,Kee Woong Park,Jeung Joo Lee 한국잡초학회·한국잔디학회 2021 Weed & Turfgrass Science Vol.10 No.1

Schoenoplectus juncoides is a noxious weed in paddy field of Korea. The excessive and unreasonable use of acetolactate synthase (ALS)-inhibiting herbicide caused the evolution of herbicide-resistant S. juncoides. As a part of the management of ALS-inhibiting herbicide resistance, early detection of the ALS-inhibiting herbicide resistance in S. juncoides is necessary. In this study, we evaluated three methods of rapidly detecting ALS-resistance in S. juncoides, including the cutting root test, cutting shoot test, and resistance in-season quick (RISQ) test. Three herbicide-resistant populations of S. juncoides (GR, 217R, and WR) and one susceptible population of S. juncoides (HS) collected from the Chungcheongnam-do and Jeollabuk-do of Korea served as experimental materials for the evaluation of three resistance test methods established by previous studies. In the three test methods, the GR and 217R were confirmed as resistant to flucetosulfuron and susceptible to penoxsulam. The WR was highly resistant to flucetosulfuron and penoxsulam. All the three resistance test methods have been successfully applied for identifying the ALS-inhibiting herbicide-resistant S. juncoides in-season. Especially, the result of cutting roots test demonstrated that the GR, 217R, and WR biotypes were highly resistant to flucetosulfuron with the R/S ratios of 34, 100, and 1852, respectively based on GR50. The WR biotype was highly resistant to penoxsulam with the R/S ratio of 912, whereas the GR and 217R biotypes were susceptible to penoxsulam. These in-season quick tests are likely to contribute to the use of effective herbicide in crops leading to decreased costs of labour and inputs in paddy field, and increased profits.

Evaluation of the Performance of the MicroScan Pos Breakpoint Combo Panel Type 28 for Susceptibility Testing of Staphylococcus aureus: Low-range Minimum Inhibitory Concentration of Vancomycin, Cefoxitin Screening, and Inducible Clindamycin Resistance Det

지미숙,이미영,노신애,김미나 대한진단검사의학회 2010 Annals of Laboratory Medicine Vol.30 No.6

Background : Susceptibility testing of Staphylococcus aureus often requires cumbersome supplementary tests. MicroScan Pos Breakpoint Combo Panel Type 28 (PBC28) (Siemens, USA) includes cefoxitin screening to detect methicillin-resistant Staphylococcus aureus (MRSA), inducible clindamycin resistance detection (ICD), and determination of low-range minimum inhibitory concentration of vancomycin (0.5-16 mg/mL). The purpose of this study was to evaluate the performance of PBC28 in comparison with that of Pos Combo Type 1A (PC1A) (Siemens). Methods : From December 2009 to March 2010, 500 non-duplicate clinical isolates of S. aureus were tested with PC1A and PBC28. Categorical agreements (CA) between the interpretations of the 2 panels were estimated. The presence of the mecA gene was determined by PCR, and double-disk diffusion test (D-test) was performed on the isolates resistant to erythromycin but susceptible or intermediately resistant to clindamycin. Ninety-six isolates representing various vancomycin minimum inhibitory concentrations (MICs) were tested in parallel with repeat PBC28, broth macrodilution, and epsilometer test (E test). Results : The CA was 99.3% with a very major error (VME) of 0.2%, major error (ME) of 0.1%, and minor error (mE) of 0.4% in total. PBC28 showed 100% CA for 1 isolate with vancomycin MIC of 4 mg/mL and 35 isolates (7.0%) with MIC of 2 mg/mL. However, only 15, 27, and 35 isolates with vancomycin MIC of 2 mg/mL showed 100% CA in repeat PBC28, broth macrodilution, and E test, respectively. PC1A and PBC28 detected all 314 mecA-positive isolates. Among the 63 isolates tested with the D-test, 58 (92.1%) were positive, and the results were 100% concordant with those of ICD. Conclusions : PBC28 can be appropriate susceptibility testing of S. aureus, including MRSA detection and ICD. However, the lower-range vancomycin MIC test was not reproducible enough to reliably differentiate MIC of 2 mg/mL from MIC≤1 mg/mL. (Korean J Lab Med 2010; 30:637-46)

재하ㆍ비재하 시험에 따른 고강도 콘크리트를 사용한 철근콘크리트 기둥부재의 내화성능평가

류동우(Ryu Dong-Woo),송영찬(Song Young-Chan),이종찬(Lee Jong-Chan),이세현(Lee Sea-Hyun),민병렬(Min Byung-Yeol) 대한건축학회 2008 大韓建築學會論文集 : 構造系 Vol.24 No.6

