Microevolution of Mycobacterium tuberculosis in a

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Microevolution of Mycobacterium tuberculosis in a

     JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 38133816 Vol. 48, No. 10 0095-1137/10/$12.00 doi:10.1128/JCM.00556-10 Copyright ? 2010, American Society for Microbiology. All Rights Reserved.

    Microevolution of Mycobacterium tuberculosis in a

    Tuberculosis Patient

    12345Sahal A. M. Al-Hajoj,* Onno Akkerman,Ida Parwati,Saad al-Gamdi,Zeaur Rahim, 667,89Dick van Soolingen,Jakko van Ingen,Philip Supply,and Adri G. M. van der Zanden

    1Department of Comparative Medicine, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia; 2Department of Pulmonology, Medical Spectrum Twente Hospital, Enschede, Netherlands; Department of Clinical Pathology, 3Dr. Hasan Sadikin Hospital, Padjadjaran University, Bandung, Indonesia; Molecular Diagnostic Unit, 4Hera General Hospital, Makkah, Saudi Arabia; International Centre for Diarrhoeal Disease Research, 5Bangladesh, 68 Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh; National Tuberculosis Reference Laboratory, National Institute for Public Health and 6the Environment, Bilthoven, Netherlands; Center for Infection and Immunity of

    Lille INSERM U 1019-CNRS UMR 8204, University Lille Nord de 78France-Institut Pasteur de Lille,and Genoscreen,Lille,

    France; and Laboratory for Medical Microbiology and 9Public Health, Enschede, Netherlands

    Received 15 March 2010/Returned for modication 12 July 2010/Accepted 28 July 2010

    Five Mycobacterium tuberculosis isolates were obtained from three body sites from a Dutch patient. The isolates displayed a single genotype by 24-locus MIRU-VNTR typing (except for a single locus not amplied from one isolate) but were differentiated by small variations in IS6110 ngerprints, spoligotypes, 6 hypervariable MIRU-VNTR loci, and/or DiversiLab proles, revealing patterns of microevolution in a clonal infection.

     namide was not available. Treatment was ended successfully CASE REPORT after 12 months. A 56-year-old woman born in the Netherlands, a low-tuber- For IS6110 restriction fragment length polymorphism (RFLP) culosis (TB)-incidence country, presented with diffuse nodular and DiversiLab analysis, DNA was extracted and puried, abnormalities on her chest X-ray. She had no history of TB. respectively, from M. tuberculosis cultures on Middlebrook The tuberculin skin test result was 0 mm. Bronchoscopy was 7H10 slants, using proteinase K and NaCl-cetyltrimethylam- performed, and sarcoidosis was diagnosed. Treatment of sar- monium bromide (24). IS6110-RFLP was performed according coidosis was started and was given for a year and a half. Five to the standardized protocol recommended by van Embden et years later, she returned with an inammatory nger. Under al. (24). DiversiLab analysis, a ngerprinting method based on suspicion of relapse of her sarcoidosis, treatment with pred- repetitive sequence-based PCR (rep-PCR), was used as de- nisone was started. At the start of treatment with prednisone, scribed by Cangelosis et al. (4) and following the manufactur- a urine sample (23 May 1986) was collected. Due to deterio- ers instructions. Analysis of the isolate patterns was performed ration of the patients condition, 2 months later, 2 more urine with the Web-based DiversiLab system software (version samples were collected on the same day (30 July 1986). The 2.1.66), using a virtual gel image and corresponding dendro- following day, samples from sputum (31 July 1986) and pus gram. from her nger (31 July 1986) were also collected. The isolates For spoligotyping and mycobacterial interspersed repetitive were cultured on Lowenstein-Jensen medium after routine unit-variable number of tandem repeats (MIRU-VNTR) typing, decontamination with 4% Na-OH. Cultures of the sputum, DNA was extracted by suspending approximately 10 mg of wet urine, and pus were all positive for Mycobacterium tuberculosis. bacterial cells in 100 l of sterile water and subsequently heating At that time, antituberculosis treatment with isoniazide (INH), at 100?C for 30 min to inactivate and lyse the cells. PCR-based rifampin, and pyrazinamide was started, after a 4-month pe- spoligotyping targets polymorphism of various spacer sequences riod of prednisone treatment. All ve isolates were sensitive to present in the direct repeat (DR) region by a reverse dot blot INH (MIC, 0.2 g/ml), ethambutol (MIC, 4 g/ml), and ri- fampin (MIC, 0.5 g/ml); the result for susceptibility to pyrazi- procedure. It was performed as described by van der Zanden et al. (23). MIRU-VNTR typing relies on PCR amplication of multiple loci containing tandemly repeated sequences using primers specic for the anking regions (6, 9, 17, 20, 21). * Corresponding author. Mailing address: Department of Com- Standard 24-locus-based MIRU-VNTR typing and 6-hyper- parative Medicine, King Faisal Specialist Hospital and Research variable-locus-based typing were performed as described pre- Centre, Riyadh 11211, Saudi Arabia. Phone: 966 1 4424992. Fax: viously (17, 19) using a quality-controlled commercial service 966 1 4427872. E-mail: (Genoscreen, Lille, France). Published ahead of print on 4 August 2010.


