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 Table of Contents  
ARTICLE
Year : 2012  |  Volume : 1  |  Issue : 1  |  Page : 40-44

Rapid molecular testing for multi-resistant tuberculosis in Mongolia: A diagnostic accuracy study


1 National Center for Communicable Diseases, Ulaanbaatar, Mongolia
2 Health Science University, Ulaanbaatar, Mongolia
3 Research Institute for Tropical Medicine, Manila, Philippines
4 Research Institute of Tuberculosis, Tokyo, Japan

Date of Web Publication28-Feb-2017

Correspondence Address:
B Buyankhishig
National Center for Communicable Diseases, MOH, Campus Nam-Yan-Ju Street, Ulaanbaatar 210648
Mongolia
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Source of Support: None, Conflict of Interest: None


DOI: 10.1016/j.ijmyco.2012.01.007

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  Abstract 


Objective: The aim of this study was to assess the performance of a molecular line probe assay, GenoType® MTBDRplus, for rapid detection of rifampicin and isoniazid resistance in the Mongolian situation. The sensitivity and specificity of GenoType® MTBDRplus to detect rifampicin and isoniazid resistance-associated mutations in culture specimens and directly in smear-positive clinical specimens was examined.
Method: 218 MDR-TB subjects aged between 14 and 75 years old from eight districts in Ulaanbaatar city (between July 2009 and May 2010) were included in this study .The GenoType Mycobacterium tuberculosis drug resistance first line (MTBDR plus) assay (Hain Life-science, Nehren, Germany) was tested on 109 clinical isolates and directly on 41 sputum specimens for the ability to detect the resistances. Results were compared with conventional culture and drug susceptibility testing on solid medium.
Results: The high correlation of the results from GenoType® MTBDRplus and conventional drug susceptibility testing was obtained from this study. The results clearly showed a high performance of GenoType® MTBDRplus with almost 100% accuracy for all the important indicators, such as sensitivity, specificity, positive and negative predictive values and detection of rifampicin resistance. Discrepancies were obtained in comparison with DNA sequencing results.
Conclusions: The Genotype174; MTBDRplus assay was demonstrated as a rapid, reliable and highly accurate tool for early detection of MDR-TB through examining smear positive cases.

Keywords: Multi-drug resistant tuberculosis (MDR-TB), Drug susceptibility testing (DST), Rapid molecular testing of MDR-TB (GenoType174; MTBDRplus)


How to cite this article:
Buyankhishig B, Oyuntuya T, Tserelmaa B, Sarantuya J, Lucero MG, Mitarai S. Rapid molecular testing for multi-resistant tuberculosis in Mongolia: A diagnostic accuracy study. Int J Mycobacteriol 2012;1:40-4

How to cite this URL:
Buyankhishig B, Oyuntuya T, Tserelmaa B, Sarantuya J, Lucero MG, Mitarai S. Rapid molecular testing for multi-resistant tuberculosis in Mongolia: A diagnostic accuracy study. Int J Mycobacteriol [serial online] 2012 [cited 2019 May 19];1:40-4. Available from: http://www.ijmyco.org/text.asp?2012/1/1/40/201198




  Introduction Top


Mongolia is one of the seven countries with a high burden of tuberculosis (TB) in the western Pacific Region [1]. Doubling of case notification rates between the years 1990 and 2007, from 79 to 166 per 100,000 population, presents a serious concern for the Mongolian health authority. The mortality rate of TB was 2.5 per 100,000 population in 2007 [2]. In 2008, the failure rate among new smear-positive cases was 6.2%, which was six times higher than the region's average. Previous studies showed a low rate of multi-drug resistant tuberculosis (MDR-TB) among new cases [1.4% (95%CI 0.7–1.6)], whereas, the rate was significantly higher [27.5% (95%CI 21.8–34.1)] in retreatment cases [3]. Importantly, around 24% of emerged MDR-TB patients were not detected in a timely manner. Therefore, rapid and accurate detection of resistant strains is essential for both efficient treatment and control strategies. Generally, drug susceptibility testing (DST) is performed by the conventional method, which takes several weeks to yield a result. Today, advances in molecular technology have provided rapid systems that shorten the time needed for MDR-TB detection. Among them, the Genotype174; MTBDR plus assay can identify the resistant strains within 1day. The MTBDR plus is a polymerase chain reaction (PCR) amplification and solid-phase reverse assay which detects mutations in the rpoB for rifampicin (RIF) resistance, the katG for high-level isoniazid (INH) resistance, and the inhA for low-level INH resistance directly from smear-positive sputum [4]. Thus, this method needs to be studied particularly in the developing world. The aim of this study was to assess the diagnostic accuracy of a molecular line probe assay (MTBDR plus assay) to rapidly detect mutations associated with RIF and INH in culture specimens and directly in smear-positive clinical specimens collected from previously treated TB patients in Mongolia.


