Emergence of New Forms of Totally Drug-Resistant Tuberculosis Bacilli: Super Extensively Drug-Resistant Tuberculosis or Totally Drug-Resistant Strains in Iran FREE TO VIEW

According to a nationwide survey¹ conducted in Iran (1999), among all Mycobacterium tuberculosis isolates tested for drug susceptibility, 10.9% were resistant to one or more antituberculosis (anti-TB) drugs and 6.7% were resistant to both isoniazid (INH) and rifampin (RF) [ie, they were multidrug-resistant (MDR) strains of M tuberculosis]. In further studies,² we documented the existence and transmission of extensively drug-resistant (XDR) tuberculosis (TB) among patients with MDR-TB. These strains were identified as belonging to the Beijing and Haarlem I superfamilies of M tuberculosis.³ By definition the XDR-TB bacilli are resistant to fluoroquinolone and to any of three injectable drugs (capreomycin [CAP], kanamycin [KAN], and amikacin [AMK]) in addition to INH and RF.^4,5 Recent investigation^6,7 on six different continents showed that 10% of MDR-TB cases became XDR-TB strains. Although the problem of XDR-TB cases remains unresolved in much of the world, here we report on more dangerous forms of the disease, which we call totally drug-resistant strains (TDR) or super XDR-TB isolates. We define TDR as MDR strains that are resistant to all second-line drug classes that our laboratory tested (ie, aminoglycosides, cyclic polypeptides, fluoroquinolones, thioamides, serine analogues, and salicylic acid derivatives). Furthermore, to gain a better appreciation of the epidemiology of these strains in Iran, classical and molecular epidemiologic techniques were utilized. To our knowledge, this is the first report that describes the prevalence of TDR among patients with MDR-TB.

The National Research Institute of Tuberculosis and Lung Diseases (NRITLD) (Tehran, Iran) acts as the sole national referral center for TB in Iran. Located inside the NRILTD is a National Reference Tuberculosis Laboratory, which is under the technical supervision of the Supranational Reference Laboratories of the Swedish Institute for Infectious Diseases Control (Solna, Sweden). Under the National Tuberculosis Control program, all the MDR and relapse cases of TB are referred to the NRITLD for evaluation and treatment. The institutional review board at the NRITLD approved the study.

According to the Iranian National Tuberculosis Control treatment protocol, all new TB patients receive the World Health Organization category I regimen or, in case of relapse or failure, the category II regimen. If category II treatment fails, patients are referred to the NRITLD. In addition to these cases, some patients were directly referred to us. These patients (12 MDR cases and 1 XDR-TB case) had no previous history of TB.

Inclusion criteria for patients with MDR-TB were a history of at least one previous period of TB treatment, two positive sputum smear test results, and a positive sputum culture result.^8–10 Patients were also required to have drug susceptibility test (DST) results that showed resistance to INH and RF, as well as chest radiograph findings and clinical symptoms that were compatible with pulmonary TB.

Because no patients with TB in Iran receive second-line drugs, MDR-TB patients are assumed to be susceptible to second-line agents, which are considered active. All the patients received at least 6 months of an injectable aminoglycoside (eg, AMK at a dose of 15 mg/kg), which was continued for at least 4 months after the culture turned negative or adverse effects to the drug developed. Patients first underwent an intermediate regimen comprising four second-line drugs: ofloxacin (OFX) [400 to 800 mg/d], cycloserine (CYC) [750 to 1,000 mg/d], prothionamide (PTH) [750 to 1,000 mg/d], and AMK (15 mg/kg/d, 5 d/wk; maximum, 1 g/d) until the results of a DST were available. The regimen was then modified on the basis of DST so that all first-line drugs to which they were susceptible were included in combination with at least four active second-line drugs. If major adverse effects occurred, the suspected agent was replaced by other drugs, such as clarithromycin or amoxicillin-clavulanate, which may be of unproven efficacy but are recommended as the fifth group of anti-TB agents by the World Health Organization for the management of MDR-TB when other agents are not available or cannot be used. After discharge from the hospital, follow-up evaluation included a sputum smear and culture every month and a chest radiograph every 3 months. For TDR cases (clinically and laboratory approved), either co-amoxiclav (625 mg/8 h) or clarithromycin (1,000 mg/d), along with a high dose of INH (15 mg/kg), were prescribed without any improvement.

