Multi Drug Resistance in Tuberculosis
and the Use of PCR for Defining
Molecular Markers of Resistance

Julio C. Mendez, M.D., Infectious and Communicable Diseases Division, Dept. of Medicine, University of Florida
and Duval County Health Department, Jacksonville, Florida

Introduction

Tuberculosis is the leading cause of mortality in adults due to an infectious agent and accounts for 26 % of all preventable adult deaths globally. According to the World Health Organization, 8 million cases of tuberculosis (TB) occur each year, resulting in 3 million deaths. 1 The strains of Mycobacterium tuberculosis resistant to anti-TB drugs have been recovered from both immunocompetent and immunocompromised patients worldwide. 2

In response to the alarming statistics and trends the Wold Health Organization declared tuberculosis to be a global public health emergency. 1 The Center for Disease Control and Prevention National Surveillance Systems registered 93,449 TB cases from 1993-1996, including 1,457 cases of multi-drug resistant tuberculosis (MDR-TB)(3). Recent data showed that about 13% of all new cases are resistant to at least one first line drug, and 1.6 % are resistant to both Isoniazid (INH) and Rifampin (RIF). 3

Multidrug-resistant Mycobacterium tuberculosis is an emerging problem of great importance to public health, with higher mortality rates than drug-sensitive TB, particularly in immunocompromised patients. MDR-TB patients require treatment with more toxic second line drugs and remain infectious for longer than patients infected with drug-sensitive strains, incurring higher costs due to prolonged hospitalization. 2

Multi-drug Resistant Tuberculosis

Multi-drug resistant tuberculosis strains are generally considered to be those resistant to at least two drugs, such as INH and Rifampin. The frequency of resistance to multi drugs, varies geographically, and acquired (secondary) resistance is more common than primary resistance. High rates of acquired MDR-TB have been reported in Nepal (48%), India (33.8%), and New York City (30%) in the early 1900s.

Although recent studies have shown an overall decrease in number of MDR-TB cases reported in New York City and in the USA, the number of states reporting these organisms have actually increased substantially since the early 1900's. 4 Table 1 summarizes the data available regarding drug-resistant tuberculosis in the USA, State of Florida and Duval County.

MDR-TB strains could arise as a consequence of sequential accumulation of mutations conferring resistance to single agents, or by a single step process such as acquisition of an MDR element. 5 A well documented example of how multi drug resistant Mycobacterium tuberculosis strains arise has been provided by the analysis of the evolution of two closely related subclones in New York City designated as a strain W and W1. These two related organisms have caused greater than 300 cases of tuberculosis in New York City and elsewhere. Automated DNA sequencing of representative organisms defined the exact series of distinct mutations conferring resistance to Rifampin, INH, Streptomycin, Ethambutol, ETH, PZA, Kanamycin, and quinolones. These drug resistant strains have spread to other US cities and Europe. Some of these MDR isolates arise because random mutations in genes that encode targets for the individual anti microbial agents are selected by sub-therapeutic drug levels resulting from treatment errors, poor adherence to treatment protocols, or other factors. 6

Molecular Basis of Antimicrobial Resistance in Tuberculosis

Substantial progress has been made in our understanding of the molecular basis of Mycobacterium tuberculosis drug resistance in the last decade. 7 Understanding the genetic events that lead to drug resistance in clinical M. tuberculosis isolates is important for the development of genetic assays, elucidation of the mechanisms of action of antimicrobial agents, and the design of novel antibiotics that are active against drug-resistant strains. 8 Drug resistant strains emerge when chemotherapy is intermittent or otherwise inadequate. Several factors influence the degree of success of treatment programs including duration and complexity of therapy, ease of healthcare access, treatment cost, patient adherence, and drug side effects. 9 The target regions of Mycobacterium tuberculosis and the mutations conferring drug resistance in patients with Tuberculosis are summarized in Table 2. Resistance mechanisms for the first line anti-tuberculous agents are discussed below.

