INT J TUBERC LUNG DIS 19(7):817–822 Q 2015 The Union http://dx.doi.org/10.5588/ijtld.14.0311

Evaluation of a novel line-probe assay for genotyping-based diagnosis of Mycobacterium tuberculosis in Thailand S. Rienthong,* C. Boonin,* B. Chaiyasirinrote,† N. Satproedprai,‡ S. Mahasirimongkol,‡ H. Yoshida,§ Y. Kondo,§# C. Namwat,* D. Rienthong* *Supra-National Tuberculosis Reference Laboratory, Department of Disease Control, Ministry of Public Health, Bangkok, †TB-HIV Research Foundation, Chiangrai, ‡Medical Life Sciences Institute, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand; §Research and Development Laboratory, Nipro Corporation, Shiga, Japan SUMMARY S E T T I N G : The Supranational Tuberculosis Reference Laboratory (NTRL), Bangkok, and Chiangrai Prachanukroh Hospital, Chiangrai, Thailand O B J E C T I V E : To evaluate the diagnostic performance of newly developed line-probe assay (LiPA) kits in tuberculosis (TB) endemic settings. D E S I G N : LiPA kits were used to evaluate 404 clinical isolates of Mycobacterium species and 163 sputum samples in Thailand. R E S U LT S : LiPA kits were able to identify M. tuberculosis, M. avium, M. intracellulare and M. kansasii with 100% sensitivity and specificity when compared with the commercially available AccuProbe assay. Testing of the LiPA kits for their ability to detect mutations in clinical isolates resistant to anti-tuberculosis drugs such

as rifampicin, isoniazid, pyrazinamide and fluoroquinolones showed that the assay had very high sensitivity (65.9–100%) and specificity (98.2–100%) compared with drug susceptibility testing and DNA sequencing. LiPA had a sensitivity of 75.0–85.7% and a specificity of 96.4–100% in testing clinical sputum samples. C O N C L U S I O N : The novel LiPA kits have high sensitivity and specificity, and may enhance the rapid detection of first- and second-line anti-tuberculosis drug resistance, improving the selection of suitable chemotherapy agents to treat multidrug-resistant and extensively drugresistant TB. K E Y W O R D S : M. tuberculosis; drug susceptibility testing; rapid test

MULTIDRUG-RESISTANT TUBERCULOSIS (MDR-TB) has become a major problem for tuberculosis (TB) control worldwide, including Thailand. The timely identification of MDR-TB is crucial to providing life-saving treatment to infected patients and to limiting the spread of MDR-TB. This approach has also been adopted for the control of extensively drug-resistant TB (XDR-TB).1,2 Although the rapid testing of rifampicin (RMP) resistance can be used as a marker for MDR-TB in countries with a prevalence of .5%,3 its diagnostic uncertainty makes it inappropriate in countries with ,5% MDR-TB prevalence, including Thailand, where highly sensitive and specific polymerase chain reaction-based tests for multiple specific mutations may be useful in managing patients with MDR- and XDR-TB. Pyrazinamide (PZA) is a key drug in the treatment of all kinds of TB, including MDR- and XDR-TB, and PZA drug susceptibility testing (DST) should be performed as early as possible. Although testing the susceptibility of strains against anti-tuberculosis

drugs, including PZA, may help identify MDR- and XDR-TB, DST results do not correlate well with patients’ clinical outcomes.4–6 Identifying mutations in genes that confer resistance to these drugs may therefore be more useful; for example, mutations in pncA have been found to correlate with PZA resistance in MDR- and XDR-TB patients.7 The newly developed line-probe assay (LiPA) kits for M. tuberculosis strains have been validated in areas of low TB prevalence.8 The present study describes our evaluation of the diagnostic performance of these kits in high TB prevalence settings, characterised by the circulation of M. tuberculosis strains with different drug-resistant phenotypes.

