Reinfection and mixed infection cause changing Mycobacterium tuberculosis drug-resistance patterns

Abstract

Rationale: Multiple infections with different strains of Mycobacterium tuberculosis may occur in settings where the infection pressure is high. The relevance of mixed infections for the patient, clinician, and control program remains unclear.

Objectives: This study aimed to describe reinfection and mixed infection as underlying mechanisms of changing drug-susceptibility patterns in serial sputum cultures.

Methods: Serial M. tuberculosis sputum cultures from patients diagnosed with multi-drug-resistant (MDR) tuberculosis were evaluated by phenotypic drug-susceptibility testing and mutation detection methods. Genotypic analysis was done by IS6110 DNA fingerprinting and a novel strain-specific polymerase chain reaction amplification method.

Measurements and main results: DNA fingerprinting analysis of serial sputum cultures from 48 patients with MDR tuberculosis attributed 10 cases to reinfection and 1 case to mixed infection. In contrast, strain-specific polymerase chain reaction amplification analysis in 9 of the 11 cases demonstrated mixed infection in 5 cases, reinfection in 3 cases, and laboratory contamination in 1 case. Analysis of clinical data suggests that first-line therapy can select for a resistant subpopulation, whereas poor adherence or second-line therapy resulted in the reemergence of the drug-susceptible subpopulations.

Conclusions: We have shown that, in some patients with MDR tuberculosis, mixed infection may be responsible for observations attributed to reinfection by DNA fingerprinting. We conclude that treatment and adherence determines which strain is dominant. We hypothesize that treatment with second-line drugs may lead to reemergence of the drug-susceptible strain in patients with mixed infection.

Figure 1.

Schematic diagram showing the grouping of patients according to DNA fingerprinting and strain-specific polymerase chain reaction (PCR) amplification methods. ^†Sputum culture from two patients could not be tested. MDR = multi-drug-resistant.

Figure 2.

Phenotypic and genotypic characterization of sputum cultures from Patients 1 and 2. Serial M. tuberculosis sputum cultures were obtained from patients diagnosed with MDR tuberculosis. Phenotypic culture-based drug-susceptibility testing was performed by the direct proportion method. Treatment regimen implemented at each visit is indicated, whereas adherence was measured for the period between each visit. Mutations conferring resistance were detected by DNA sequencing or PCR dot blot (23). All sputum cultures were genotyped by IS6110 DNA fingerprinting (2), and the strain(s) present was randomly assigned an alphabetic designation according to its strain family classification. Presence of multiple strains in each sputum culture was determined using strain-specific PCR amplification (20). D = default (stopped therapy for a period of > 2 months); Emb = ethambutol; Eth = ethionamide; Inat = isoniazid and thiacetazone; Inh = isoniazid; Kana = kanamycin; nd = not determined; neg = negative; Oflox = ofloxacin; pos = positive; Pza = pyrazinamide; R = drug resistant; Rif = rifampin; S = drug sensitive; Sm = streptomycin; Teri = terizidone; thia = thiacetazone; U = unknown; + = mutation present; − = mutation absent; *internal positive PCR control (Rv3875).