Rifampin is a first line TB medication. The highly effective bactericidal action of this drug against M. tuberculosis has made it a key component of the initial anti-tuberculous regimen. Analysis of approximately 500 Rifampin strains from global sources has found that 96% of Rifampin resistant clinical isolates of Mycobacterium tuberculosis have mutations in the 81-bp core region of rpoB gene, which encodes the B subunit of RNA polymerase. 7,10 These mutations are absent in susceptible organisms. Although minor discrepancies have been reported, in general there have been a strong correlation of a specific amino acid substitutions and MIC. Missense mutations in codons 513, 526, or 531 result in high level Rifampin resistance, whereas amino acid changes at position 514 or 533 usually result in low levels of Rifampin resistance. The molecular mechanism of resistance in 4% of Rifampin resistant tuberculosis isolates that lack RRDR changes is unknown. 5 It is estimated that 90% of rifampin-resistant isolates in some areas are also resistant to isoniazid, making rifampin resistance a useful surrogate marker for multidrug resistance and indicating that second and third line drugs to which these isolates are susceptible are urgently required. 11,12

INH is a synthetic, bactericidal agent, used as a first line TB drug. Despite its widespread application to tuberculosis therapy and prophylaxis and intensive laboratory investigation, there is much that is not yet understood about the bacteria targets and mode of action of INH. Investigators on several continents have reported that many (50-60%) INH resistant patient isolates have mutations, small deletions or insertions that are not represented among INH sensitive control strains. 7 Mutations leading to INH resistance have been identified in different gene targets including KatgG, inha, ahpc and other genes that remain to be established. 5 Telenti et al analyzed Mycobacterium tuberculosis isolates by PCR and found that the mutation frequencies were as follows for INH resistant strains KatG (36.8%), inhA (31.6%), KatG-inhA (2.6%), ahpC (13.2%) and KatG-ahpC (2.6%). 13

Amino acid replacements in the NADH binding site of InhA apparently result in INH resistance by preventing the inhibition of mycolic acid biosynthesis. Mutations in the KatG or inhA do not account for all INH resistant strains since 15-25% INH resistance clinical isolates have both wild-type KatG and inhA genes. The mechanism of INH resistance in some strains remains to be determined.

Ethionamide is a second line TB drug that is thought to inhibit mycolic acid biosynthesis in Mycobacterium tuberculosis. Studies have shown that for certain strains, low level of INH resistance is correlated with co-acquisition of Ethionamide resistance, suggesting that INH and Ethionamide share a common molecular target and most likely the mab-inhA genes. 14

Streptomycin is another first line TB drug. Mutations associated with streptomycin resistance in tuberculosis have been identified in the 16S rRNA gene (rrs) and rpsL gene. In contrast to other bacteria that have multiple copies of rRNA genes, mycobacterium tuberculosis complex members have only one copy. Therefore, single nucleoside changes can potentially produce antibiotic resistance. Mutations in the rrs are clustered in two regions around nucleotides 530 and 951. The 530 loop 16S rRNA is highly conserved and is located adjacent to the 915 region in secondary structure models. The majority of mutations producing streptomycin resistance occur in rpsL. The most common mutation is at the codon 43. Mutations also have occurred in codon 88. About 65-75% of streptomycin resistant isolates have resistance-associated changes in rpsL or rrs. Failure to identify resistance-associated variations in these genes in 25-35% of organisms indicates that other molecular mechanisms of streptomycin resistance exist. 15

Pyrazinamide (PZA) is a structural analogue of nicotinamide that is used as a first line TB drug. PZA kills semi-dormant tubercle bacilli under acidic conditions. It is believed that in the acidic environment of phagolysosomes the tubercle bacilli produce pyrazinamidase, an enzyme that converts PZA to pyrazinoic acid, the active derivative of this compound. To define the molecular mechanism of PZA resistance the mycobacterium tuberculosis pncA gene encoding pyrazinamidase has been sequenced. The results have provided evidence that pncA mutations conferred PZA resistance. DNA sequencing of PZA resistance clinical isolates identified mutations at codons, 63, 138, 141, and 162. In contrast, susceptible organisms had wild type sequences. Lack of pncA mutations in 28%of PZA resistant isolates suggested the existence of at least one additional gene participating in resistance. A remarkably wide array of pncA mutations resulting in structural changes in the PncA has been identified in greater than 70% of drug resistant isolates. It is presumed that these structural changes detrimentally change enzyme function, thereby altering conversion of PZA to its bioactive form. 5,16