MATERIALS AND METHODS Clinical isolates A total of 304 stored M. tuberculosis and 100 stored non-tuberculosis mycobacteria (NTM) clinical isolates were obtained from the Supranational Tubercu-

Correspondence to: Yuji Kondo, Research and Development Laboratory, Nipro Corporation, 3023 Nojicho, Kusatsu, Shiga 525-0055, Japan. Tel: (þ81) 77 564 9463. Fax: (þ81) 77 565 1642. e-mail: [email protected]. Article submitted 15 April 2014. Final version accepted 2 March 2015.

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Table 1

Description of clinical isolates used in this study Categorised isolates, n Drug-susceptible RMP-resistant INH-resistant LVX-resistant PZA-resistant M. tuberculosis MDR-TB M. tuberculosis M. tuberculosis M. tuberculosis M. tuberculosis LiPA tests (n ¼ 105) (n ¼ 54) (n ¼ 49) (n ¼ 52) (n ¼ 30) (n ¼ 14) n

NTM/MDR-TB detection kit INH-resistant TB detection kit FQ-resistant TB detection kit PZA-resistant TB detection kit

105 105 61* 61*

54 54

49

52 52 30 14

260 211 91 75

* The remaining 44/105 isolates did not show results on FQ and PZA DST tests and were not assayed using FQ- and PZA-resistant TB detection kits. MDR-TB ¼ multidrug-resistant tuberculosis; RMP ¼ rifampicin; INH ¼ isoniazid; LVX ¼ levofloxacin; PZA ¼ pyrazinamide; LiPA ¼ line-probe assay; NTM ¼ nontuberculous mycobacteria; FQ ¼ fluoroquinolone.

losis Reference Laboratory (NTRL) of Bangkok, and from Chiangrai Prachanukroh Hospital, Chiangrai, Thailand. Of the 304 M. tuberculosis isolates, 105 were categorised as drug-susceptible M. tuberculosis, 54 as MDR-TB, 49 as RMP-resistant M. tuberculosis, 52 as isoniazid (INH) resistant M. tuberculosis, 30 as levofloxacin (LVX) resistant M. tuberculosis and 14 as PZA-resistant M. tuberculosis (Table 1). The species of each NTM isolate was identified using the AccuProbe Mycobacterium avium Complex Culture Identification Test,9 the AccuProbe Mycobacterium intracellulare Culture Identification Test10 and the AccuProbe Mycobacterium kansasii Culture Identification Test11 (Gen-Probe, San Diego, CA, USA), according to the manufacturer’s instructions. DNA was later extracted by heating isolates diluted in 1.0 ml Tris-ethylenediaminetetraacetic acid buffer, pH 8.0, at 95–1008C for 15–30 min. The DNA samples were stored at 208C if not analysed immediately. Sputum specimens All of the 163 sputum samples collected from Chiangrai Prachanukroh Hospital between 2011 and 2013 were decontaminated using the N-acetylL-cysteine-sodium hydroxide (NALC-NaOH) method, with a final NaOH concentration of 1%. Concentrated sputum sediments were subjected to acid-fast bacilli (AFB) staining, MGITe (Mycobacteria Growth Indicator Tube; BD, Sparks, MD, USA) and Lowenstein-Jensen (LJ) culture. Standard bio¨ chemical tests, such as SD TB Ag MPT 64 Rapid assays (Standard Diagnostics, Seoul, South Korea) and AccuProbe assays, were performed to identify M. tuberculosis complex and NTM. The remaining sediment was stored overnight at 2–88C; the following day, DNA was extracted as described above. Drug susceptibility testing DST of the culture isolates against RMP and INH was performed using BD BACTEC MGIT 960 SIRE (BD, Sparks, MD, USA); PZA susceptibility was determined using BD BACTEC MGIT PZA (BD), and LVX susceptibility was determined using the proportion method with LJ medium. DST of sputum specimen