  Methods and materials Top


Study setting and population

This study was conducted at the National Reference TB Laboratory located at the National Center for Communicable Diseases, Ulaanbaatar (UB). This laboratory serves a population of approximately 1million people in the capital city of Ulaanbaatar. All diagnostic centers in eight districts of UB participated in the survey. The calculation of the sample size followed the principles outlined in the WHO/IUATLD guidelines [5]. Participant recruitment was based on the results of sputum smear examination for acid-fast bacillus smear and culture and sensitivity (AFB). Specimens with culture negative results, contamination, or growth with other Mycobacterium spp. were excluded from further investigation. Overall, 180 smear-positive specimens from relapse, failure or default of the treatment outcomes were included in this study.

Specimen

Two specimens with the highest count of bacilli upon Ziehl–Neelsen examination were submitted for culture and susceptibility testing on solid media and direct MTBDR plus tests. Specimens were processed by the conventional N-acetyl-l-cysteine-NaOH method (1% final NaOH concentration). After decontamination, the concentrated sediments were suspended in 1.0–1.5ml sterile phosphate buffer (pH 7.0) and smears were prepared by the Ziehl–Neelsen staining method. After inoculation onto solid medium, the leftover sediments of the decontaminated sputum specimens were stored at −20 °C. The leftover sediments of the chosen specimens were thawed and used for MTBDR plus testing. Sample volumes of 500microliters (μl) were centrifuged for 15min at 10,000g, the supernatant was discarded, and the pellet was re-suspended in100μl of distilled water. Subsequently, the suspension was boiled for 20min and incubated in a sonic water bath at room temperature for 15min [6].

Drug susceptibility testing

Sputum smear microscopy positive samples were cultured on Löwenstein-Jensen (LJ) culture medium. Mycobacterium tuberculosis complex was identified by culturing on LJ medium containing p-nitrobenzoic acid. Drug susceptibility testing (DST) was performed by proportion method on LJ medium impregnated with isoniazid, rifampicin, streptomycin, and ethambutol according to the proportion method as recommended by WHO/IUATL [7].

GenoType174; MTBDRplus assay

The strip assay was performed as recommended by the manufacturer [6]. Briefly, for amplification, 35μl of a primer-nucleotide mixture (provided with the kit), 5μl of buffer containing 2.5mM MgCl2, 1.25 U Taq DNA polymerase, and 5μl of the preparation of mycobacterial DNA in a final volume of 50μl were used. The amplification protocol consisted of 15min of denaturing at 95 °C; 10 cycles comprising 30s at 95 °C and 120s at 58 °C; an additional 20 cycles comprising 25s at 95 °C, 40s at 53 °C, and 40s at 70 °C; and a final extension at 70 °C for 8min. Hybridization and detection were performed in a shaking incubator, and this procedure was performed at 45 °C for 30min, followed by two washing steps. Steps taken to avoid amplicon contamination were manual pipetting of the amplicon, use of separate wells and tubes for each strip, and extensive rinsing after each use. After a final washing, strips were air dried and fixed on paper. For the sputum specimens, an altered amplification protocol was applied which consisted of 15min of denaturing at 95 °C; 10 cycles comprising 30s at 95 °C and 120s at 58 °C; an additional 30 cycles comprising 25s at 95 °C, 40s at 53 °C, and 40s at 70 °C; and a final extension at 70 °C for 8min. Hybridization and detection were performed as described above. Each strip consists of 27 reaction zones (bands), including six controls (conjugate, amplification, M. tuberculosis complex, rpoB, katG, and inhA controls), eight rpoB wild-type (WT) and four mutant probes (rpoB MUT D516V, rpoB MUT H526Y, rpoB MUT H526D, and rpoB MUT S531L), one katG wild-type and two mutant probes (katG MUT T1S315T1 and MUT T2S315T2), and two inhA WTand four mutant probes (inhA MUT1C15T, inhAMUT2A16G, inhA MUT3A TBC, inhA TBA) ([Figure 1]). Results were interpreted according to the manufacturer's instructions.
Figure 1: Results for patterns of Genotype MTBDR plus strips (Hain Lifescience GmbH, Nehren, Germany). No. 1 – Negative control. No. 2 – INH monoresistant (inhAC15T mutation). No. 3 – MDR TB (rpoB mutation in 530–533 region, katG S315T1 mutation). No. 4 – MDR (rpoB S531L, katG S315T1 mutation). No. 5 – MDR (rpoB S531L, inhAC15T mutation). No. 6 –INHmono-resistant (inhAC15T mutation). No. 7 – MDR (rpoB S531L, katG S315T1 mutation).

