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Year : 2013  |  Volume : 2  |  Issue : 1  |  Page : 14-17

Inconsistencies in drug susceptibility testing of Mycobacterium tuberculosis: Current riddles and recommendations

Mycobacteriology Research Section, Department of Infection and Immunity, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia

Date of Web Publication27-Feb-2017

Correspondence Address:
Sahal Al-Hajoj
Mycobacteriology Research Section, Department of Infection and Immunity, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211
Saudi Arabia
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Source of Support: None, Conflict of Interest: None

DOI: 10.1016/j.ijmyco.2012.11.003

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Drug susceptibility testing (DST) of Mycobacterium tuberculosis is a crucial procedure to determine the effective drug regimen for patients' treatment. Reporting of erroneous DST results to the treating physician has adulterous effects on patients. As a first study of its type, the inconsistencies in reporting DST results of rifampicin and isoniazid from Saudi Arabia were assessed. An automated liquid culture-based DST and a molecular mutation detection technique were used. Performance of first-line drug susceptibility testing of 1904 clinical isolates showed 44 inconsistent results. The majority of the cases reported as MDR-TB from the referral laboratories could not reproduce the same results at a different site (Mycobacteriology Research Section). Of the 44 cases, 16 (36.3%) showed false resistance to isoniazid and rifampicin and on the other hand, 14 (31.8%) cases showed false susceptibility to the same drugs. The possible causes for the inconsistencies and recommendations to overcome the biases based on this experience are discussed.

Keywords: Tuberculosis, Drug susceptibility testing, Genotyping

How to cite this article:
Varghese B, Al-Omari R, Al-Hajoj S. Inconsistencies in drug susceptibility testing of Mycobacterium tuberculosis: Current riddles and recommendations. Int J Mycobacteriol 2013;2:14-7

How to cite this URL:
Varghese B, Al-Omari R, Al-Hajoj S. Inconsistencies in drug susceptibility testing of Mycobacterium tuberculosis: Current riddles and recommendations. Int J Mycobacteriol [serial online] 2013 [cited 2017 Dec 11];2:14-7. Available from: http://www.ijmyco.org/text.asp?2013/2/1/14/201039

  Introduction Top

Tuberculosis (TB) is one of the most ancient diseases of mankind caused by Mycobacterium tuberculosis [1]. Despite the existence of drugs and vaccines for the last six decades, still TB continues to be a major threat worldwide with high morbidity and mortality. The most substantial reason for this phenomenon is the emergence of drug resistant strains, particularly the multidrug resistance (resistance to isoniazid and rifampicin). Multidrug-resistant TB (MDR-TB) has become a serious threat to global TB control as a result of the difficulties in diagnosis, treatment and associated high cost to TB control programs. The emergence of extensively drug-resistant TB (XDR-TB), with its poor treatment outcome and the extraordinary potential of high mortality in HIV-infected patients and rapid transmission, has been alarmed public health officials. MDR-TB does not respond to treatment with first-line drugs, and its management using second-line drugs has not yet been properly organized by most TB control programs [2],[3]. Recent findings of the World Health Organization showed globally the rate of any drug resistance is 20% and multidrug resistance is 5.3% of the total notified TB cases [4].

TB drug susceptibility testing (DST) has been documented to be one of the most complex procedures to perform in the mycobacteriology laboratory; it requires technical expertise to produce valid and reliable results, and it requires 4–6 weeks to get the final results. Valid and reliable DST is important to design appropriate drug regimens. Since the clinical decisions are made based on the laboratory findings, the reliability of results and the potential of the laboratory to test the drug susceptibility are imperative.

Saudi Arabia is a moderate TB burden country with an established national tuberculosis control program (NTP). The National Tuberculosis Program (NTP) of the country created nine regional laboratories that are serving as referral laboratories to confirm the diagnosis and perform drug susceptibility testing. All the laboratories are following the WHO guidelines and using the same technology (MGIT 960, Becton Dickinson, Maryland, USA) for mycobacterial culture and DST. A recent report from WHO showed that the new case rate is 24/100,000 population with a 2.1% of MDR-TB cases. However, the drug resistant rates in Saudi Arabia were found to be fluctuating from 2–44% [5],[6],[7]. Recently while conducting a nationwide drug surveillance survey, a considerable inconsistency on reporting DST results was observed. This study focused on the two most important drugs isoniazid and rifampicin as they are the key drugs for primary TB treatment and predictors for MDR-TB confirmation. The inconsistencies found in the reports led to the design of this study to compare the DST results using the same culture technique and a rapid molecular assay. This study attempts to explain the possibilities of misclassification of MDR-TB, which holds very high clinical implications. Possible reasons for such inconsistencies and recommendations to overcome the biases are discussed.