isolates grown in MGIT was determined using MGIT 960 SIRE (BD). Line-probe assay Four different LiPA kits (Nipro Corporation, Osaka, Japan) were used as described in the manufacturer’s instructions and in a previous study.8 Briefly, NTM/ MDR-TB detection kits were designed to detect M. tuberculosis complex, M. avium, M. intracellulare and M. kansasii, in addition to mutations in the rpoB gene associated with RMP resistance, and in both the katG (S315) gene and the promoter region of the fabG1-inhA operon (PfabG1-inhA), commonly found in INH-resistant strains.12 INH-resistant TB detection kits are designed to detect spanning mutations in PfabG1-inhA(c-15t and t-8c), fabG1 (g609a [L203L]) and many mutations in katG that confer INH resistance. Fluoroquinolone (FQ) and PZA-resistant TB detection kits are also designed to detect gyrA mutations associated with FQ resistance and pncA mutations associated with PZA resistance. Of the 304 M. tuberculosis isolates, the 260 categorised as drug-susceptible, MDR, RMP-resistant or INH-resistant M. tuberculosis were assayed using NTM/MDR-TB detection kits; the 211 categorised as drug-susceptible, MDR or INH-resistant M. tuberculosis were assayed using INH-resistant TB detection kits; of the 119 isolates categorised as drugsusceptible or PZA-resistant M. tuberculosis, 75 were assayed using PZA-resistant TB detection kits (the remaining 44 isolates did not show results on PZA DST and were not assayed using PZA-resistant TB detection kits). Of the 135 isolates categorised as drug-susceptible or LVX-resistant M. tuberculosis, 91 were assayed using FQ-resistant TB detection kits (the remaining 44 isolates did not show results on LVX DST and were not assayed using FQ-resistant TB detection kits) (Table 1). Samples with no consensus results when compared with DST were subjected to DNA sequencing, and their sequences compared with that of M. tuberculosis H37Rv. Ethical consideration This study protocol was reviewed and approved by the institutional review board of the Department of

Evaluation of a novel LiPA in Thailand

Table 2

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Diagnostic performance of LiPA in comparison with DST* M. tuberculosis isolates n

DST RMP Resistant Susceptible

LiPA results, n Resistant

Susceptible

Sensitivity % Specificity % 92.8

97 163

NTM/MDR-TB detection kit 90 7† 3‡ 160

98.2

INH Resistant Susceptible

NTM/MDR-TB detection kit 98 9§ 0 153

91.6

100

107 153

INH Resistant Susceptible

INH-resistant TB detection kit 100 6¶ 0 105

94.3

100

106 105

LVX Resistant Susceptible

FQ-resistant TB detection kit 29 15# 0 47

65.9

100

44 47

PZA Resistant Susceptible

PZA-resistant TB detection kit 5 9** 0 61

35.7

100

14 61

* The diagnostic performance of NTM/MDR-TB, INH-resistant TB, FQ-resistant TB and PZA-resistant TB detection kits was compared with that of DST. † Six isolates had no mutation on rpoB sequencing, one was a mixed isolate of wild-type and rpoB (H526R). ‡ These three isolates had substitutions in rpoB, one each with S531L, L533P and H526N substitutions. § Three isolates had no mutations in inhA, fabG1 and katG; one had a silent mutation in katG (F565F); four had substitutions in katG, including one each with D189H, T618M, G279D and A379D, G699R; and one had a mutation in fabG1(g690a). ¶ Three isolates had no mutations in inhA, fabG1 and katG. The other three had substitutions in katG, including one each with D189H, T618M and G279D. # All 15 isolates had a lineage-specific polymorphism in gyrA (S95T), with one isolate also having a silent mutation in gyrA (g105c V35V). None had mutations in gyrB. ** Nine isolates were false-resistant on initial MGIT PZA testing and had no mutations in pncA. Six of the nine isolates resistant on initial MGIT PZA were susceptible on repeat MGIT PZA testing (Tables 3 and 4). LiPA ¼ line-probe assay; DST ¼ drug susceptibility testing; RMP ¼ rifampicin; NTM ¼ non-tuberculous mycobacteria; MDR-TB ¼ multidrug-resistant TB; INH ¼ isoniazid; LVX ¼ levofloxacin; PZA ¼ pyrazinamide; FQ ¼ fluoroquinolone; TB ¼ tuberculosis; MGIT ¼ Mycobacteria Growth Indicator Tube.

