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Co-infection with drug-susceptible and reactivated latent multidrug-resistant Mycobacterium tuberculosis.

To the Editor: Genotyping of Mycobacterium tuberculosis (MTB) has identified mixed infections involving >1 MTB strain (1-4), which are clinically relevant when different susceptibility patterns are involved (4-7). We describe a tuberculosis (TB) case-patient with mixed infection in an area of moderate incidence. In a low-resistance setting (monoresistance 4.6%; multiresistance 1.7%), 1 of the strains was drug susceptible and the other was multidrug-resistant (MDR). Molecular fingerprinting and epidemiologic research revealed that the infection corresponded to a recent infection by a susceptible strain and reactivation of an MDR TB strain. The patient was an HIV-negative woman, 47 years of age, who had immigrated to Spain from Romania and had been living in Almeria for >3 years. TB was diagnosed in May 2014; she had experienced symptoms for 2 months. Her diagnosis was confirmed 3 years after being studied in Almeria as a close contact of her husband, also from Romania, who had tested positive for MDR TB (resistant to rifampin and isoniazid). When she was observed in the contact trace, she tested positive for purified protein derivative, had been vaccinated against the M. bovis bacillus Calmette-Guerin strain, and had no radiologic findings or clinical symptoms. Based on the susceptibility profile of her husband, prophylaxis was not prescribed. Her husband adhered to anti-TB treatment for 20 months; all microbiological control test results had been negative since 2 months after starting therapy.

Because her infection was thought to originate from previous contact with an MDR TB case-patient, we assessed her sputum samples for resistance using GenoType-MTBDR-plus (Hain-Lifescience, Nehren, Rhineland-Paltinate, Germany). The test showed hybridization with the same mutant probes (r/>oB-MUT3; katG-MUT1) as those of her husband, the assumed index MDR TB case-patient (Figure, panel A). The pattern was indeterminate because the hybridization for the mutant probes was faint, and intense hybridization was observed for all the wt probes (Figure, panel A). Results suggested the simultaneous presence of an MDR strain and a susceptible strain in a respiratory specimen. The presence of the MDR TB strain was confirmed by the phenotypic antibiogram in a BBL MGIT mycobacteria growth indicator tube (Becton Dickinson, Franklin Lakes, NJ, USA) after the isolate had been cultured. GeneXpert (Cepheid, Sunnyvale, CA, USA) was used to analyze 2 respiratory specimens. Results indicated susceptibility to rifampin, revealing the limitations of this test: the use of probes targeting the wt sequences failed to detect resistant strains that coexist with a susceptible strain (7).

To ascertain the likelihood of 2 co-infecting strains, we analyzed the specimen and the cultured isolate by mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR), which is highly sensitive for detecting complex infections (8,9). Double alleles were found at 12 loci (Figure, panel B), confirming co-infection.

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Peaks in the electropherograms suggested that 1 of the 2 strains was under-represented and its proportion was lower in the cultured isolates (Figure, panel C), indicating that culturing diminished its representation of the minority strain. This finding was consistent with the inability of the GenoType test to detect the MDR strain when applied to the cultured isolate (Figure, panel A). We also detected lower fitness for the MDR strain compared to the susceptible strain (p<0.01) (online Technical Appendix Table, http://wwwnc.cdc.gov/EID/article/21/11/15-0683 Techapp1.pdf).

Proportions of the resistant and susceptible strains were determined by plating on Middlebrook 7H11 +/-isoniazid (0.4 [micro]L/mL) and counting single colonies. Because 2% of the colonies were of the resistant strain, we separated the strains and concluded: 1) the strain cultured in the presence of an antimicrobial drug coincided in the population exclusively with the MDR strain from the husband (as shown by MIRU-VNTR); and 2) the co-infecting MDR strain was a minority strain that was under-represented in the cultured isolate.

To analyze the origin of the susceptible strain, we investigated its MIRU-VNTR type in the population-based molecular epidemiology survey (10) and found another 4 cases (from 2008, 2011 [2 cases], and 2014). Three case-patients had emigrated from Romania, and all 5 case-patients lived in the same area of Almeria. These data indicated that the susceptible strain was circulating in the geographic/epidemiological context of the current case-patient before and when she tested positive for that strain; therefore, she likely acquired the susceptible strain through recent transmission.

The presence of susceptible and resistant strains in a patient should be considered even in moderate incidence settings and where resistance rates are not high. Underdetection of these cases could lead to misinterpretation when MDR became apparent after treatment of susceptible strains. Diagnostic laboratories could easily screen for mixed infections by applying MIRU-VNTR. However, only by integrating clonal analysis, refined molecular typing, and epidemiologic data from universal genotyping programs can we clarify the reasons underlying complex MTB infections. For this case-patient, a recent infection with a susceptible strain coincided with or could have triggered reactivation of a latent infection involving an MDR strain acquired through close contact years previously. We emphasize the alteration of the true clonal complexity of an infection induced by culturing specimens and that some commercial tests do not identify complex MTB infections. These findings are particularly relevant when the infection involves resistant strains such as those found in this case-patient.

