In recent years, the assumption that infections with Mycobacterium tuberculosis (MTB) are caused by single strains has been weakened. Episodes caused by more than one MTB strain have been described and different MTB clones have been found in recurrences,1,2 in single episodes3,4 and in disseminated tuberculosis (TB).5 The focus of this report is to describe the highly complex clonal features found in the MTB isolates cultured from a child.
As a part of a study searching for clonal heterogeneity in primary respiratory infections by MTB,6 we found a case (1 of 12 of the children analyzed) with clonal differences between 2 coinfecting MTB isolates. Our aim in this report was to study more thoroughly this child as a representative of clonal heterogeneity in MTB primary infection.
The patient was a 2-year-old girl with tuberculous meningitis in which MTB was isolated from gastric fluid and cerebrospinal fluid (CSF) (samples separated by 2 days). Both isolates were susceptible to anti-TB drugs. The girl was human immunodeficiency virus-negative and she had no risk factors for TB. In an attempt to find a source-case for the patient, her family was screened. All her relatives were Mantoux negative except for her mother and grandmother. None of her relatives had symptoms of TB, radiographic studies did not reveal any abnormalities suggestive of active TB and sputum cultures were negative. The day-care facility was also studied and all personnel were Mantoux-negative and none of the workers had missed work because of illness in the previous months. The epidemiologic surveillance was maintained for 6 months after the detection of the case and the index-case was not found.
Despite the absence of risk factors for tuberculosis in this child and the fact that her socioepidemiologic circumstances did not favor multiple exposure to infectious cases, we detected the presence of 2 distinct MTB clones after analyzing by spoligotying 30 colonies from the gastric fluid culture (Fig. 1). Spoligotyping explores potential genotypic differences in 43 spacers, which are located in a region of the MTB chromosome constituted by directed repeats.7 The spoligotypes for the 2 coinfecting MTB isolates were highly similar and the subtle differences between the 2 MTB clones were also confirmed by IS6110-RFLP, the method considered to be the standard for genotyping MTB8 (Fig. 1). The possibility of laboratory cross-contamination as an explanation of this finding was ruled out because all isolates processed in the laboratory during the same week had completely different genotypes.
The situation was revealed to be even more complex when we studied the molecular composition of the MTB culture obtained from the CSF specimen (obtained 2 days apart from the gastric fluid specimen). Although the analysis of 30 independent colonies from the gastric fluid culture revealed the presence of 2 different MTB strains (proportion 24:6; Fig. 1), only one of the strains that were coinfecting the gastric fluid (the minority strain; Fig. 1) was found in CSF after genotyping the 30 colonies. These data indicate that this case was coinfected by 2 MTB clones but each of them was infecting independent anatomic sites (respiratory and extrarespiratory sites), which led to a compartimentalized infection. The presence of several MTB clones coinfecting a patient has been described before,3,4,9,10 but what is new is to describe simultaneous coinfection and compartmentalization in a child in whom the genetic differences between the coinfecting strains are subtle.
To test whether the coinfecting MTB strains had different infectivities that could explain their different efficiencies to infect respiratory and extrarespiratory sites, an ex vivo coinfection assay was performed. A 50:50 mixture of the 2 MTB strains was used to competitively coinfect human monocyte-derived macrophages. The infected culture was lysed after 24 hours and 7 days, and the supernatants were plated to grow the bacilli from the infected macrophages. Fifty single colonies from the cultured lysates were spoligotyped and the relative proportion between the 2 strains in the postinfection cultures was equivalent to that in the preinfection inoculum. These results indicated that the 2 MTB isolates infected macrophages with equivalent efficiency.
We have previously described compartmentalized infections with involvement of 2 different MTB clones in 3 adults,5 but the present case shows relevant differences with these cases. It is difficult to identify the cause for this clonal complexity. First, the adults had epidemiologic risk factors that could explain the potential multiple exposure to other infectious cases, which could lead to mixed infection, whereas the child had no socioepidemiologic circumstances to explain it. The possibility of transmission of multiple strains from a single source, although improbable, could not be fully ruled out. Second, in adults the possibility of microevolution (apparition of subtle genetic changes in an MTB subpopulation during the infection) leading to clonal heterogeneity exists because the course of the infection by MTB is usually long between exposure and clinical symptoms, whereas microevolution in a 2-year-old child is unlikely because of the short course of the infection. Third, the MTB strains involved in the compartmentalized infection reported in adults were clearly genetically different, whereas those in the child in this case were similar, which indicates that subtle genetic rearrangements could be responsible for very different infecting behaviors. Finally, we have recently found that the MTB strains involved in compartmentalization in the adults had different infectivity and that the strains with a higher efficiency to infect extrarespiratory sites also showed a higher infectivity in vitro and in vivo,11 whereas the infectivity of the 2 strains in this child was equivalent.
We believe that the case presented here constitutes one of the most complex situations described for an infection by MTB: a case of primary infection by MTB where 2 coinfecting MTB clones were involved. These 2 clones showed only subtle genetic differences and shared an equivalent infectivity in vitro, but they were infecting independent anatomic sites, leading to a compartmentalized infection.
Molecular tools are essential for a full understanding of the true microbiological clonal complexity of tuberculosis. Further in-depth analysis of clonally complex cases such as that presented here could be useful in understanding the role of new phenomena in TB, such us microevolution, polyclonality and compartmentalization and their potential impacts on the course of this infection.
We are indebted to Thomas O'Boyle for his revision of this manuscript.
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