Disease Control, Ministry of Public Health, Nonthaburi, Thailand. All clinical isolates and specimens were from specimens banked at the NTRL and Chiangrai Prachanukroh Hospital. All samples were studied blind and cannot be traced back to specific patients or any other biological data. Only the clinical diagnostic results relevant to this study, such as AFB, TB culture and DST results, were used for the study.

RESULTS Identification of NTM using the NTM/MDR-TB detection kit To assess the ability of the NTM/MDR-TB detection kits to identify NTM species, we compared the results on 100 NTM culture isolates with those of the AccuProbe assays. The NTM/MDR-TB detecTable 3 MGIT 960 PZA retesting results and pncA sequencing on isolates that were PZA-resistant on initial MGIT PZA but PZAsusceptible using LiPA Isolates n

MGIT DST (first result)

MGIT DST (second result)

pncA sequencing

LiPA

6 3

Resistance Resistance

Susceptible Resistance

WT WT

WT WT

MGIT ¼ Mycobacteria Growth Indicator Tube; PZA ¼ pyrazinamide; LiPA¼ lineprobe assay.

tion kits showed that, of the 100 NTM culture isolates, 52 were M. avium, 19 were M. intracellulare and 25 were M. kansasii, in agreement with the results of the AccuProbe assays. In contrast, four isolates were negative using the NTM/MDR-TB detection kit and invalid using the AccuProbe assay, i.e., the samples producing signals ranged between positive and negative. Correlation between LiPA and standard DST results of clinical isolates To assess the diagnostic performance of the NTM/ MDR-TB, INH-resistant TB, FQ-resistant TB and PZA-resistant TB detection kits, we compared the results of these kits with the corresponding DST results (Table 2). PZA-resistant TB detection kits showed five isolates as resistant compared to 14 in the MGIT 960 PZA assays. As MGIT PZA assays have been reported to be unreliable in determining PZA resistance,4–6 we repeated the MGIT PZA assays and sequenced pncA in nine samples that were PZA-resistant on MGIT 960 PZA but PZAsusceptible on LiPA. Of these nine samples, six originally found to be resistant were found to be susceptible, and none of the nine isolates had mutations in the pncA gene (Table 3). The PZAresistant TB detection kit showed an overall sensitivity and specificity of 100% (Table 4).

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Table 4

Diagnostic performance of the PZA-resistant TB detection kit in comparison with DST*

DST Probably resistant Probably susceptible

LiPA result of PZA kit

M. tuberculosis isolates n

Resistant

Susceptible

5 70

5 0

0 70†

Sensitivity % Specificity % 100

100

* DST results were regarded as probably resistant and probably susceptible in combination with the results of repeat MGIT PZA testing and pncA sequencing. † Nine of 70 isolates were false-resistant on initial MGIT PZA testing and had no mutations in pncA. Six of the nine isolates resistant on initial MGIT PZA were susceptible on repeat MGIT PZA testing (see Table 3). PZA ¼ pyrazinamide; TB ¼ tuberculosis; DST ¼ drug susceptibility testing; LiPA ¼ line-probe assay; MGIT ¼ Mycobacteria Growth Indicator Tube.

Direct detection of drug-resistant M. tuberculosis in sputum specimens The clinical sensitivity of LiPA kits was tested on 163 sputum samples. The overall sensitivity of the NTM/ MDR-TB detection kit was 84.0% (Table 5), and the overall sensitivities of the INH-, FQ- and PZAresistant TB detection kits were respectively 42.3%, 85.3% and 86.5% (data not shown). All sputum samples were confirmed as culture-positive and were identified as M. tuberculosis. To assess the clinical diagnostic performance of NTM/MDR-TB detection kits, we used 127/163 sputum samples for which both RMP and INH DST results were available. The NTM/MDR-TB detection kits detected 6/8 RMP-resistant isolates (sensitivity 75.0%), and all 119 susceptible isolates (specificity 100%). When the NTM/MDR-TB detection kits were used to test 112 INH-susceptible and 15 INH-resistant sputum specimens, we found a sensitivity of 86.7% (13/15) and a specificity of 96.4% (108/112), comparable with the results of culture isolates (Table 6).