  Materials and Methods Top

Study samples

A nationwide survey of drug-resistant tuberculosis obtained 2235 non-repetitive mycobacterial clinical isolates from nine regional laboratories during the period 2009–2010. Of the total, 256 (11.4%) were found to be non tuberculous Mycobacteria (NTM) and 75 (3.4%) isolates were lost because of contamination. The remaining 1904 isolates were subjected to first-line DST. When the primary data were analyzed, 44 inconsistent results were observed and enrolled into the study.

Drug Susceptibility Testing

All the isolates enrolled in the study were subjected to first-line DST by using the commercially available kit BACTEC MGIT SIRE (Becton Dickinson, Maryland, USA) as per the recommendation of the manufacturer. The concentrations of the drugs used were 0.1 μ;g/ml for INH and 1 μ;g/ml for RIF. Isolates with inconsistent results were subjected to DST twice at different intervals to reproduce similar results in the mycobacterial research section of King Faisal Hospital and Research Centre [MRS-KFSHRC]. Moreover, the kits used each time belong to different lots and batches.

Quality Assurance

At MRS-KFSHRC strict and regular practices of internal and External Quality Assurance (EQA) were followed. In brief, bimonthly internal quality assurance testing was carried out for the MGIT SIRE (streptomycin, isoniazid, rifampicin, and ethambutol) kit with the standard strain of M. tuberculosis (ATCC 27294). In addition, the center regularly participates in the EQA surveys conducted by the College of American Pathologists (Illinois, USA). Furthermore, the section participates in DST EQA analysis coordinated by the World Health Organization (Supranational Reference Laboratory, Cairo, Egypt).

Detection of mutations conferring resistance to rifampicin and isoniazid

Genomic DNA was extracted by using the standard spin column technique (QIAAmp DNA Mini kit, QIAGEN, GmbH, Germany) as per the manufacturer's protocol. All the isolates were genotyped to determine the mutations conferring INH or RIF resistance by using the PCR-based reverse blot hybridization assay, Genotype MTBDR Plus® (Hain Lifescience, Nehren, Germany) according to the manufacturer's recommendations. In brief, a PCR was conducted by using the primers provided by the manufacturer, and reverse blot hybridization based on strips was carried out by using the automated system GT Blot 48 (Hain Life Science, Nehren, Germany). The strips after hybridization were scanned by using the automated system Geno-Scan (Hain Life Science, Nehren, Germany). The results were interpreted by using the software Blotrix (Hain Life Science, Nehren, Germany). Isolates with mutations in the rpoB gene and katG/inhA were considered to be RIF and INH resistant respectively. Strains possessing mutations in both rpoB and katG/inhA genes were regarded as MDR-TB.

Data analysis

The statistical analysis of the results of susceptibility testing and genotyping were carried out by using the software Epi-info V6.0 (CDC, Atlanta, USA).

  Results Top

Out of 1904 isolates, 44 (2.3%) isolates showed discrepant susceptibility results obtained from referral laboratories for either INH or RIF or both when tested at MRS-KFSHRC. Of the 44 isolates, 25 of them were reported as MDR-TB, 14 as susceptible to both drugs, and 5 of them resistant to INH by the regional laboratories. On the other hand, DST results at MRS-KFSHRC showed 19 as MDR-TB, 17 as susceptible to both drugs, 6 as INH resistant and 2 RIF resistant, respectively. Molecular genotyping results showed 19 as MDR-TB, 5 as INH resistant and 2 as RIF resistant, respectively. The remaining 18 isolates were observed as susceptible to both drugs; one phenotypically INH-resistant isolate showed susceptibility by the mutation detection assay ([Table 1]). Surprisingly, from the referral laboratories, 38.6% of the pan susceptible isolates were classified as MDR-TB, and on the other hand, 31.8% of isolates were misclassified vice versa.
Table 1: Phenotypic and genotypic drug susceptibility patterns of the 44 study samples.

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  Discussion Top

The goal of this study was to evaluate the inconsistencies on reporting rifampicin and isoniazid susceptibility at the regional TB laboratories emphasizing on the misidentification of MDR-TB. An attempt was made to formulate the current problems that may be faced by TB diagnostic facilities anywhere in the world. The limitations of current practices and recommendations to improve the performances were discussed.

