International Association of Physicians in AIDS Care, February 2000 Journal
Gaby E. Pfyffer, PhD
Swiss National Center for Mycobacteria Department of Medical Microbiology, University of Zurich
Zurich, Switzerland

Presented at Diagnostic Technologies in the Management of HIV/AIDS and Other Life-Threatening Coinfectious Diseases: An IAPAC Symposium, October 10, 1999, Vienna, Austria

Introduction

According to the World Health Organization (WHO), more people will die of tuberculosis (TB) this year than in any other year in history. Of equal concern, however, are the emergence and nosocomial transmission of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis. In light of this frightening global scenario, development of efficient laboratory strategies for rapid and reliable antimicrobial susceptibility testing of M tuberculosis to antituberculous drugs is of prime importance.

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The HIV-infected patient's vulnerability to TB

In areas where both HIV infection and TB are common, HIV has exerted a dramatic impact on the course of TB in the recent past.¹ A vast number of published reports clearly demonstrates that HIV infection is a major risk factor not only for reactivation of a latent TB infection but also for rapid progression of a recent infection to manifest disease.^2,3 The estimated annual risk for development of active TB in patients infected with both HIV and M tuberculosis is 7 to 10 percent annually, compared with a chance of 5 to 10 percent in the lifetime in immunocompetent individuals.² In other words, the annual risk of active TB is 170 times greater in patients with AIDS and 113 times higher in HIV-infected individuals than in persons with no known risk factors.⁴

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Drug resistance and M tuberculosis

Resistance of M tuberculosis to anti-TB drugs (Table 1) is the result of a spontaneous genetic event and, worse, "a man-made amplification of the natural phenomenon".⁵ In M tuberculosis spontaneous mutations occur at a frequency of approximately 10^-5 to 10^-8.^6-8 Since resistance to various drugs arises independently, the likelihood of spontaneous mutation to isoniazid and rifampin, for instance, is 1 in 10¹⁴ (10⁶ x 10⁸). At first sight, the probability of a dual mutation seems minimal. However, since pulmonary TB is always associated with enormous bacterial masses (cavities contain as many as 10⁷ to 10⁹ organisms), dual mutations will be seen with a certain frequency. This threat of multidrug resistance is one reason why combination regimens must always be used for TB.

The main reason for the global emergence of resistant strains, particularly MDR strains, is, however, unsuccessful treatment leading to acquired resistance. This phenomenon has been well known since the first antituberculous agent appeared on the market, that is, as early as 1946, when streptomycin was administered as a monotherapy.⁹ Besides poorly designed drug regimens, other reasons for treatment failure are (1) poor patient compliance with therapy, (2) mediocre drug quality, and/or (3) inadequate supervision of the patient by health authorities. As a consequence, drug-resistant organisms will by and large predominate the bacterial population in the lung and ultimately cause any therapy to fail.

Recently, results of the first phase of the Global Project on Anti-TB Drug Resistance Surveillance, initiated by the WHO and the International Union Against TB and Lung Disease, have become available.¹⁰ Although preliminary, the data generated in 35 countries on five continents clearly demonstrate that in certain areas the proportion of resistant strains has reached an alarming extent (Table 2).

In general, manifest disease with an MDR strain of M tuberculosis--a strain resistant to both isoniazid and rifampin, and possibly to additional drugs--has a poor clinical outcome since efficient therapeutic strategies are still lacking. In the recent past several outbreaks of MDR strains of M tuberculosis have been reported in hospitals, affecting both patients^11,12 and health care workers,¹³ and in prisons.¹⁴ In most of these outbreaks most patients were HIV infected. This high rate of coinfection is of utmost concern because, as these studies found, fatality rates among HIV-positive individuals with an active MDR TB are extremely high (72 to 89 percent); the median interval from diagnosis to death lies between four and 16 weeks.

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Traditional laboratory methods for antimicrobial susceptibility testing

Initially, antimicrobial susceptibility testing of M tuberculosis is carried out with a primary set of drugs, consisting of the front-line drugs isoniazid, rifampin, ethambutol, pyrazinamide, and, optionally, streptomycin. If resistance to one or several of these drugs is detected, it is common practice to test an extended spectrum of antimicrobial compounds.

