Combination of Xpert MTB/RIF and MTBDRplus for Diagnosing Tuberculosis in a Chinese District.

BACKGROUND The incidence of tuberculosis (TB) remains high in many countries, including some middle- and high-income countries without financial constraints for diagnosis and treatment. The implementation of an improved algorithm for diagnosis using 2 rapid molecular tests should help reduce the TB burden. MATERIAL AND METHODS Between April 2018 and March 2019, sputum samples from 711 patients suspected of TB in Nanshan, Shenzhen, China, were included in this prospective study. All sputum samples were examined by smear microscopy, Mycobacterium Growth Indicator Tube (MGIT) 960 culture, and Xpert MTB/RIF. The sputum remnants of Xpert MTB/RIF were used for MTBDRplus to confirm the Xpert results both for the presence of TB bacilli and for resistance to rifampicin (RIF), and also to diagnose multidrug-resistant tuberculosis (MDR-TB). RESULTS In total, 200 (28.1%) of the 711 sputa were positive for TB by Xpert MTB/RIF, and the sputum remnants were used for MTBDRplus. The simultaneous use of Xpert MTB/RIF and MTBDRplus directly on sputum samples permitted accurate bacteriologic confirmation of TB in 64% (119/187) of cases and detection of 70% (7/10) of strains that were MDR. CONCLUSIONS The implementation of 2 rapid nucleic acid-based tests on sputum samples could facilitate the prompt and appropriate treatment of most TB cases.

Use of GeneXpert Remnants for Drug Resistance Profiling and Molecular Epidemiology of Tuberculosis in Libreville, Gabon.

Multidrug-resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis pose major problems for global health. The GeneXpert MTB/RIF (Xpert) assay rapidly detects resistance to rifampin (RIFr), but for detection of the additional resistance that defines MDR-TB (MDR tuberculosis) and XDR-TB, and for molecular epidemiology, specimen cultures and a biosafe infrastructure are generally required. We sought to determine whether the remnants of sputa prepared for the Xpert assay could be used directly to find mutations associated with drug resistance and to study molecular epidemiology, thus providing precise characterization of MDR-TB cases in countries lacking biosafety level 3 (BSL3) facilities for M. tuberculosis cultures. After sputa were processed and run on the Xpert instrument, the leftovers of the samples prepared for the Xpert assay were used for PCR amplification and sequencing or for a line probe assay to detect mutations associated with resistance to additional drugs, as well as for molecular epidemiology with spoligotyping and selective mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. Of 130 sputum samples from Gabon tested with the Xpert assay, 124 yielded interpretable results; 21 (17%) of these were determined to be RIFr Amplification and sequencing or a line probe assay of the Xpert remnants confirmed 18/21 samples as MDR, corresponding to 12/116 (9.5%) new and 6/8 (75%) previously treated TB patients. Spoligotyping and MIRU typing with hypervariable loci identified an MDR Beijing strain present in five samples. We conclude that the remnants of samples processed for the Xpert assay can be used in PCRs to find mutations associated with the resistance to the additional drugs that defines MDR and XDR-TB and to study molecular epidemiology without the need for culturing or a biosafe infrastructure.

Drug resistance, fitness and compensatory mutations in Mycobacterium tuberculosis.

For tuberculosis to be eradicated, the transmission of Multi-Drug-Resistant and eXtensively Drug Resistant strains of Mycobacterium tuberculosis (MDR and XDR-TB) must be considerably reduced. Drug resistant strains were initially thought to have reduced fitness, and the majority of resistant strains may actually have compromised fitness because they are found in only one or a few patients. In contrast, some MDR/XDR-TB strains are highly transmitted and cause large outbreaks. Most antibiotics target essential bacterial functions and the mutations that confer resistance to anti-TB drugs can incur fitness costs manifested as slower growth and reduced viability. The fitness costs vary with different resistance mutations and the bacilli can also accumulate secondary mutations that compensate for the compromised functions and partially or fully restore lost fitness. The compensatory mutations (CM) are different for each antibiotic, as they mitigate the deleterious effects of the specific functions compromised by the resistance mutations. CM are generally more common in strains with resistance mutations incurring the greatest fitness costs, but for RIF resistance, CM are most frequent in strains with the mutation carrying the least fitness cost, Ser450Leu. Here, we review what is known about fitness costs, CM and mechanisms of resistance to the drugs that define a strain as MDR or XDR-TB. The relative fitness costs of the resistance mutations and the mitigating effects of CM largely explain why certain mutations are frequently found in highly transmitted clusters while others are less frequently, rarely or never found in clinical isolates. The CM illustrate how drug resistance affects bacteria and how bacteria evolve to overcome the effects of the antibiotics, and thus a paradigm for how mycobacteria can evolve in response to stress.