DISCUSSION This study compared the accuracy of the NTM/MDRTB detection kits with that of AccuProbe assays in identifying various species of mycobacteria in clinical NTM isolates. The two assays were in 100% agreement for all but four isolates, which were invalid on the AccuProbe assay and negative on the NTM/ MDR-TB detection kit. The poor results of both assays on these four isolates may be due to poor quality or the low concentration of extracted DNA, or the four isolates may have belonged to NTM species other than Table 5 Sensitivity of the NTM/MDR-TB detection kit in detecting M. tuberculosis in the sputum samples and in the study Sputum samples n

Sputum status AFB

113 50 Total (n ¼ 163)

þ 

Sensitivity NTM/MDR-TB detection kit n/N (%) 108/113 (95.6) 29/50 (58.0) 137/163 (84.0)

NTM ¼ non-tuberculous mycobacteria; MDR-TB ¼ multidrug-resistant tuberculosis; AFB ¼ acid-fast bacilli; þ¼ positive;  ¼ negative.

M. avium, M. intracellulare or M. kansasii. Nevertheless, the use of these kits may be beneficial in a country endemic for the human immunodeficiency virus (HIV), as HIV infection increases the number of patients infected with NTM.13–15 The sensitivity and specificity of NTM/MDR-TB detection kits for RMP susceptibility were respectively 92.8% (90/97) and 98.2% (160/163), similar to previous findings.16–18 Of the seven isolates found to be RMP-resistant on DST but RMP-susceptible on LiPA, six had no mutations in the RMP resistancedetermining region (RRDR) of the rpoB gene, but may have had mutations in other regions of rpoB. The remaining isolate was a mixture of wild-type (wt) and rpoB (H526R) mutant. These kits can distinguish among common rpoB mutants, including D516L, H526Y, H526D and S531L, even when mixed with wt, because they have probes detecting these mutants. However, when used to assay a mixture of wt and an rpoB mutant not recognised by this kit, the isolate is interpreted as wt. Three isolates were found to be RMP-susceptible on DST but RMP-resistant on LiPA. One isolate each had S531L, L533P and H526Y substitutions in rpoB, substitutions known to confer resistance to RMP.19 In addition, the kits can detect INH resistance due to mutations in the katG (S315) and PfabG1-inhA genes. The sensitivity of the NTM/ MDR-TB detection kits in detecting INH resistance strains was 91.6% (98/107), higher than in a previous report from Japan (61.6%).8 This result indicated that the distribution of the most frequent INH resistance-conferring mutations differed in isolates from Thailand and Japan. Many other genes are associated with INH resistance, including ahpC, fabG1, the furA-katG intergenic region and all regions of katG.19–22 The INH-resistant TB detection kit is designed to detect mutations in many of these genes, including PfabG1-inhA(c-15t and t-8c), fabG1 (g609a [L203L]) and katG. The sensitivity of the INH-resistant TB detection kits in identifying INH resistance mutations was 94.3% (100/106), higher than that of the NTM/MDR-TB detection kits. Of the six isolates found to be INH-resistant using DST but INH-susceptible on LiPA, three had no mutations in any of the genes assayed, indicating that mutations in other genes may be associated with INH

Evaluation of a novel LiPA in Thailand

Table 6

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Comparison of the diagnostic performance of LiPA with DST in clinical specimens Culture-positive M. tuberculosis isolates tested n

Resistant

Susceptible

Sensitivity %

RMP Resistant Susceptible

8 119

6 0

2 119

75.0

INH Resistant Susceptible

15 112

13 4

2 108

86.7

DST

NTM/MDR-TB detection kit LiPA result, n Specificity % 100

96.4

LiPA ¼ line-probe assay; DST ¼ drug susceptibility testing; NTM ¼ non-tuberculous mycobacteria; MDR-TB ¼ multidrugresistant tuberculosis; RMP ¼ rifampicin; INH ¼ isoniazid.