Ethambutol is a bactericidal first line TB drug. This agent has been proposed to be an arabinose analog; the specific target is likely to be an arabinosyl transferase, presumably a functionally important site. To understand the mechanism of resistance of Ethambutol a two gene locus (embAB) that encodes arabinosyl transfer has been established. Automated sequencing of these regions in clinical isolates from diverse geographical areas, discovered that 69% of Ethambutol resistance isolates had an amino acid substitution in EmbB that was not found in Ethambutol susceptible strains. The great majority (98%) of strains had mutations in codon 306, however, mutations were also identified in 3 additional codon 285, 330, and 630. These mutations were also uniquely represented among Ethambutol resistance organisms. The data are consistent with the idea that specific amino acid substitutions in EmbB detrimentally affect the interaction between Ethambutol, a putative anabinose analogue and EmbB likely to be an arabinosyl transferase. EmbB mutations are associated with Ethambutol resistance in roughly 70% of Ethambutol isolates of Mycobacterium tuberculosis. The cause of Ethambutol resistance in many organisms lacking mutations in ERDR of EmbB is unknown. 17

Rapid Detection of Drug-Resistant TB Strains

Delineation of the molecular mechanisms of antimicrobial agent resistance has resulted in the development and application of several PCR-based strategies designed to rapidly detect mutations associated with resistance. 18 Molecular Assays are potentially the most rapid and sensitive methods for the detection of drug resistance and are theoretically able to provide a same-day diagnosis from clinical samples. The utility of these assays is dependent on their ability to detect all common drug resistance mutations. Some of these methods are summarized in table 3 and include direct sequencing of PCR products, SSCP analysis, heteroduplex analysis, dideoxy fingerprinting, an RNA/RNA duplex, base pair-mismatch assay, a luciferase mycobacteriophage strategy, a rRNA/DNA-bioluminescence- labeled probe method, a reverse hybridization-based line probe assay, and other strategies. 7

These methods are all designed to exploit the observation that specific polymorphisms found in resistant strains are absent in susceptible organisms. The fact that natural populations of drugs-susceptible Mycobacterium tuberculosis isolates recovered globally have remarkably few polymorphisms in structural genes greatly simplifies interpretation of these assay. In essence, the M. tuberculosis complex is an ideal situation for application of certain kinds of molecular diagnostic testing strategies. 5 Each molecular strategy has advantages and disadvantages, and a full discussion of this important topic is beyond the scope of this review.

Among the many techniques used to identify drug resistance-associated mutations, automated DNA sequencing of PCR products has been the most widely applied. Previous studies have suggested that the DNA sequence of M. tuberculosis is extraordinarily well conserved and that mutations in the M. tuberculosis genome are almost always associated with drug resistance. One important advantage of sequence-based approaches is that the resulting data are virtually unambiguous because the resistance associated mutation is either present or absent. Automated sequencing has been used by several groups in routine clinical setting and has been found to give excellent benefit to patient care activities. For example, sequencing of the rpoB gene could generate data bearing rifampin-resistance within a few days from growth obtained in early BACTEC- positive culture. 19 Similarly, other groups have demonstrated good performance of SSCP-based interrogation of target sequences. 13 The line-probe assay strategy has the advantage of relatively reliable performance, and potential commercial availability. A new technique recently described is the molecular beacons assay. This is ideally suited for genotypic assays, since it is able to detect amplicons or PCR products as they are synthesized during real-time PCR and to discriminate between DNA sequences that differ from one another by as little as a single nucleotide substitution. 8

All strategies suffer from the fact that for no antimycobacterial agent do we understand the molecular of resistance for 100% of organisms. Hence, identification of a resistance-associated mutation is clinically informative whereas lack of a mutation in the target sequence must be interpreted with considerable caution.

