This is the first national survey of drug resistance among people with TB in Timor-Leste; most TB cases identified were male and under 55 years of age. Notably, children <15 years old account for 11% of TB globally, but only represented 2% in Timor-Leste during this 9-month period. The WHO Global TB Report 202011 estimated that 8% of notified TB cases were children in Timor-Leste, including both clinically diagnosed and bacteriologically confirmed pulmonary and extra-pulmonary TB. This difference may be partially explained by the DRS enrolling only the bacteriologically confirmed pulmonary TB cases. Obtaining bacteriological confirmation of TB in children remains a challenge.
The overall prevalence of RR-TB was 0.8% (95% CI 0.3–1.5%), with 0.6% (95% CI 0.2–1.3%) among new TB patients and 2.7% (95% CI 0.5–8.2%) among previously treated TB patients. This is notably lower than previous modelled estimates prior to the survey. The results of this survey show a lower prevalence than in neighbouring Indonesia (2.4% (1.8-3.3) among new cases and 13% (9-18) among previously treated cases) and in the WHO Southeast Asia Region (2.5% (1.9-3.3) among new cases and 14% (7.7-21) among previously treated cases)12. The lower levels of rifampicin resistance in Timor-Leste may relate to the fact that rifampicin was only introduced into the four-month continuation phase of the first-line treatment regimen from the second quarter of 2015 (in line with the WHO-recommended regimen of 2HRZE+4RH), which was much later than in neighbouring countries. The lower level of rifampicin resistance found in this study in Timor-Leste might also be attributed to a good treatment success rate among TB patients who were enrolled on treatment (88% in new and relapse TB cases in 2018). However, treatment coverage is low (63%) compared to the estimated number of incident TB cases in Timor-Leste, and this also may have led to a lower selection pressure on DR-TB strains. Transmission rates of TB remain high, with the second highest incidence rate (498 cases per 100,000 population) in the WHO South-East Asia Region and among the top ten in the world.
This survey did not find any significant risk factors associated with resistance to rifampicin, likely as a result of the small number of RR-TB cases identified (7 cases only).
Although this study did not formally evaluate resistance to drugs other than rifampicin due to the limited number of sequenced isolates, mutations conferring resistance to isoniazid, ethambutol and streptomycin were identified, with 7/64 isolates displaying genotypic resistance to isoniazid. No resistance-conferring mutations for pyrazinamide or fluoroquinolones were observed among sequenced samples, which may bode well for the management of DR-TB in the country. However, WGS was not systematically performed for all samples.
The WHO End TB Strategy calls for the early diagnosis of TB and universal drug susceptibility testing (DST), highlighting the critical role of laboratories for rapidly and accurately detecting TB and drug resistance. DR-TB is a public health crisis and a major threat to global TB control. WHO recommends the use of Line Probe Assay (LPA) as a rapid diagnostic test for the detection of rifampicin, isoniazid, fluoroquinolone and pyrazinamide resistance12. A Regional Green Light Committee (r-GLC) mission in 2018 in Timor-Leste recommended investment by the country in LPA13, to rapidly detect isoniazid and fluroquinolone resistance to inform appropriate patient management. The TB Joint Monitoring Mission (JMM) conducted in Timor-Leste in 2019 also recommended that the NTP consider introducing LPA capacity into the NTRL. By mid-2021, it is anticipated that all bacteriologically-confirmed TB patients will be offered DST for rifampicin and isoniazid, as well as fluoroquinolones among cases of RR-TB, thus facilitating timely clinical management of TB patients and appropriate implementation of WHO guidelines on use of all-oral shorter regimen.
This study showed good agreement between Xpert MTB/RIF and WGS results (97%), suggesting that nationwide diagnosis of RR-TB through Xpert MTB/RIF is adequate in this setting. However, WGS also identified an unusual double mutation in the RRDR region of the genome which was missed by Xpert MTB/RIF. The occurrence of mutations missed by routine diagnostic tools should be regularly monitored in the region through collaborations with supranational referral laboratories, SNRLs, for quality assurance purposes. One RR-TB case by Xpert MTB/RIF, later found to be susceptible to rifampicin by WGS, may be explained by the analysis of an heteroresistant sample by Xpert MTB/RIF and/or the preferential selection of a susceptible strain upon culturing a sputum sample harbouring a mixed infection, prior to WGS. We also found that of the 40 samples that tested negative to M. tuberculosis by Xpert MTB/RIF at NTRL, 4 were positive by culture. It is possible that the bacterial load may have been too low for detection by Xpert MTB/RIF. Similar findings have been reported in other studies14.
Currently, eight GeneXpert machines are functional in Timor-Leste. An additional two machines of 16 modules have been received for the Covid-19 response, which can also be used by the NTP for TB diagnosis. Linkages with the GeneXpert machines will also be prioritised for intensified case finding at all facilities with a high outpatient caseload 15. During the survey, sputum samples were transported from all the municipalities in the country to the GeneXpert sites and NTRL with minimal delay (median of 2 days) through human carriers who were adequately incentivized for this work. Delay was observed in sample shipment from NTRL, Dili, to the SNRL, Chennai, and the availability of the courier agency to ship biohazard samples, which led to decreased viability and contamination of transported samples. Any discordance between Xpert and LPA tests could be resolved by phenotypic testing, once capacity is established in-country, and appropriate clinical evaluation of the patient
For routine patient management and surveillance of drug resistance, it is planned that sputum specimen samples will be routinely transported from all the municipalities to the eight GeneXpert sites, as per the revised NTP guidelines. An efficient specimen referral system is expected to be developed in the next Global Fund cycle with the support of partners, including linking with active TB case finding initiatives led by the NTP and community volunteers The same mechanism could be replicated for routinely transporting samples from the microscopy labs to the GeneXpert sites and could address barriers to accessing TB diagnosis and care from remote, hilly terrains in this island nation.
In conclusion, the survey highlighted the need for strengthening sputum specimen transportation, establishing electronic recording and reporting, achieving NTRL accreditation, and ultimately implementing universal DST in Timor-Leste for both first- and second-line drugs. The relatively low prevalence of RR-TB in Timor-Leste is an encouraging finding, but gaps remain in obtaining bacteriological confirmation of TB and routine rifampicin testing among bacteriologically confirmed cases. This study showed that the highest burden of TB was in economically productive age groups and predominantly in male gender, as well as reproductive age groups, as in other countries. Thus, the survey highlights the significant negative impact of TB on the country’s economic growth, and therefore addressing TB and DR-TB in particular, merits sustained investment.