resistance. Of the remaining three isolates, one each had D189H, T618M and G279D substitutions in katG. These substitutions were located outside the detection region of the kit and, to our knowledge, have never been reported previously. These mutations may be associated with INH resistance and should be incorporated into future INH-resistant TB detection kits. Mutations in the gyrA gene have been reported to account for 42–100% of FQ-resistant M. tuberculosis isolates, with most of these mutations found in codons 90, 91 and 94.19,23–25 However, FQ-resistant clinical isolates with wt gyrA were reported to have gyrB mutations.26 The FQ-resistant TB detection kit only detects mutations in gyrA, and has shown some results that are inconsistent with those of conventional DST. DNA sequencing results revealed that the clinical isolates with discordant results had the evolutionrelated variant S95T substitution in gyrA. This polymorphism at codon 95 was found to have no effect on the FQ resistance phenotype,19 and is considered a variation specific to the Beijing lineage.27 FQ-resistant TB detection kits are designed to identify the polymorphism with wt. The use of FQ-resistant TB detection kits is beneficial in a TB-endemic country such as Thailand, where FQ resistance status is important for selecting treatment regimens. PZA resistance is largely due to mutations in the pncA gene, which encodes the enzyme pyrazinamidase (PZase) that converts PZA to its active form, pyrazinoic acid (POA). PZase activity is required for PZA to be metabolised to POA, the active metabolite with microbicidal activity.28,29 Approximately 72–100% of PZA-resistant M. tuberculosis isolates have mutations in the pncA gene or its upstream region.19,30,31 Further phenotypic characterisation has correlated mutations in the pncA gene with loss of PZase activity. As the minimum inhibitory concentrations of these PZase-positive PZA-resistant isolates with wt pncA were very low using the BACTEC 460 method, they may have shown false resistance due to the acidity of the medium used for PZA susceptibility assays, which inhibited the growth of M. tuberculosis.4–6 In addition, a previous study in Thailand reported that the sensitivity and specificity of PZase activity assays were

respectively 65.4% and 100% compared with MGIT 960 PZA assays, whereas the sensitivity and specificity of pncA sequencing were respectively 75% and 89.8% relative to MGIT 960 PZA assays.32 Isolates reported as PZA-resistant using MGIT 960 PZA assay should therefore be retested for susceptibility and the pncA gene sequenced. We found that nine isolates showed discordant results on PZA DST and LiPA. We therefore sequenced their pncA genes and retested their PZA susceptibility using MGIT 960 PZA assays. Of these nine isolates, six that were originally found to be resistant were susceptible on MGIT 960 PZA tests, and none of the nine had mutations in the pncA gene. These results indicate that the initial MGIT 960 PZA results yielded nine false-positives. The PZA-resistant TB detection kits showed an overall sensitivity and specificity of 100% (Table 4). When testing sputum samples, the NTM/MDR-TB detection kits showed the same ability to detect RMPand INH-resistant M. tuberculosis strains as conventional DST, although the sensitivity of the former was lower on sputum samples than in isolates. This finding was in good agreement with a systematic review and meta-analysis of commercial LiPA in the detection of MDR-TB.13 We also found that the sensitivity of LiPA kits was better with smear-positive than with smear-negative sputum.

CONCLUSION Rapid MDR-TB detection is a key strategy in the global control of the TB epidemic. Although M. tuberculosis culture and conventional DST are costeffective diagnostic tools, their longer turnaround times are an obstacle to clinical care and control of MDR- and XDR-TB. The newly developed LiPA kit evaluated in this study could deliver MDR-TB results within hours, accelerating the delivery of life-saving treatment to these patients. By rapidly detecting RMPand INH-resistant M. tuberculosis, these kits can help clinicians select suitable treatment plans more quickly, especially for MDR-TB patients. Moreover, with the increase in numbers of MDR-TB patients each year, information on resistance to FQ and PZA may prevent prescription of unnecessary treatment with ineffective

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medications, which carry the risk of adverse drug effects without enhancing efficacy. Acknowledgements This study was supported by a grant from Nipro Corporation, Shiga, Japan (Grant No. 3R11010). Conflicts of interest: none declared.