References

  1. Raviglione M., Sinder D., Kochi A. 1995. Global epidemiology of tuberculosis-morbidity and mortality of a worldwide epidemic. JAMA 273:220-226.
  2. Cohn D., Bustreo F., and Raviglione M. Drug-Resistant Tuberculosis: Review of the Worldwide Situation and the WHO/IUATLD Global Surveillance Project. Clinical Infectious Diseases, 1997; 24 (Suppl 1): S121-30
  3. Moore M., Onorato I., McCray E., et al. Trends in drug-resistant tuberculosis in the United States, 1993-1996. JAMA 1997; 278:833-837.
  4. Bloch A., Cauthen G., Onorato I., et al. Nationwide survey of drug-resistance tuberculosis in the United States. JAMA 1994; 271:665-671.
  5. Ramaswamy S., Musser J. Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 Update. Tubercle and Lung Disease, 1998; 79(1): 3-29.
  6. Agerton T., Valway S., Blinkhorn R., et al. Spread of Strain W, a Highly Drug-Resistant Strain of Mycobacterium tuberculosis, Across the United States. Clinical Infectious Diseases, 1999; 29:85-92.
  7. Musser J. Antimicrobial Agent Resistance in mycobacteria: molecular genetic insights. Clin Microbiol Rev 1995; 8:496-514.
  8. Piatek A.,Telenti A.,Murray M., et al. Genotypic Analysis of Mycobacterium tuberculosis in Two Distinct Populations Using Molecular Beacons: Implications for Rapid Susceptibility Testing. Antimicrobial Agents and Chemotherapy, Jan 2000; 44(1): 103-110.
  9. Mahmoudi A., Iseman M. Pitfalls in the care of patients with tuberculosis: common errors and their association with the acquisition of drug resistance. JAMA 1993; 270:65-68.
  10. Valim A., Rossetti M., Ribeiro M, et al. Mutations in the rpoB Gene of Multidrug-Resistant Mycobacterium tuberculosis Isolates from Brazil. J. Clin. Microbiology; Aug. 2000, 3119-3122
  11. Yuen L., Leslie D., Coloe P. Bacteriological and molecular analysis of rifampin-resistant Mycobacterium tuberculosis strains isolated in Australia. J. Clin. Microbiology; 1999; 37(12): 3844-50
  12. Watterson S., Wilson S., Yates M., et al. Comparison of three molecular assays for rapid detection of rifampin resistance in Mycobacterium tuberculosis. J. Clin. Microbiology, 1998; 36(7): 1969-73.
  13. Telenti A., Honore N., Bernasconi C., et al. Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at reference laboratory level. J. Clin Microbiol 1997; 35:719-723
  14. Banerjee A., Dubnan E., Quemard A., et al. InhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 1994; 263:227-230
  15. Douglas J., Steyn L. A ribosomal gene mutation in streptomycin-resistant Mycobacterium tuberculosis isolates. J. Infect. Dis. 1993; 167:1505-1506
  16. Cynamon M., Klemens S. Antimycobacterial activity of a series of pyrazinoic acid esters. J. Med. Chem. 1992; 35:1212-1215
  17. Telenti A., Philipp W., Sreevatsan S., et al. The emb operon, a gene cluster of Mycobacterium tuberculosis involved in Resistance to ethambutol. Nature Med. 1997; 3:567-570
  18. Riska P., Jacobs W., Alland D. Molecular determinants of drug resistance in Tuberculosis. Int. J. Tuberc. Lung Dis; 2000 Feb; 4(2 Suppl 1): S4-10
  19. Pai S., Esen N., Pan X., et al. Routine rapid Mycobacterium species assignment based on species-specific allelic variation in the 65-kilodalton heat shock protein gene (hsp65). Arch. Pathol. Lab. Med.1997, 121:859-864.
  20. Cingolani A.,Antinori A., Sanguinetti M., et al. Application of Molecular Methods for Detection and Transmission Analysis of Mycobacterium tuberculosis Drug Resistance in Patients Attending a Reference Hospital in Italy. The Journal of Infectious Diseases, 1999;179:1025-9
  21. Williams D., Spring L., Gillis T., et al. Evaluation of a polymerase chain reaction _based universal heteroduplex generator assay for direct detection of rifampin susceptibility of Mycobacterium tuberculosis from sputum specimens. Clin. Infect. Dis.1998;26:446-450.
  22. Nash K., Gaytan A., Inderlied C. Detection of rifampin resistance in Mycobacterium tuberculosis by use of a rapid, simple, and specific RNA/RNA mismatch assay. J. Infect. Dis. 1997; 176:533-536.
  23. Nachamkin I.,Kang C.,Weinstein M. Detection of resistance to isoniazid, rifampin, and streptomycin in clinical isolates of Mycobacterium tuberculosis by molecular methods. Clin. Infect. Dis. 1997;24:894-900.
  24. Jacobs W., Barletta R.,Udani R., et al. Rapid assessment of drug susceptibilities by means of luciferase reporter phages. Science 1993:260:819-822.
  25. Cangelosi G., Brabant W., Britschgl T., et al. Detection of rifampin- and ciprofloxacin- resistance Mycobacterium tuberculosis by using species-specific assays for precursor rRNA. Antimicrob. Agents Chemother 1996;40: 1790-1795.
  26. De Beenhouwer H., Lhiang Z., Jannes G., et al. Rapid detection of rifampin resistance in sputum and biopsy specimens from tuberculosis patients by PCR and line probe assay. Tuber. Lung Dis. 1995;76:425-430.
February, 2001/ Jacksonville Medicine

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