For quite some time three different growth-based laboratory methods have been accepted for determining antimicrobial susceptibility of M tuberculosis: (1) the resistance ratio method, (2) the absolute concentration method, and (3) the proportion method.⁸ Most laboratories in the Western hemisphere utilize a modified proportion method on solid medium, according to the proposed standard of the National Committee for Clinical and Laboratory Standards.¹⁵ For most of the major antituberculous agents, this technique defines resistance of M tuberculosis as a percentage of resistant organisms larger than 1 percent in a given population of bacilli.⁸ TB therapy will be successful if (1) the critical proportion of organisms and the critical concentration of a drug8 are strictly adhered to, and (2) the strain is fully susceptible to front-line drugs in vitro.

Because antimicrobial susceptibility testing on solid media requires visible growth of the organisms (which requires three weeks of incubation), testing is preferentially done in liquid media today. The radiometric BACTEC 460 TB technique, established more than 20 years ago, is particularly efficient because it generates results for front-line drugs within a few days.¹⁶ To facilitate rapid therapeutic decisions for patients harboring MDR TB organisms, critical concentrations have recently been established for second-line drugs as well as for newer drugs such as capreomycin, ethionamide, amikacin, kanamycin, clofazimine, ofloxacin, and rifabutin.¹⁷

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More recent developments

In the last decade antimicrobial susceptibility testing has become a dynamic field spawning many new technologies that may one day prove successful in a clinical mycobacteriology laboratory. They all comply with the standard set by the Centers for Disease Control and Prevention that susceptibility testing results for M tuberculosis have to be available within 28 days of the time the specimen arrives in the laboratory.¹⁸

• Nonmolecular techniques. Among the leading novel strategies are several new growth-based, nonradiometric methods developed mainly to overcome the limitations of radiolabeled substrates (in particular, safety and regulatory principles). These methods are either manual or fully automated, including the Mycobacteria Growth Indicator Tube (MGIT, Becton Dickinson),¹⁹ the ESP Culture System II (Difco),²⁰ and the MB/BacT (Organon-Teknika).²¹ Preliminary studies with isoniazid, rifampin, ethambutol, and streptomycin show excellent overall agreement with the proportion method on solid and liquid media as well as promising turnaround times comparable to those of radiometry.

  Another strategy to detect drug resistance involves determining minimal inhibitory concentrations (MICs) of drugs. Well known as labor intensive, this procedure has been considerably facilitated by the Etest.²² After an Etest strip containing a gradient of the desired drug is applied to an agar plate, MICs are easily and rapidly determined at the point where the ellipse formed by inhibited growth crosses the strip. Although convenient, especially for determining resistance in rapidly growing mycobacteria, use of Etest strips for susceptibility testing of M tuberculosis remains limited because of the potential hazard arising from the high inoculum needed to inoculate agar plates and slow growth of the microorganism.

  In addition to strictly growth-based strategies, an increasing number of approaches assess drug susceptibility by identifying alternative markers of drug-resistant metabolic activities. Although some of these alternatives require time-consuming pregrown cultures, most of these novel techniques are straightforward. Among those are colorimetry,²³ flow cytometry,²⁴ bioluminescence assay of mycobacterial adenosine triphosphate,²⁵ and quantitation of mycobacterial antigens.²⁶ Mycobacteriophage-based methods, for example, with luciferase reporter phages²⁷ or PhaB phages,²⁸ appear to be promising as well. However, the complexity of these technologies and high cost have largely hampered their wider application in the clinical mycobacteriology laboratory.

• Molecular biology as a tool to detect resistant TB. Although the targets of the major anti-TB drugs within the cell of M tuberculosis have largely been known for some time, progress in understanding resistance mechanisms at the molecular level has been made only recently. M tuberculosis resistance to drugs always results from mutations. These mutations are either deleterious for the bacterial cell or, conversely, alter the structure of a protein targeted by a drug without compromising the protein's function for the microorganism. In M tuberculosis these mutations appear to be confined to chromosomal DNA and do not involve mobile genetic elements (such as plasmids), as mutations do in other bacteria.

  In the past few years several comprehensive reviews focused on resistance mechanisms at the molecular level.^29,30 At the same time a large array of techniques has become available to detect mutations in genes associated with resistance mechanisms in mycobacteria. Among them are, in particular, DNA sequencing (mostly 16S rDNA), but also other techniques such as gel electrophoresis (single-stranded conformation polymorphism [SSCP]-PCR, dideoxy fingerprinting) and hybridization on solid phase (line probe assay, DNA chip technology) or on liquid phase (heteroduplex analysis, mismatch cleaving assay, molecular beacon).