     3814 CASE REPORTS J. CLIN. MICROBIOL. a TABLE 1. MIRU typing for ve isolates using 24 loci MIRU-VNTR allele in locus: Isolate 154 424 577 580 802 960 1644 1955 2059 2163b 2165 2347 2401 2461 2531 2687 2996 3007 3171 3192 3690 4052 4156 4348 1 NA 2 3 2 2 5 2 3 2 5 3 4 4 2 5 1 5 3 2 3 3 5 3 2 2 2 2 3 2 2 5 2 3 2 5 3 4 4 2 5 1 5 3 2 3 3 5 3 2 3 2 2 3 2 2 5 2 3 2 5 3 4 4 2 5 1 5 3 2 3 3 5 3 2 4 2 2 3 2 2 5 2 3 2 5 3 4 4 2 5 1 5 3 2 3 3 5 3 2 5 2 2 3 2 2 5 2 3 2 5 3 4 4 2 5 1 5 3 2 3 3 5 3 2

    a Correspondences with body sites and dates of isolation are given in Fig. 1. MIRU-VNTR markers are named according to a standardized designation (i.e., position on H37Rv chromosome). NA, nonamplied marker after one multiplex and two simplex PCR rounds.

     All ve isolates showed identical patterns by standard MIRU- urine isolates. Two spoligotypes were observed for the ve

    VNTR typing, parsimoniously accounting the missing data in isolates, differing in the presence of spacer 13 and distinguish- locus 154 (MIRU 02) of isolate 1 as noninformative (Table 1). ing sputum (isolate 2), pus (isolate 4), and the rst urine isolate

    Submission of this 24-locus-based genotype to the MIRU- (isolate 3) from urine isolates 1 and 5. Finally, three different VNTRPlus database according to the strategy described by patterns sharing more than 93% similarity were obtained by Allix-Beguec et al. (1) predicted that the corresponding geno- using the DiversiLab typing system (Fig. 2). Isolate 1 (urine) type belongs to the Haarlem family, a member of the Euro- and isolate 2 (sputum) were indistinguishable, since they were American superlineage. This was fully supported by the typical above the 99% similarity threshold. Likewise, isolate 4 (pus) signatures of the two variant spoligotypes detected among the and isolate 5 (urine) displayed other patterns that were con- ve isolates (3) (Fig. 1). sidered the same, since they were above 98% similarity. Isolate In contrast, small variations were detected among iso- 3 (urine) displayed a unique pattern, albeit closely related to lates with each of the four other genotyping methods. Three those of the two others.

    different patterns were obtained by using 6 hypervariable Despite these variations, both the above genotyping results MIRU-VNTR loci, mutually differing by single-locus varia- and the TB patients history strongly indicate that the ve

    tions (SLVs) (Table 2). Urine isolates 1 and 3 showed different isolates are not independent but represent the progeny of a alleles at loci 3232 and 3336, respectively, compared to the single original strain and infection. The patient was born in the genotype shared by the other three isolates. Three different Netherlands, a country with one of the lowest TB incidences IS6110 RFLP patterns differing by one-band changes were worldwide, the isolates had a Haarlem genotype (as seen by detected. Sputum (isolate 2) and pus (isolate 4) isolates, re- 24-locus based MIRU-VNTR typing and spoligotyping; see spectively, showed an additional band above or at an interme- above) typically associated with this geographic region, and the diate position compared to the 6 bands shared with the three patient had no known high risk of TB exposure. These features

     FIG. 1. RFLP and spoligotyping patterns. Correspondences with isolate identication numbers, body sites, and dates of isolation are indi- cated.