Primary isolation and culturing of Mycobacterium isolates from sputum specimens were followed in accordance with the procedures manual.¹¹ All isolates were identified as M tuberculosis by using biochemical tests, including production of niacin, catalase activity, nitrate reduction, pigment production, and growth rate. DST against INH, RF, streptomycin (SM), and ethambutol (ETB) were performed by the proportional method on Löwenstein- Jensen media at a concentration of 0.2, 40, 4.0, and 2.0 μg/mL, respectively.¹¹ Susceptibility to pyrazinamide (PZA) [900 and 1,200 μg/mL] was tested using a two-phase medium where the strain was reported to be resistant to PZA if, on day 21, the proportion of drug-resistant colonies was higher than the defined critical proportion. DST against second-line drugs (CAP, 10 μg/mL; KAN, 20 μg/mL; ethionamide [ETH], 20 μg/mL; OFX, 2 μg/mL; ciprofloxacin [Cip], 2 μg/mL; CYC, 30 μg/mL; AMK, 4 μg/mL; and para-aminosalicylic acid [PAS], 5 μg/mL) was performed on all MDR strains using two critical proportions of 1% and 10%.¹² TDR strains were resistant to all first- and second-line drugs tested. Overall, the DST against first- and second-line drugs was performed on the first culture-positive specimen that was collected from MDR-TB patients prior to starting treatment at the NRITLD. In the case of XDR or TDR reports, the DST was repeated 6 to 8 months after starting the treatment with second-line drugs. (The XDR or TDR strains discussed here had a similar pattern of resistance in the first and second DST).

The extraction of bacterial DNA was performed with standard protocols.¹³ For spoligotyping, the direct repeat region was amplified by polymerase chain reaction (PCR) using primers derived from the direct repeat sequence (Isogen Bioscience; Maarssen, the Netherlands). The amplified DNA was hybridized to a set of 43 immobilized oligonucleotides derived from the spacer sequences of M tuberculosis H37RV and Myobacterium bovis BCG P3 by reverse line blotting.

Variable numbers of tandem repeats (VNTR)-PCR primers were designed as described by Frothingham and Meeker-O'Connell.¹⁴ PCRs were run in DNA thermal cyclers (model 480; Perkin Elmer; Golden Valley, MN) under the following conditions: 95°C for 12 min, 40 cycles of 94°C for 30 s, 60°C for 1 min, and 72°C for 2 min, followed by final extension at 72°C for 7 min. PCR products were analyzed by agarose gel electrophoresis. The number and size of repeat units in H₃₇RV(bp) for MPTR-A, ETR-A, ETR-B, ETR-C, ETR-D, ETR-E, and ETR-F were as follows: 16 × 15, (3 × 75) + 23, (3 × 57) + 8, (4 × 58)−21, (3 × 77) + 7, (3 × 53) −1, (3 × 79) −13, respectively.¹⁴

The autoradiograph of spoligotyping was scanned with the Snap Scan 1236 Scanner (AGFA; Sint-Martens-Latem, Belgium). Bionumerics Software, version 2.5 (Applied Maths; Kortrijk, Belgium) was used to analyze the molecular patterns generated by spoligotyping. Strains were classified as a cluster if they shared similar spoligopatterns and VNTR profiles.

The continuous variables were expressed as group means (± SD). The variables included sex, age, the pattern of drug resistance, the result of a purified protein derivative, and HIV between XDR- and TDR-TB patients.