Amidst increasingly rising skyscrapers, higher than 30 stories, the concrete also tends to be strengthened day by day. Then the high strength concrete becoming more popular for the high-rise buildings, when exposed to high temperature heat in fire, has caused sectional damage to the structure, thereby resulting in deterioration of bearing capacity since the reinforcement is directly exposed to high temperature, and likely having impact on structural stability of the building. However, according to current laws and regulations, reinforced concrete structure are designated as fire-resistance structure as long as they maintain a certain level of thickness (25㎝ in diameter for column structure), irrespective of type of concrete material (standard or high strength), and it doesn't require any separate fire resistance test. Thus it's questionable, if they substantially meet the performance requirements as a fire resistance structure, depending on use and material of structural members. The study hence was intended to evaluate and compare the fire resistance efficiency of high strength concrete after assembling the column specimens with the same mixing ratio and conducting the loading and non-loading tests in accordance with ISO 834 standard fire heat curve. Furthermore, they were compared with non-loading test in small scale conducted using a floor heat furnace available in domestic labs in a bid to present the basic data for evaluating the fire resistance of concrete column. The fire resistance test result is described as follows. 1) Explosive spalling on high strength concrete started in 7 minutes after heating and lasted fro 30 minutes. 2) As a result of comparing the time for fire resistance at the loading test and unloading test, unloading test was found to be disadvantageous in destruction and deflection test. 3) Domestic unloading test was found to have been in between Japanese loading test and unloading test.

LRFD Resistance Factors for Auger Cast-in-Place Piles using the t-z Model

Sung Won Park,Anil Misra 한국지반공학회 2012 international journal of geo-engineering Vol.4 No.3

In the recent years, there has been a move to shift the design approach from working stress design to load resistance factor design (LRFD) methods. With the increasing use of augered cast-in-place (ACIP) piles in new construction, it is important that proper resistance factors be developed for the LRFD method. This paper focuses upon the development of the ACIP pile resistance factors using the t-z model. The t-z model provides the ability to enhance the load test data and to interpret field data for loading conditions beyond those measured during test. Ten static load tests consisting of load-settlement and load transfer data for ACIP piles were analyzed using the t-z model. A series of hack-calculations were performed to enhance the static load test data, which were then analyzed to obtain resistance bias factors for the currently used ACIP pile design methods. In addition, the back-calculated t-z model parameters were compared to those predicted using correlations for standard field investigation data, including standard penetration test (SPT), cone penetration test (CPT) and effective stress of the ground, Subsequently, the resistance biases were then used to obtain the resistance factors based upon the First-Order Second-Moment (FOSM) method Although, the LRFD of AC1P piles can be performed using the obtained resistance factors, the current design methods arc found to predict the side and tip behavior inconsistently and, therefore, the need for improved methods for resistance factor calibration are suggested.

전해부식시험을 이용한 니켈- 크롬도금강판 및 아연도금강판의 내식성 비교평가시험

이재봉 ( Jae Bong Lee ),김경욱 ( Kyung Wook Kim ),박민우 ( Nun Woo Park ),송태준 ( Tae Jun Song ),이채승 ( Chae Seung Lee ),이의종 ( Eui Jong Lee ),김상열 ( Sang Yeol Kim ) 한국부식방식학회(구 한국부식학회) 2013 Corrosion Science and Technology Vol.12 No.1

An Eectmlytie Corrosion( EC) test method as evaluated by the comparison with Copper Accelerated Acetic Sak Spray(CASS) and Neutral Salt Spniy(SS) tests. Those methods re applied in order to evaluate corrosion resistance of Ni-Cr plated and Zn-plated Fe substrates. The correlations between results obtained by different test methods were investigated. Results showed that the electrochemical method such as the EU test method was superior to the conventional methods such as (``ASS and SS, in terms of the quantitative accuracy and the test-time span. Furthermore, the EC test method provided the useful means to estimate the initiation of corrosion of each layer by monitoring the rest potentials of the coated layers such as Ni. Cr, and Zn on Fe substrate. With regard to test time spans. the EC test provided the 78 times and 182 times faster results than the CASS test in cases of Fe + 5um Ni ÷ 0.5 in Cr and Fe + 20 tn Ni + 0.5 to Cr respectively. while the EC test was 85 times faster results than the Salt Spray test in the case of Fe + 20 g/m2 Zn. Therefore, the EC test can be the better method to evaluate the resistance to corrosion of coated layers than the conventional methods such as the SS test and the CASS.

Demonstration Testing of Boiler Tubes in a Clean Power Test-Bed Plant

Yeon kwan Kang,Yinsheng He,이한상,김범수,Liming Xu,신기삼 대한금속·재료학회 2019 대한금속·재료학회지 Vol.57 No.12

The use of locally developed high performance alloys in power plants has become essential in the past ten years. Application of these alloys has been difficult because of a shortage of field demonstration testing, and the lack of lab-based test data. In this work, three types of locally developed heat resistant alloys were demonstration tested in boiler tubes in a power plant. The test procedures included a pre-test, a field demonstration and a post-test. The pre-test revealed the quality of the local tubes was the same level as the originally imported tubes. This suggested the potential outcomes of further demonstration testing. The demonstration was carried out by replacing parts of the original components with newly manufactured test components, made of local and imported tubes. There were no failures in the test components after 13,000h of operation. A portion of the tubes were sampled for a post-test, while the remaining parts were operated in the boiler for an even longer time. The post-test results revealed no significant degradation of the properties of the local tubes. The works no matter set up the properties database, but also set-up the performance and reliability, as well as test procedures for locally developed tubes.