Laura Perez-Lago, Miguel Martinez Lirola, Yurena Navarro, Marta Herranz, Maria Jesus Ruiz-Serrano, Emilio Bouza, Dario Garcia-de-Viedma

Author affiliations: Gregorio Maranon University General Hospital, Madrid, Spain (L. Perez-Lago, Y. Navarro, M. Herranz, M.J. Ruiz-Serrano, E. Bouza, D. Garcia-de-Viedma); Gregorio Maranon Health Research Institute, Madrid (L. Perez-Lago, Y. Navarro, M. Herranz, M.J. Ruiz-Serrano, E. Bouza, D. Garcia-de-Viedma); Centro de Investigacion Biomedica en Red en Enfermedades Respiratorias, Madrid (L. Perez-Lago, Y. Navarro, M. Herranz, M.J. Ruiz-Serrano, E. Bouza, D. Garcia-de-Viedma); Torrecardenas Hospital, Almeria, Spain (M. Martinez Lirola); Complutense University of Madrid, Spain (E. Bouza); Moncloa International Campus of Excellence, Complutense University-Politecnica University, Madrid (Y. Navarro, D. Garcia-de-Viedma)

DOI: http://dx.doi.org/10.3201/eid2111.150683

Acknowledgment

We thank Thomas O'Boyle for proofreading the manuscript.

This study was supported by Plan Estatal de I? 2013-2016, ISCIII (PI 12/02080; 13/01207) and Fondo Europeo de Desarrollo Regional FEDER "Una manera de hacer Europa."

YN. was supported by a PICATA predoctoral fellowship (BE55/11) from the Moncloa Campus of International Excellence (UCM-UPM, Instituto de Investigation Sanitaria Gregorio Maranon).

References

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(2.) Shin SS, Modongo C, Ncube R, Sepako E, Klausner JD, Zetola NM. Advanced immune suppression is associated with increased prevalence of mixed-strain Mycobacterium tuberculosis infections among persons at high risk for drug-resistant tuberculosis in Botswana. J Infect Dis. 2015;211:347-51. http://dx.doi.org/10.1093/infdis/jiu421

(3.) Dickman KR, Nabyonga L, Kateete DP, Katabazi FA, Asiimwe BB, Mayanja HK, et al. Detection of multiple strains of Mycobacterium tuberculosis using MIRU-VNTR in patients with pulmonary tuberculosis in Kampala, Uganda. BMC Infect Dis. 2010;10:349. http://dx.doi.org/10.1186/1471-2334-10-349

(4.) Niemann S, Richter E, Rusch-Gerdes S, Schlaak M, Greinert U. Double infection with a resistant and a multidrug-resistant strain of Mycobacterium tuberculosis. Emerg Infect Dis. 2000;6:548-51. http://dx.doi.org/10.3201/eid0605.000518

(5.) Hingley-Wilson SM, Casey R, Connell D, Bremang S, Evans JT, Hawkey PM, et al. Undetected multidrug-resistant tuberculosis amplified by first-line therapy in mixed infection. Emerg Infect Dis. 2013;19:1138-41. http://dx.doi.org/10.3201/eid1907.130313

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(7.) Zetola NM, Shin SS, Tumedi KA, Moeti K, Ncube R, Nicol M, et al. Mixed Mycobacterium tuberculosis complex infections and false-negative results for rifampin resistance by GeneXpert MTB/RIF are associated with poor clinical outcomes. J Clin Microbiol. 2014;52:2422-9. http://dx.doi.org/10.1128/JCM.02489-13

(8.) Garcia de Viedma D, Alonso Rodriguez N, Andres S, Ruiz Serrano MJ, Bouza E. Characterization of clonal complexity in tuberculosis by mycobacterial interspersed repetitive unit-variable-number tandem repeat typing. J Clin Microbiol. 2005;43:5660-4. http://dx.doi.org/10.1128/JCM.43.11.5660-5664.2005

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(10.) Alonso-Rodriguez N, Martinez-Lirola M, Sanchez ML, Herranz M, Penafiel T, Bonillo Mdel C, et al. Prospective universal application of mycobacterial interspersed repetitive-unit-variable-number tandem-repeat genotyping to characterize Mycobacterium tuberculosis isolates for fast identification of clustered and orphan cases. J Clin Microbiol. 2009;47:2026-32. http://dx.doi.org/10.1128/JCM.02308-08

Address for correspondence: Dario Garcia de Viedma, Servicio de Microbiologia y Enfermedades Infecciosas, Hospital General Universitario Gregorio Maranon, Instituto de Investigation Sanitaria Gregorio Maranon, C/Dr Esquerdo, 46, 28007 Madrid, Spain; email:dgviedma2@gmail.com
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Title Annotation:LETTERS
Author:Perez-Lago, Laura; Lirola, Miguel Martinez; Navarro, Yurena; Herranz, Marta; Ruiz-Serrano, Maria Jes
Publication:Emerging Infectious Diseases
Article Type:Letter to the editor
Date:Nov 1, 2015
Words:1530
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