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Laboratory quality control

Procedures for external quality assurance for smear and culture were based on the WHO guidelines [5]. The National Reference Laboratory before the start of the survey had participated in external quality assurance proficiency tests. The efficiency for determining INH and RIF resistance in 2009 was 100% using the conventional proportion method as a standard by SRL of Research Institute of Tuberculosis, Japan (RIT) [7]. The efficiency for identifying INH- and RIF-resistant strains using MTBDR plus assay was 99.0% and 91.6%, respectively, considering the conventional proportion DST as a standard.

Data analysis

The data were analyzed using SPSS 17.0 software. Sensitivity, specificity, positive predictive and negative predictive values of the molecular line probe assay were assessed compared with conventional testing [8]. Reporting time was calculated from the date of inoculation and the date of reporting of the conventional DST result or the date of availability of the molecular line probe assay.


  Results Top


Study subjects

The study covered 218 MDR-TB subjects within the age range of 14–75 years old from 8 districts of UB registered from 1 July 2009 to 30 May 2010. One hundred twenty-seven (127; 58.3%) were males and the remaining were female (91; 41.7%). A total of 68 patients with exclusive criteria were excluded from the study. A total of 150 patients with both results of MTBDR assay and conventional DST are subjects of this analysis.

GenoType MTBDRplus testing from smear-positive sputum

The GenoType174; M. tuberculosis drug resistance first line (MTBDRplus) assay (Hain Life-science, Nehren, Germany) was used in the study. As shown in [Table 1], of the total specimens with conventional DST results, 75 (50.0%) were fully susceptible strains, 61 (40.7%) were MDR, 5 (3.3%) were RIF mono-resistant, 10 (6.7%) were INH mono-resistant. The results were comparatively analyzed with MTBDR plus assay. A total of three discrepant results between LPA and DST were observed. For detection of discrepancies, the samples were compared with DNA sequencing results. The overall, sensitivity, specificity and positive and negative predictive values of the specimens with rapid and conventional results are shown in [Table 2]. Comparing the different banding patterns in drug-resistant isolates, including MDR, RIF-mono-resistant and INH-mono-resistant strains showed S531L mutation (MUT3 band) was more frequent and occurred in all RIF-resistant strains (78.8%). Of all INH-resistant strains, 33.8% (37.7% of MDR strains and 10% of INH-mono-resistant strains) had a mutation (MUT1) in the katG, and 67.6% (63.9% of MDR strains and 90% of INH-mono-resistant strains) had a mutation (MUT1) in the inhA. Three strains had mutations in both the katG and inhA. Nine of 10 (90%) INH-mono-resistant strains were detected by the presence of a mutation in inhA only ([Table 3]).
Table 1: Genotype MTBDR and conventional drug susceptibility testing (DST) results of M. tuberculosis samples surveyed from July 2009 to May 2010 in Ulaanbaatar, Mongolia.

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Table 2: Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of Genotype MTBDR plus in detecting rifampicin and isoniazid resistance in smear and isolate samples.

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Table 3: Pattern of Gene mutations in resistant Mycobacterium tuberculosis strains using Genotype MTBDR plus assay.