Drug susceptibility testing in mycobacteriology needs significant technical proficiency to produce reliable and accurate results. Indeed, the fact is that even in most competent laboratories discrepant results can occur for many reasons. A certain proportion of drug-resistant bacteria exists in all populations of drug-susceptible M. tuberculosis. In addition, current methods for susceptibility testing of M. tuberculosis complex (MTBC) are based on proportion methods, which rely on a bacteriological definition of drug resistance that was developed in recognition of the difficulties in defining clinical resistance for mycobacteria. These methods provide qualitative results of “susceptible” or “resistant,” defining resistance as growth of greater than 1% of an inoculum of bacterial cells in the presence of a “critical” concentration of the drug. The critical concentrations of drugs were established on an empiric basis and adopted by international convention. The critical concentration represents the lowest concentration of drug that inhibit 95% of “wild strains” of M. tuberculosis and not inhibiting strains of M. tuberculosis isolated from patients unresponsive to therapy and considered resistant. The other key factors which may influence the inter-lab and intra-lab results are: bacterial population (repeated sub-culturing may lead to losing the slow-growing resistant population and false susceptible result), different growth kinetics, cross-contamination, growth difficulties of some strains, and minimum inhibitory concentration (MIC) of some isolates which is closer to the critical concentration [8],[9].

In this study, the drug susceptibility results of the regional referral laboratories were compared with the MRS-KFSHRC results. Molecular assay to determine the mutations responsible for drug resistance was performed for a reconfirmation of results. However, low-grade discrepancies (2.3%) were found among MRS-KFSHRC rsults, even on defining MDR-TB. Of the total (44 cases), 24 (54.5%) cases were reported as MDR-TB from the regional laboratories which could not reproduce the same results at MRS-KFSHRC. On the other hand, 14 (31.8%) cases reported as susceptible TB by the regional laboratories showed up as MDR-TB results in molecular testing as well as DST at MRS-KFSHRC. Both events of misclassification of MDR-TB have serious clinical implications; either the patient receive a highly complex wrong drug regimen or do not receive an adequate therapy. In addition, one phenotypically INH resistant isolate showed no mutation in the genotypic assay, which could possibly be due to an uncommon mutation in a gene other than katG or inhA as it was found in a low level in some isolates previously [10].

Interestingly a new phenomenon of TB transmission is also underway in the country as per the recent study (Varghese B. et al., Publication In Press) which showed that there is an endogenous reactivation of TB infection followed by an exogenous reinfection caused by ongoing drug-resistant strain transmission, mainly among African migrants. However, migrants from Asia and Saudi nationals also possess a low-level risk of this new phenomenon. In addition concurrent infection by multiple strains was also noticed. This could present two different DST results at the same time or within a short interval depending on the drug susceptibility profile of concurrently infected strains, particularly if the patient received a drug-resistant strain recently. Nevertheless, these phenomena could also be contributing factors in inconsistent results of DST as described previously [11],[12].

  Conclusion and Recommendations Top

The crucial TB-DST particularly defining MDR-TB in peripheral diagnostic laboratories of Saudi Arabia has a noticeable level (35.9%) of inconsistencies. The obtained results from the referral laboratories showed discrepancies which have serious consequences on patient management. An urgent reevaluation and training are needed for the staff involved in the TB-DST. Even though the discrepancies are common in low grade around the globe, false reporting of MDR-TB cases has serious clinical implications. Rapid molecular testing can be preferred for screening suspected high risk groups for drug resistance. High level, inconsistent results propose an immediate action to standardize the guidelines and reevaluate the existing policies.

It is recommended that the following practices be implemented to improve and to make highly reproducible results in concordance with international guidelines and this study's experiences [8]:

