Double Infection with a Resistant and a Multidrug-Resistant Strain of Mycobacterium tuberculosis.

An immunocompetent patient was dually infected with a resistant and a multidrug-resistant strain of Mycobacterium tuberculosis (TB). The multidrug-resistant strain, which belongs to the W-strain/Beijing family, was first isolated after 3 months of therapy. Inappropriate treatment led to further drug resistance and unsuccessful therapy. Thus, additional infections with resistant M. tuberculosis strains should be considered when tuberculosis therapy fails.

During the last decade, drug-resistant Mycobacterium tuberculosis (TB) strains have emerged, posing a major threat to global TB control efforts. The incidence of drug-resistant TB has increased in many parts of the world, not only in developing countries but also in industrialized countries, where the prevalence of drug-resistant TB had been low (1). The emergence of drug resistance during antituberculosis therapy results mainly from inadequate therapy, i.e., improper prescription of treatment regimens, addition of single drugs to failing treatment regimens, and patient noncompliance. However, inconsistent drug-susceptibility patterns or delayed responses to TB therapy may also indicate exogenous reinfection with a strain resistant to multiple drugs or mixed infection with a sensitive and a multidrug-resistant TB strain. Such infections occur in immunocompromised and immunocompetent persons (2-7) and may be more common in areas with high prevalence of resistant TB.

We report the case of an immunocompetent patient initially infected with an isoniazid- and streptomycin-resistant TB strain, who after the first 3 months of TB therapy was found to be infected with a second multidrug-resistant TB strain, resulting in treatment failure. In all, the patient's cultures were resistant to nine anti-TB drugs.

The Study

A 24-year-old man from Kazakhstan was admitted to hospital A in February 1996, 2 weeks after his arrival in Germany for further diagnosis of a cavernous lesion in the upper lobe of his left lung. The patient reported coughing, but no weight loss, night sweats, or hemoptysis. An elevated erythrocyte sedimentation rate was the only abnormal laboratory finding. He was seronegative for HIV. A tuberculin skin test was positive (12 mm), acid-fast bacilli were detected in gastric aspirates, and two sputum cultures were positive for TB. Antituberculosis therapy was started with isoniazid, rifampin, ethambutol, and pyrazinamide. The patient was then transferred to hospital B, where therapy was continued with three drugs only (ethambutol was discontinued). After 2 months of therapy, the patient showed clinical improvement, resulting in a sputum smear- and culture-negative phase of approximately 4 weeks (Table). When susceptibility testing of the first culture obtained 2 months previously revealed resistance to isoniazid and streptomycin, ethambutol was re-added to the treatment regimen. Isolates were identified as M. tuberculosis complex by using gene probes (ACCUProbe, GenProbe, San Diego, California, USA). Drug susceptibility was determined by the proportion method on Lowenstein-Jensen medium, the modified proportion method in BACTEC 460TB, or both.

Suspicion of nonadherence to therapy during the first 3 months led to transfer of the patient to hospital C and 3 weeks later to hospital D, where treatment was administered on a closed ward (Table). At that time, cultures tested positive again, and the chest X-ray showed slight deterioration. On August 1, 1996, the latest susceptibility tests showed resistance to isoniazid, rifampin, and streptomycin, and an intermediate result for rifabutin and ethambutol. Therapy was switched to ethambutol, pyrazinamide, rifabutin, and ofloxacin. Subsequently, treatment regimens were changed several times, but cultures continued to be positive for mycobacteria. In early 1997, atelectasis of the left lower lobe and thickening of the wall of the cavernous lesion in the left upper lobe became apparent. Lung resection was suggested, but thoracic surgeons declined to operate because of the extensive lung involvement. By year-end, susceptibility tests showed resistance to seven drugs: isoniazid, rifampin, ethambutol, pyrazinamide, protionamide, rifabutin, and streptomycin. A regimen with rifabutin, ciprofloxacin, amikacin, para-aminosalicylic acid, and clofazimine led to a short phase of negative sputum cultures, but a chest X-ray showed no improvement. Later, additional resistance to para-aminosalicylic acid and amikacin was documented.

Table. Treatment regimens and characteristics of Mycobacterium tuberculosis isolates(a)

Therapy               Treatment
(months)   Hospital   regimen              Culture

0          A/B        H, R, E,(c) Z        pos.
1          B          H, R, Z              pos.
2          B          H, R, E, Z           neg.
3          B          H, R, E, Z           neg./
                                            pos.
4          C          H, E, Pt             pos.
5          D          R, E, Z              pos.
6          D          E, Z, Rb, Of         pos.
7-12       D          E, Z, Rb, Pt         pos.
13         D          E, Z, Rb, Pt         pos.
14-18      D          Z, Pt, Ci            pos.
19         D          Z, Pt, Rb, Ci        pos.
20-21      D          Rb, Ci, Am, Pa       pos.
22-24      D          Rb, Ci, Am, Pa, Cl   pos.
25-28      D          Rb, Ci, Am, Pa, Cl   pos./
                                            neg.
29-32      D          Rb, Ci, Am, Pa, Cl   pos.
33         D          Rb, Ci, Cl, Ca       pos.