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  Discussion Top


The prevalence of identified MDR-TB cases was higher (40, 7%) in Mongolian retreated cases. Using MTBDRplus assay, most common mutations were rapidly and specifically detected along with their resistance not only to RIF, but also to INH in M. tuberculosis isolates ([Table 2] and [Table 3]). The probes used in this assay combine targeting the 81-bp “hot-spot” region in the rpoB gene, and two mutations at position 315 of the katG gene. Mutations affecting this codon are responsible for INH resistance in 60% of the cases worldwide [4]. In this current research, of all INH-resistant strains, 33.8% (37.7% of MDR strains and 10% of INH-mono-resistant strains) had a mutation of MUT1 in the katG. High frequency of mutations were found in accordance with most available reports at codons 531, 526, and 516. Although a high frequency of S531L mutation (MUT3 band) was observed in all RIF-resistant strains (83.6% of MDR), other mutations in the 530–533 regions also occurred; as detected by the lack of binding to the WT8 probe in the absence of S531L mutation.

Based on previous review literature, frequencies of katG mutation among INH resistant M. tuberculosis isolates in three South American countries were as follows: Brazil (81.3%), Peru (82.4%) and Argentina (71.4%). In contrast, the frequencies for the katG S315T mutation in INH resistant M. tuberculosis isolates for patients diagnosed in Kuwait and the Netherlands (65% and 55%, respectively) was lower than what was described in Russia (95%). Mutations in the inhA promoter region was variable (15–35%) among INH-resistant M. tuberculosis strains that were collected from the same geographical region [9]. In this study, mutations in the promoter region of the inhA gene played a major role (67.6% [63.9% of MDR strains and 90% of INH-mono-resistant strains]). Based on this finding, inhA mutation may increase the capability of predicting INH resistance. At present, the manual method was used for the GenoType174; MTBDRplus assay, which needs one day for amplification and another day for hybridization. Therefore, it will take two to three days to interpret the results.


  Conclusions Top


Molecular-based rapid MDR screening with the GenoType174; MTBDRplus test can be routinely used in a reference laboratory setting when rapid sensitivity testing is required for the proper management of patients or for the contacts of drug resistant cases. However, because only some of the mutations are targeted, this molecular test cannot be considered to perform alone in the laboratory.


  Acknowledgements Top


We would like to express our gratitude to: our colleagues in the TB clinics in eight districts; the TB surveillance department of the National Center for Communicable Diseases; the WHO representative office in Mongolia; the Research Institute for Tropical Medicine in Manila, Philippines, for their excellent technical assistance; the Research Institute of Tuberculosis in Japan; and the Japan Supranational Reference Laboratory for their great contributions with regard to initiating the survey.

Fund source: This project was given financial support by the WHO/TDR foundation.



 
  References Top

1.
Joint Review of the National Tuberculosis Programme, Mongolia September 2008, 23p.  Back to cited text no. 1
    
2.
TB Surveillance department annual report, NCCD, 2008.  Back to cited text no. 2
    
3.
B. Buyankhishig, N. Naranbat, S. Mitaria, H.L. Reider, Nationwide survey of anti-TB drug resistance in Mongolia, The International Journal of Tuberculosis and Lung Disease 15 (9) (2011) 1–5.  Back to cited text no. 3
    
4.
D. Hilleman, S. Rüsch-Gerdes, E. Richter, Evaluation of the GenoType MTBDRplus Assay for Rifampin and Isoniazid Susceptibility Testing of Mycobacterium tuberculosis strains and in clinical specimens, Journal of Clinical Microbiology 45 (2007) 2635–2640.  Back to cited text no. 4
    
5.
World Health Organization, Guidelines for surveillance of drug resistance in tuberculosis, 4th ed, World Health Organization Document WHO/HTM/TB/2009.422 (2009) 1–83.  Back to cited text no. 5
    
6.
GenoType MTBDRplus, version 1.0 [product insert]. (Internet) Feb 2007 (updated 2007 Apr 3; accessed 2007 May 9), Hain Lifescience, GmbH, Nehren, Germany. Available from: http:// www.hain-lifescience. com/pdf/304xx_pbl.pdf.  Back to cited text no. 6
    
7.
Panel test result of SRL from Research Institute of Japan, 2009.  Back to cited text no. 7
    
8.
P. Bossuyt, J. Reitsma, et al, The STARD statement for reporting studies of diagnostic accuracy: explanation and elaboration, Clinical Chemistry 49 (2003) 7–18.  Back to cited text no. 8
    
9.
F. Brossier, N. Veziris, et al, Performance of the Genotype MTBDR line probe assay for detection of resistance to rifampin and isoniazid in strains of mycobacterium tuberculosis with lowand high-level resistance, Clinical microbiology (2006) 3659–3664.  Back to cited text no. 9
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


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