Endogenous relapse and exogenous reinfection in recurrent pulmonary tuberculosis: A retrospective study revealed by whole genome sequencing

Abstract

Background: Tuberculosis may reoccur due to reinfection or relapse after initially successful treatment. Distinguishing the cause of TB recurrence is crucial to guide TB control and treatment. This study aimed to investigate the source of TB recurrence and risk factors related to relapse in Hunan province, a high TB burden region in southern China.

Methods: A population-based retrospective study was conducted on all culture-positive TB cases in Hunan province, China from 2013 to 2020. Phenotypic drug susceptibility testing and whole-genome sequencing were used to detect drug resistance and distinguish between relapse and reinfection. Pearson chi-square test and Fisher exact test were applied to compare differences in categorical variables between relapse and reinfection. The Kaplan-Meier curve was generated in R studio (4.0.4) to describe and compare the time to recurrence between different groups. p < 0.05 was considered statistically significant.

Results: Of 36 recurrent events, 27 (75.0%, 27/36) paired isolates were caused by relapse, and reinfection accounted for 25.0% (9/36) of recurrent cases. No significant difference in characteristics was observed between relapse and reinfection (all p > 0.05). In addition, TB relapse occurs earlier in patients of Tu ethnicity compared to patients of Han ethnicity (p < 0.0001), whereas no significant differences in the time interval to relapse were noted in other groups. Moreover, 83.3% (30/36) of TB recurrence occurred within 3 years. Overall, these recurrent TB isolates were predominantly pan-susceptible strains (71.0%, 49/69), followed by DR-TB (17.4%, 12/69) and MDR-TB (11.6%, 8/69), with mutations mainly in codon 450 of the rpoB gene and codon 315 of the katG gene. 11.1% (3/27) of relapse cases had acquired new resistance during treatment, with fluoroquinolone resistance occurring most frequently (7.4%, 2/27), both with mutations in codon 94 of gyrA.

Conclusion: Endogenous relapse is the main mechanism leading to TB recurrences in Hunan province. Given that TB recurrences can occur more than 4 years after treatment completion, it is necessary to extend the post-treatment follow-up period to achieve better management of TB patients. Moreover, the relatively high frequency of fluoroquinolone resistance in the second episode of relapse suggests that fluoroquinolones should be used with caution when treating TB cases with relapse, preferably guided by DST results.

Keywords: recurrence; reinfection; relapse; tuberculosis; whole genome sequencing.

Figure 1

Flowchart of recurrent TB cases included and excluded from the study.

Figure 2

The distribution of SNP differences between paired isolates. Reinfection was defined as a recurrent disease episode caused by a new TB strain with a genetic distance of more than 12 SNPs compared with the strain that caused the original episode. Relapse was defined as a genetic distance of 12 or fewer SNPs between paired strains isolated from two episodes in TB recurrence. The SNP differences between paired isolates were calculated by using Snp-dists (v.0.8.2).

Figure 3

Phylogenetic tree and drug-resistant profile of 69 MTB strains from 34 recurrent patients. Inner band indicates TB recurrence classification (reinfection represents strain pairs differences >12 SNPs, whereas relapse represents strain pairs differences ≤12 SNPs) and the outer band suggests phenotypic drug-resistant type (see legend). Solid circles indicate genetic drug resistance detected by TB-profiler. Reinfected patients are highlighted with different colors and curves connecting patients’ samples in the phylogeny indicating paired strains isolated from the same patient.

Figure 4

Kaplan–Meier survival estimates for TB recurrence. Comparison of the time interval required for relapse and reinfection (A). The time interval required for relapse to occur by gender (B), nationality (C), pulmonary cavity (D), MDR-TB (E), and Lineage of TB strains (F). dashed line indicate that the time interval required for 50% of TB recurrence or relapse to occur. p-value was calculated by Log-rank test.