                      Susceptibility testing

                      Current                         Time
Therapy    Culture    resistance                      delay
(months)   obtained   pattern                         (months)(b)

0          02/28/96   H, S                            2
1          03/17/96   H, S                            2
2          --         --                              --
3          --         n.d.                            --

4          06/06/96   H, S, R, (E), (Rb)              2
5          07/12/96   H, S, R, (E), (Rb)              2
6          --         n.d.                            --
7-12       --         n.d.                            --
13         03/15/97   H, S, R, E, Rb, Pt              1
14-18      04/25/97   H, S, R, E, Rb, Pt, Z           5
19         --         n.d.                            --
20-21      --         n.d.                            --
22-24      --         n.d.                            --
25-28      02/18/98   H, S, R, E, Rb, Pt Z, Pa        4

29-32      06/23/98   H, S, R, E, Rb, Pt, Z, Pa, Am   4
33         --         n.d.                            --

Therapy
(months)   Spoligotype

0          type I
1          type I
2          --
3          n.d.

4          type II
5          type II
6          n.d.
7-12       n.d.
13         type II
14-18      type II
19         n.d.
20-21      n.d.
22-24      n.d.
25-28      type II

29-32      type II
33         n.d.

(a) Abbreviations and symbols: H, isoniazid; S, streptomycin; R, rifampin; E, ethambutol; Rb, rifabutin; Pt, protionamide; Z, pyrazinamide; Pa, p=aminosalicylic acid; Am, amikacin; Of, ofloxacin, Ci, ciprofloxacin; Cl, clofazimine; Ca, capreomycin; borderline results are shown in parentheses; pos., positive culture result; neg., negative culture result; n.d., not determined.

(b) Time between the date on which the specimen was obtained and the date on which the drug-susceptibility pattern was available for the clinician (drug-susceptibility tests were not always performed directly after the cultures were grown but were done retrospectively).

(c) E was given in the first 4 days of therapy only.

To elucidate the reasons for therapy failure, we compared the current susceptibility patterns with the treatment regimens applied earlier. We found that during several phases of treatment, this patient was treated with only one effective drug. During extended periods, he was culture positive, but no susceptibility tests were performed, even after relapse. Additionally, treatment regimens were changed, and single drugs were added several times without determination of the actual resistance pattern. Treatment regimens, drug-susceptibility patterns, and detailed information on the history of the case are summarized in the Table.

All isolates obtained were submitted to spoligotyping and IS6110 fingerprinting (5,8). Spoligotypes (Figure), as well as the IS6110 restriction fragment length polymorphism (RFLP) patterns (data not shown) of the first two isoniazid- and streptomycin-resistant cultures, were identical (type I) but differed clearly (IS6110 identity of less than 30%) from those of later multidrug-resistant isolates (second phase of sputum culture positivity, type II; see Table). These results indicate that the patient was infected with a second TB strain, which showed initial resistance to isoniazid, streptomycin, and rifampin, and borderline resistance to ethambutol and rifabutin. IS6110 RFLP patterns of multidrug-resistant isolates from the patient were compared with those from other patients who had been treated in hospitals B and C during the same period and with IS6110 RFLP patterns from resistant TB strains isolated from unrelated patients living in other areas of Germany. Gelcompar software was used for this analysis (Windows 95, version 4.0; Applied Maths, Kortrijk, Belgium) (5). No isolate showing an identical IS6110 RFLP pattern was identified (data not shown). Spoligotype and the IS6110 RFLP patterns of the patient's multidrug-resistant strain were similar to those of the W-strain or Beijing family, which have been found in New York, USA, and Beijing, China (9,10).

[Figure ILLUSTRATION OMITTED]

Conclusions

We report an immunocompetent patient with pulmonary TB who had double infection with a resistant and a multidrug-resistant TB strain, leading to therapy failure. After 2 years of treatment, resistance to eight antituberculosis drugs--including the most potent first- and second-line treatments--occurred, despite clinically supervised hospital therapy. Four months later, resistance to a ninth drug occurred. Progressive disease caused by a second multidrug-resistant TB strain, as demonstrated by molecular strain typing methods, was the initial cause for this occurrence. A possible variation of the initial strain has been excluded since the spoligotype patterns of the multidrug-resistant isolates completely differed from the two isolates of the first TB period (spoligotype patterns have been shown to be highly stable among serial patient isolates [11]).