     VOL. 48, 2010 CASE REPORTS 3815

    TABLE 2. MIRU-VNTR typing for ve isolates using six lected at the same day or at a 2-month interval, as monitored ahypervariable loci by using ve independent genotyping methods. As suggested for cases of pulmonary TB with mixed infection (14), this might MIRU-VNTR allele in locus: Isolate reect some compartmentalized infection even at the suborgan 1982 2163a 3232 3336 3820 4120 level, the lesions of which could then be stochastically sampled, 1 4 7 9 7 3 5 leading to isolation of different variants. 2 4 7 7 7 3 5 It is also noteworthy that the changes in the different mark- 3 4 7 7 1 3 5 ers were generally not associated. For instance, spoligotype 4 4 7 7 7 3 5 5 4 7 7 7 3 5 changes affected two isolates that were different from those

    affected by RFLP changes. Likewise, the SLV in hypervariable a Correspondences with body sites and dates of isolation are given in Fig. 1. MIRU-VNTR markers are named according to a standardized designation (i.e., MIRU-VNTR locus 3336 occurred in isolate 3, which was position on M. tuberculosis H37Rv chromosome). affected neither by RFLP nor by spoligotype change. This lack of association reveals stochastic evolution of independent and render very unlikely the scenario of mixed or superinfection by relatively high-turnover markers in the short term. Because of two different strains, typically seen with various frequencies in this stochastic nature, no general conclusion on the relative high-incidence or high-risk settings (2, 12, 14, 16, 18). More- evolutionary rates or discriminatory power of the markers can over, the 24 standard MIRU-VNTR loci were identical among be drawn from observations on a limited number of isolates. the 5 isolates (accounting for missing data in one locus; see Nonetheless, such stochastic evolution can explain some de- above). In line with the well-documented clonal stability of gree of discordance between results of different genotyping these markers, complete conservation of these standard methods among very closely related strains over short evolu- MIRU-VNTR loci is the most likely situation among serial tionary distances (8). Over longer genetic distances, in popu- isolates from a single infection (1, 11, 13, 19). Changes in one lations of more distantly related strains, a higher correlation is band or one spacer and variations limited to an SLV are apparent as a reection of the clonal evolution of M. tubercu- hallmarks of genetic drift in clonal subpopulations, as seen in losis (15, 22). (hypervariable) MIRU-VNTR loci, IS6110 RFLP, and spoli- In conclusion, this report constitutes additional but puzzling gotyping, respectively, (1, 5, 10, 19). Likewise, rep-PCR pat- evidence against the dogma of a systematically homogenous terns were highly similar if not identical in some cases. Alto- within-host population of M. tuberculosis. Besides mixed infec- gether, these data thus strongly support that the observed tions, previous studies have shown the existence of clonal sub- changes result from microevolution in a clonal population of populations in some patients with a single infection by a single M. tuberculosis. strain, as reected by small genotypic heterogeneity detected in the isolate mycobacterial population (e.g., see references 5, 10, 13, 14, and 16). In almost all cases, observations were The relative extent of such microevolution is unprecedented. limited to two or three isolates from the same body site (i.e., Except for the 24 standard MIRU-VNTR loci, the combina- pulmonary), and genotypic variations were limited to small tion of the other markers (even without including rep-PCR changes seen in a single genotyping method. Our data, based results) systematically dened a unique clonal variant for each on ve isolates from three different body sites of a same TB of the ve isolates obtained from this single patient, regardless patient, as monitored by using ve independent genotyping of body site origins. This extent may result from the probably methods, suggest that microevolution might be of a greater long course between initial infection and TB diagnosis in the extent than previously assumed, at least in some cases. In the present case, leading to some differentiation in the clonal pop- present case, this phenomenon was probably driven by genetic ulation. Infection with two probable clonal variants compart- drift favored both by a long disease incubation period leading mentalized in two different body sites has been described (7). to a large infectious population size and random sampling/ Here, however, we report microevolution by an unusual extent

    compartmentalization effects linked to extrapulmonary dis- of distinct clonal variants among ve isolates from three dif-

    semination. ferent body sites and one same site/uid sample (urine) col-

     FIG. 2. Dendrogram and similarity matrix for ve M. tuberculosis isolates analyzed using the DiversiLab system. Correspondences with body sites and dates of isolation are given in Fig. 1.


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