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Evaluation of a novel LiPA in Thailand

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RESUME C O N T E X T E : Laboratoire Supranational de R´ef´erence de la Tuberculose (NTRL) de Bangkok, et l’Hopital ˆ Prachanukroh de Chiangrai, a` Chiangrai, Tha¨ılande. O B J E C T I F : Evaluer les performances diagnostiques des kits de tests par sonde en ligne (LiPA) r´ecemment e´ labor´es dans des r´egions d’end´emie. S C H E´ M A : Les kits LiPA ont e´ t´e utilis´es afin d’´evaluer 404 isolats cliniques d’esp`eces de Mycobacterium et 163 e´ chantillons de crachats en Tha¨ılande. R E´ S U LT A T S : Les kits LiPA ont permis d’identifier les souches de M. tuberculosis, M. avium, M. intracellulaire et M. kansasii avec une sensibilit´e et une sp´ecificit´e de 100% par comparaison aux tests AccuProbe disponibles dans le commerce. La v´erification de la capacit´e de ces kits LiPA a` d´etecter des mutations dans des isolats cliniques r´esistants aux

m´edicaments antituberculeux comme la rifampicine, l’isoniazide, le pyrazinamide et les fluoroquinolones, a montr´e que ces tests avaient une sensibilit´e (65,9–100%) et une spe´ cificit e´ (98,2–100%) tre` s e´ lev e´ es par comparaison au test de pharmacosensibilite´ et au s´equen¸cage de l’ADN. Les LiPA ont eu une sensibilit´e de 75,0–85,7% et une sp´ecificit´e de 96,4–100% lors des tests effectu´es sur des e´ chantillons de crachats. C O N C L U S I O N S : Les nouveaux kits LiPA ont une sensibilit´e et une sp´ecificit´e e´ lev´ees et ils pourraient ame´ liorer la de´ tection rapide des re´ sistances aux antituberculeux de premi`ere et deuxi`eme ligne, ce qui contribuerait a` une meilleure s´election des m´edicaments les plus approprie´ s afin de traiter la tuberculose multir´esistante et ultra-r´esistante.

RESUMEN DE R E F E R E N C I A: El Laboratorio Supranacional de Referencia de la Tuberculosis en Bangkok y el Hospital Chiangrai Prachanukroh, de Chiangrai, en Tailandia. O B J E T I V O: Evaluar el rendimiento diagnostico ´ de varios estuches de desarrollo reciente de hibridacion ´ por sondas en tiras (LiPA) en los entornos end´emicos. M E T O D O: Se utilizaron los estuches de LiPA con el fin de evaluar 404 aislados cl´ınicos del ge´ nero Mycobacterium y 163 muestras de esputo en Tailandia. R E S U LT A D O S: Mediante las pruebas LiPA se detectaron M. tuberculosis, M. avium, M. intracellulare y M. kansasii con una sensibilidad y una especificidad de 100%, cuando se compararon con la prueba AccuProbe comercialmente disponible. Cuando se analiz o´ la capacidad de los estuches LiPA para detectar mutaciones en los aislados cl´ınicos con cepas MARCO

resistentes a rifampicina, isoniazida, pirazinamida y fluoroquinolonas, se observo´ que esta prueba ofrece un muy alto grado de sensibilidad (65,9–100%) y especificidad (98,2–100%), cuando se compara con las pruebas corrientes de sensibilidad y con la secuenciacion ´ del ADN. Los estuches LiPA ofrecieron una sensibilidad de 75,0 a 85,7% y una especificidad de 96,4% a 100% cuando se analizaron las muestras cl´ınicas de esputo. C O N C L U S I O N: Los nuevos estuches diagnosticos ´ LiPA ofrecen un alto grado de sensibilidad y especificidad y pueden reforzar la deteccion ´ ra´pida de la resistencia a los medicamentos antituberculosos de primera y segunda l´ınea y mejorar as´ı la eleccion ´ de un tratamiento adecuado en los casos de tuberculosis multidrogorresistente y extremadamente drogorresistente.

Evaluation of a novel line-probe assay for genotyping-based diagnosis of Mycobacterium tuberculosis in Thailand.

The Supranational Tuberculosis Reference Laboratory (NTRL), Bangkok, and Chiangrai Prachanukroh Hospital, Chiangrai, Thailand...
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