  1. Existing quality control programs need immediate enhancement. Strict practice of internal quality assurance must be practiced in all the regional laboratories. This would include testing of kits and media used with standard strains (ATCC-American Type Culture collection, VA, USA) and strict adherence to standard operating protocols.
  2. An intra-laboratory quality assurance program must be run between the laboratories that are involved in performing DST in a frequency of at least twice in a year. A central laboratory must coordinate with the sampling, result analysis and grading of the participating laboratories with proper auditable documentations.
  3. Participation in international EQA programs should be implemented at all regional laboratories. To date, the most trusted international proficiency testing programs for TB-DST available are CAP-E Survey (College of American Pathologists, Illinois, USA), and MTB-DST proficiency testing (WHO/IUATLD, Supranational Reference Laboratory; Center for Disease Control, Atlanta, GA, USA and American Proficiency Institute, Traverse City, MI, USA).
  4. Training and education of the staff in the laboratories which perform DST; and clinicians interpreting the test results should be available on a recurring basis.
  5. A clinical database of all the reported patients must be created and used as a reference for studying complicated cases.
  6. Indeed, considering molecular testing as an alternative for confirming drug susceptibility results or as a screening for MDR-TB or XDR-TB is new, therefore it needs to be standardized and adapted to practice in all the referral laboratories. There are commercially available kits for first-line and second-line drug resistance screening based on mutation detection from different manufacturers.
  7. Tuberculosis research in Saudi Arabia or any developing country should be focused on the following: adaptability (cost and capability) of rapid molecular methods for the screening of drug resistance in high-risk groups, the role of molecular methods to detect primary resistance and in treatment failures; assessment of current practices in the laboratories and referral strategies to develop optimal strategies to improve TB detection and DST turnaround times; and clinical utility of MIC testing.
  8. A central reference laboratory with all the sophisticated technologies should be designated to be involved in handling all complicated cases and coordinating with all the intra-lab quality control assessments.

  Acknowledgements Top

This study was funded by the King Abdul-Aziz City for Science and Technology (Project AT-26-110), Riyadh, Saudi Arabia. The sponsor has no role in study design, execution, data management and publication.

  References Top

B.M. Rothschild, L.D. Martin, G. Lev, H. Bercovier, G.K. Bar-Gal, C. Greenblatt, et al, Mycobacterium tuberculosis complex DNA from an extinct bison dated 17,000 years before the present, Clin. Infect. Dis. 33 (2001) 305–311.  Back to cited text no. 1
World Health Organization, The WHO/IUATLD Global Project on Anti-Tuberculosis Drug Resistance Surveillance. Anti-Tuberculosis Drug Resistance in the World, WHO, Geneva, Switzerland, 2008.  Back to cited text no. 2
L.W. Riley, Drug-resistant tuberculosis, Clin. Infect. Dis. 17 (1993) S442–S446.  Back to cited text no. 3
World Health Organization, Global TB Epidemiology Report, WHO, Geneva, 2010.  Back to cited text no. 4
K.K. Abu-Amero, Status of antituberculosis drug resistance in Saudi Arabia 1979–98, East. Mediterr. Health J. 8 (2002) 664–670.  Back to cited text no. 5
F.N. Kordy, S. Al-Thawadi, A.A. Alrajhi, Drug resistance patterns of Mycobacterium tuberculosis in Riyadh, Saudi Arabia, Int. J. Tuberc. Lung Dis. 8 (2004) 1007–1011.  Back to cited text no. 6
A.A. Alrajhi, A.M. Al-Barrak, Mycobacterium tuberculosis susceptibility in Saudi Arabia, Saudi Med. J. 23 (2002) 1227–1231.  Back to cited text no. 7
Association of Public Health Laboratories, TB Drug Susceptibility Testing, Association of Public Health Laboratories, Atlanta, USA, 2007.  Back to cited text no. 8
A. Van Deun, L. Barrera, I. Bastian, L. Fattorini, H. Hoffmann, K.M. Kam, et al, Mycobacterium tuberculosis strains with highly discordant rifampin susceptibility test results, J. Clin. Microbiol. 47 (2009) 3501–3506.  Back to cited text no. 9
A. Van Rie, R. Warren, I. Mshanga, A.M. Jordaan, G.D. van der Spuy, M. Richardson, et al, Analysis for a limited number of gene codons can predict drug resistance of Mycobacterium tuberculosis in a high-incidence community, J. Clin. Microbiol. 39 (2001) 636–641.  Back to cited text no. 10
A. Van Rie, T.C. Victor, M. Richardson, R. Johnson, G.D. van der Spuy, E.J. Murray, et al, Reinfection and mixed infection cause changing Mycobacterium tuberculosis drug-resistance patterns, Am. J. Respir. Crit. Care Med. 172 (2005) 636–642.  Back to cited text no. 11
X. Li, Y. Zhang, X. Shen, G. Shen, X. Gui, B. Sun, et al, Transmission of drug-resistant tuberculosis among treated patients in Shanghai, China, J. Infect. Dis. 195 (2007) 864–869.  Back to cited text no. 12


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