From October 2006 to October 2008, 166 MDR-TB patients were referred to the NRITLD for treatment and diagnosis. Of these patients, 20 patients (12%) had infection with mycobacterium other than tuberculosis. The results of susceptibility against first-line drugs were improved resistance to INH and RF in the remaining MTB isolates (n = 146). The results of susceptibility against second-line drugs were as follows: 65 strains (44.5%) were susceptible to all drugs tested, 8 strains were XDR (5.4%), and 15 strains were TDR isolates (10.2%). The remaining strains (n = 58; 36.7%) had other resistance. Table 1 shows the demographic characteristics of XDR and TDR TB. The majority of them (95.6%) had a previous history of TB, and all of them had negative HIV test results (100%). The male-to-female ratio was more than threefold in TDR cases (p < 0.05). The median ages in MDR, XDR, and TDR patients were 49.12 ± 27.9, 56.7 ± 24.3, and 50.2 ± 29 years, respectively. Although the immigrants with XDR or TDR-TB isolates were much younger than Iranian patients (average age, 34.5 vs 61.2 years, respectively), the differences were statistically significant (p < 0.05). The XDR and TDR Afghan immigrants were living in Iran and had been frequently traveling in and out of the country. The other TDR immigrants (two Azerbaijani and one Iraqi) had been referred to the NRITLD for diagnosis and treatment.

Overall, the medians of treatments and drugs previously received by the MDR-TB patients was two courses of therapy in 15 months with five drugs (INH, RF, SM, ETB, and PZA), respectively. The medians of in vitro drug resistance for all studied cases were INH and RF. In the TDR cases, the smear and cultures remained positive after 18 months of treatment with second-line drugs. Changing the treatment to co-amoxiclav (625 mg/8 h) or clarithromycin (1,000 mg/d), along with high dose of INH (15 mg/kg), made no improvement in them.

When the spoligotypes from the isolated XDR and TDR M tuberculosis strains were compared with earlier published spoligotypes, our isolates could be identified as members of the superfamilies Haarlem I (n = 9; 39.1%), Beijing (n = 5; 21.7%), EAI (n = 5; 21.7%), and CAS (n = 4; 17.5%) [Table 2]. The VNTR profiles of spoligotypes strains were different in each superfamily (Table 2). The five isolated Beijing families had 6424353, 6425353, 6324321, 5214431, and 6322322 profiles. Similarly, the Haarlem I family had different profiles (6314332, 6435322, 5324332, 6435322, 6425333, 6424353, 5114332, 5314332, and 5314331). The different VNTR profiles patterns dismissed the possibility of recent transmission among XDR- or TDR-TB cases.

This study represents the first report on the existence and prevalence of TDR strains in Iran. TDR strains not only constitute a deadly threat to the affected patients with TB but also hamper the TB-control program. Previously, we identified two clusters of XDR-TB isolates in both family and community outbreaks.² These strains were fully capable of being transmitted and causing active diseases in individuals with secondary cases. In the present report, 95% of XDR and TDR strains were isolated from patients with a previous history of TB.

In this study, XDR-TB was defined as MDR isolates with a further resistance to fluoroquinolone and to at least one of the three injectable drugs used in anti-TB treatment. TDR was defined as M tuberculosis isolates that were resistant to all first-line (INH, RF, SM, ETB, and PZA) and second-line drugs tested (OFX, CYC, PTH, AMK, KAN, ETH, PAS, and CAP). Generally, the spectrum of resistance reflects the drugs that the patients have used and the way in which therapy was controlled.^9,15 The Iranian TDR-TB patients had not received second-line drugs before being admitted to the NRITLD. But to our surprise, 10.2% of these strains showed resistance to all the second-line drugs tested. In Iran, some of the second-line drugs (ie, aminoglycosides and fluoroquinolones) are routinely used for the treatment of respiratory diseases other than TB. It is most likely that these patients had been treated previously with aminoglycosides and fluoroquinolones in a poorly controlled manner. In contrast, the other drugs (ie, CAP and PAS) were introduced to Iran beginning in 2002, and none of the Iranian TDR patients had a previous history of receiving such drugs. At present, we do not know why some of the MDR-TB strains showed resistance to second-line drugs, but the possibility of sequential mutations is highlighted and is under investigation in our laboratory.