  For most of the front-line anti-TB drugs and some secondary antimicrobial agents, these techniques have unveiled the mutations responsible for resistance (Table 3). Resistance to rifampin, the most important component of current treatment regimens, is associated with a short core region consisting of 27 amino acids in the rpoB gene, which codes for the ß subunit of RNA polymerase.³¹ More than 97 percent of all rifampin-resistant TB isolates carry a mutation in this specific core region, where a total of 15 distinct mutations have been identified. The ethambutol resistance-determining region (ERDR) has been proposed as a mutational hot spot in the embB gene,^32,33 whereas the situation with pyrazinamide resistance is less clear.³⁴

  Table 3. Cellular targets for antimicrobial drugs and gene mutations responsible
  for resistance in M tuberculosis

  Drug	Cellular target	Gene	Gene product/functional role
  Isoniazid	Cell wall	katG inhA kasA oxyR-ahpc	Catalase-peroxidase/activation of prodrug Enol reductase/mycolic acid biosynthesis ß-ketoacyl acyl carrier protein synthase/mycolic acid biosynthesis Hydroreductase/unknown
  Ethambutol	Cell wall	embB	Arabinosyl transferase/arabinan polymerization
  Streptomycin	Protein synthesis	rpsL rrs	Ribosomal protein S12/translation 16S rRNA / translation
  Rifampin	Nucleic acids	rpoB	ß subunit of RNA polymerase/transcription
  Fluoroquinolones	Nucleic acids	gyrA	DNA gyrase subunit/DNA replication
  Pyrazinamide	Unknown	pncA	Pyrazinamidase/activation of prodrug

  Molecular research has also shown that, often, more than a single mutation is responsible for drug resistance. Resistance to isoniazid, for instance, appears to be the complex result of single or multiple mutations (deletions and missense mutations) in the katG, inhA, oxyR-ahpC, and/or kasA gene(s).^35-37 Similarly, mutations in the rpsL³⁸ and/or rrs³⁹ gene(s) correlate with resistance in approximately 80 percent of streptomycin-resistant strains.

  Among the techniques currently available for detecting resistance to anti-TB drugs, the molecular methods are by far the most rapid; results are available within one to two days. However, despite this enormous advantage, most molecular techniques are not yet ready for clinical diagnostic laboratories. Rifampin represents the only notable exception in this respect. The Inno LiPA Rif. TB Assay (Innogenetics), a reverse hybridized line probe assay, can easily and reliably detect resistance to rifampin via an oligonucleotide probe that detects M tuberculosis complex and nine probes that detect nucleotide changes in the relevant part of the rpoB gene.⁴⁰

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The limits and opportunities of susceptibility testing today

In light of the worsening global TB epidemic and the extreme vulnerability of HIV-infected individuals to TB, rapid and reliable antimicrobial susceptibility testing in the laboratory is paramount for proper management of patients, particularly those with MDR TB.⁴¹ Principally, susceptibility testing must be performed for each new patient. If resistance to isoniazid, rifampin, or ethambutol is detected, an extended spectrum of anti-TB drugs should be tested immediately. Further emergence of resistance must be carefully monitored by repeating susceptibility testing with subsequent isolates within a short time frame, for example, every two months.

Rapid, growth-based, and molecular biological methods are available for antimicrobial susceptibility testing. Liquid media (radiometric or nonradiometric) yield susceptibility results within five to 10 days. Molecular susceptibility testing, however, is in its infancy and still done mainly by research laboratories. The only exception is rifampin resistance, which can easily be detected in a clinical mycobacteriology laboratory by using a line probe assay.

Nevertheless, even the most refined laboratory methods cannot prevent M tuberculosis from becoming MDR without a concerted effort involving patient, physician, and laboratory. With cooperation from all sides, the benefits of a straightforward strategy in managing MDR TB patients are more than obvious:

"The total cost for testing all initial isolates from approximately 20,000 new [TB] patients diagnosed annually in the US . . . would have been roughly $1 million. This is equivalent to the cost of managing fewer than ten patients with MDR-TB."⁴²

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