Highly transmitted M. tuberculosis strains are more likely to evolve MDR/XDR and cause outbreaks, but what makes them highly transmitted?

Multi-Drug-Resistant strains of Mycobacterium tuberculosis (MDR-TB) are a serious obstacle to global TB eradication. While most MDR-TB strains are infrequently transmitted, a few cause large transmission clusters that contribute substantially to local MDR-TB burdens. Here we examine whether the known mutations in these strains can explain their success. Drug resistance mutations differ in fitness costs and strains can also acquire compensatory mutations (CM) to restore fitness, but some highly transmitted MDR strains have no CM. The acquisition of resistance mutations that maintain high transmissibility seems to occur by chance and are more likely in strains that are intrinsically highly transmitted and cause many cases. Modern Beijing lineage strains have caused several large outbreaks, but MDR outbreaks are also caused by ancient Beijing and lineage 4 strains, suggesting the lineage is less important than the characteristics of the individual strain. The development of fluoroquinolone resistance appears to represent another level of selection, in which strains must surmount unknown fitness costs of gyrA mutations. The genetic determinants of high transmission are poorly defined but may involve genes encoding proteins involved in molybdenum acquisition and the Esx systems. In addition, strains eliciting lower cytokine responses and producing more caseating granulomas may have advantages for transmission. Successful MDR/XDR strains generally evolve from highly transmitted drug sensitive parent strains due to selection pressures from deficiencies in local TB control programs. Until TB incidence is considerably reduced, there will likely be highly transmitted strains that develop resistance to any new antibiotic.

Publisher Correction: M. tuberculosis infection and antigen specific cytokine response in healthcare workers frequently exposed to tuberculosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

M. tuberculosis infection and antigen specific cytokine response in healthcare workers frequently exposed to tuberculosis.

Tuberculosis (TB) is the leading cause of death due to an infectious agent, but only a small fraction of those infected develop the disease. Cytokines are involved in the mediation and regulation of immunity, and their secretion patterns may reflect the infection status. To increase our understanding of immune response to M. tuberculosis infection, we conducted a cross-sectional study investigating M. tuberculosis infection status and comparing the release profiles of cytokines GM-CSF, IFN-γ, IL-1ß, IL-10, IL-12 (p70), IL-2, IL-4, IL-5, IL-6, IL-8, TNF-α, in community controls (CCs) and healthy healthcare workers (HCWs) highly exposed to TB. Among HCWs and CCs, the probability of latent M. tuberculosis (LTB+) infection was respectively 5.4 (p = 0.002) and 3.4 (p = 0.006) times higher in men than women. The odds ratio of LTB infection was 4 times higher among HCWs in direct contact with active TB patients than other HCW (p = 0.01). Whole blood supernatant cytokine responses to M. tuberculosis antigens showed differential pro-inflammatory responses between HCWs and CCs. CCsLTB- had higher IL-1ß responses than HCWsLTB- (p = 0.002). HCWsLTB+ had significantly higher IL-8 responses to M. tuberculosis antigens than HCWsLTB- (p = 0.003) and CCsLTB- (p = 0.015). HCWsLTB+/- showed weak but positive TNF-α responses to M. tuberculosis antigen stimulation compared to CCsLTB+/- (p ≤ 0.015). Looking at T-helper (1 and 2) responses, HCWsLTB+ and CCsLTB+ had significantly higher IFN-γ and IL-2 responses compared to HCWsLTB- and CCsLTB- (p < [0.0001-0.003]). Also, TB antigen induced IL-5 secretion was significantly higher in HCWsLTB+ and CCsLTB+ than in non-infected CCsLTB- (p < [0.005-0.04]). M. tuberculosis antigen specific responses in HCWsLTB+ varied based on active TB exposure gradient. HCWsLTB+ who were highly exposed to active TB (≥3 hours per day) had significantly higher IFN-γ and IL-8 responses (p ≤ 0.02) than HCWs LTB+ not in direct contact with active TB patients. HCWsLTB+ working with active TB patients for 5 to 31 years had a significantly enhanced secretion of proinflammatory cytokines (GM-CSF, IFN-γ, IL-1ß, IL-2, IL-6, IL-8, IL-12p70, TNF-α) compared to HCWsLTB- (p < [0.0001-0.01]). Secretion of anti-inflammatory/Th2 cytokines IL-5 and IL-10 was also higher in HCWsLTB+ than HCWsLTB-. In conclusion, LTBI individuals controlling the M. tuberculosis infection have an enhanced TB specific Th1-cytokines/proinflammatory response combined with selected Th2 type/anti-inflammatory cytokines induction.