In this study, 43% of XDR and TDR strains belonged to immigrants living in Iran or who had visited the country. These patients did not have a proper clinical history, and the possibility they had received second-line drugs could not be ignored. We have already shown that 32% of the initial TB patients referred to our unit were Afghan-born immigrants.¹⁶ The majority of them (58%) had either resistance to any drug or to a drug combination including MDR-TB.¹⁶ The incidence of intracommunity transmission between Iranian and Afghan patients rose significantly, from 13 to 41%, from 2005 to 2007.¹⁷ These findings highlight the need to adopt new strategies with regard to screening immigrants from neighboring countries, which is absent in the current system.

The isolated strains belonged to different superfamilies of M tuberculosis, that is, Haarlem (39.1%), Beijing (21.7%), EAI (21.7%), and CAS (17.3%). The Haarlem I and Beijing were the most frequent superfamilies among MDR-TB patients.^16,17 The Haarlem I and Beijing strains have been reported in different geographic regions of the world, and they are thought to possess selective advantages in comparison with other M tuberculosis.^17,18 In the present study, 60% of patients were infected with the Haarlem I or Beijing superfamily. Therefore, it is clear that both these superfamilies can cause an epidemic, and from an epidemiologic point of view, it is necessary to conduct more extensive surveillance of MDR-TB strains because they might cause serious outbreaks.² Further epidemiologic studies¹⁷ using VNTR techniques showed different VNTR profiles among identified superfamilies. This means the studied strains probably were not transmitted via recent transmission but rather seemed to have developed due to the failure of the current recommended policies and protocols initially to diagnose, treat, and cure such cases adequately.

This assertion is supported by the finding of only a single patient with new XDR-TB (not receiving previous treatment) among the studied cases (Table 2). The isolation of TDR strains from MDR-TB patients who belonged to different regional countries is alarming, and it underlines the possible dissemination of such strains in Asian countries. A 2004 population-based study⁴ of drug susceptibility among isolates from patients with TB showed that 4%, 19%, and 15% of MDR-TB cases in the United States (1993 to 2004), Latvia (2000 to 2002), and South Korea (2004), respectively, were XDR TB. However, what percentages of MDRTB are TDR is not known. Today, the most important question is how to control and prevent the transmission of such deadly bacilli regionally and globally.¹⁹ And what combination of drugs has to be used for these patients? Based on standard protocols, if a patient has an isolate that is resistant to all but two or three relatively weak drugs, the patient should undergo surgery. However, in our study, surgery was not applicable (because of extensive and diffused lung damage in such cases), and the patients were receiving either co-amoxiclav (625 mg/8 h) or clarithromycin (1,000 mg/d), along with a high dose of INH (15 mg/kg) without any improvement. This problematic situation illustrates an urgent need to find an effective medicine for treating such complicated cases.

Our study was limited to patients who were referred to the NRITLD, and it cannot represent the overall situation in the country. Possibly the condition is worse in provinces near the borders (until now we have found no recent transmission of TDR-TB cases within the country). Therefore, further research is required to determine the prevalence of such type of TB bacilli not only in Iran but also in nearby countries (ie, Afghanistan, Pakistan, Iraq, and the former Soviet Union). Indeed, we have to achieve a clear picture about the spread and transmission of TDR bacilli within these countries. In conclusion, the emergence of TDR bacilli is a worrisome development, and it clearly underlines the urgent need to reinforce the Iranian TB-control policy, with special attention to the prompt and reliable laboratory detection of drug-resistant TB, as well as the need for efficient infection-control measures to stop or strongly limit the spread of TDR M tuberculosis.

Drs. Velayati and Masjedi participated in the study conception and manuscript review. Dr. Farnia designed and implemented the study, and wrote the manuscript. Drs. Tabarsi, Ghanavi, ZiaZarifi, and Hoffner reviewed the data and manuscript.

The authors have reported to the ACCP that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

We thank the TB patients and their families who helped us complete the required information.