In this case, TB therapy was often based on drug-resistance data not representing the current drug-resistance pattern, resulting in improper treatment and many periods in which the patient received only one effective drug; the second TB strain could have acquired further resistance during these many periods of monotherapy. Earlier identification of the second infection might have led to treatment with a more appropriate drug regimen, resulting in a more successful outcome. The second multidrug-resistant TB strain could have been acquired by mixed-strain infection or exogenous reinfection (2-7). However, our investigation did not identify a possible index patient. Moreover, the fact that the patient was an immigrant from Kazakhstan, a country with high rates of resistant TB (1), suggests that he may have been infected with the second multidrug-resistant strain in his homeland.

This patient was seronegative for HIV and had no clinical measurements indicative of immunosuppression, suggesting that additional infection with multidrug-resistant TB during treatment can be largely independent of a host's immune status. These mixed-strain infections with at least one resistant strain may lead to unsuccessful TB therapy. Although few have been reported (6, 7), such cases may become more frequent in areas with high rates of drug-resistant TB. Standard TB treatment apparently is not sufficient to protect patients from infection with a second multidrug-resistant TB strain.

Clinicians should consider the possibility of additional infection with multidrug-resistant TB in cases when TB therapy fails. In such cases, inappropriate treatment regimens and delayed follow-up of susceptibility tests can permit additional resistance to develop, which can dramatically complicate TB therapy. However, regardless of the cause, when a clinical course is abnormal, adding single drugs to failing treatment regimens should be avoided, and retreatment programs should not be initiated before culture sensitivity results are available.

Acknowledgments

We thank I. Radzio, B. Schluter, and A. Zyzik for excellent technical assistance, and S. Schwander and D. van Soolingen for critical reading of the manuscript.

Parts of this work were supported by the Robert Koch Institut, Berlin, Germany.

References

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(2.) Horn DL, Hewlett D, Haas WH, Butler WR, Alfalla C, Tan E, et al. Superinfection with rifampin-isoniazid-streptomycin-ethambutol (RISE)-resistant tuberculosis in three patients with AIDS: confirmation by polymerase chain reaction fingerprinting. Ann Intern Med 1994;121:115-6.

(3.) Small PM, Shafer RW, Hopewell PC. Exogenous reinfection with multidrug-resistant M. tuberculosis in patients with advanced HIV infection. N Engl J Med 1993;328:1137-44.

(4.) Shafer RW, Singh SP, Larkin C, Small PM. Exogenous reinfection with multidrug-resistant Mycobacterium tuberculosis in an immunocompetent patient. Tuber Lung Dis 1995;76:575-7.

(5.) Niemann S, Rusch-Gerdes S. Richter E. IS6110 fingerprinting of drug-resistant Mycobacterium tuberculosis strains isolated in Germany during 1995. J Clin Microbiol 1997;35:3015-20.

(6.) Glynn JR, Jenkins PA, Fine PE, Ponnighaus JM, Sterne JA, Mkandwire PK, et al. Patterns of initial and acquired antituberculosis drug resistance in Kaonga District, Malawi. Lancet 1995;345:907-10.

(7.) Theisen A, Reichel C, Rusch-Gerdes S, Haas WH, Rockstroh JK, Spengler U, et al. Mixed-strain infection with drug-sensitive and multidrug-resistant strain of Mycobacterium tuberculosis. Lancet 1995;345:1512.

(8.) Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 1997;35:907-14.

(9.) Van Soolingen D, Qian L, de Haas PE, Douglas JT, Traore H, Portaels F, et al. Predominance of a single genotype of Mycobacterium tuberculosis in countries of east Asia. J Clin Microbiol 1995;33:3234-8.

(10.) Moss AR, Alland D, Telzak E, Hewlett D Jr, Sharp V, Chiliade P, et al. A city-wide outbreak of a multipledrug-resistant strain of Mycobacterium tuberculosis in New York. Int J Tuberc Lung Dis 1998;1:115-21.

(11.) Niemann S, Richter E, Rusch-Gerdes S. Stability of Mycobacterium tuberculosis IS6110 RFLP patterns and spoligotypes determined by analyzing serial isolates of patients with drug-resistant tuberculosis. J Clin Microbiol 1999;37:409-12

Address for correspondence: S. Rusch-Gerdes, Forschungszentrum Borstel, National Reference Center for Mycobacteria, Parkallee 18, D-23845 Borstel, Germany; fax: [phone]; e-mail: [e-mail].

Dr. Niemann is working in a postdoctoral position at the German National Reference Center for Mycobacteria, Research Center Borstel, Borstel, Germany. He is responsible for molecular characterization and typing of mycobacteria. Research interests include the characterization of the MTB complex by molecular techniques and the epidemiology of TB by DNA fingerprinting.