M. tuberculosis families in Lao PDR
This is the first study on the genetic structure of the M. tuberculosis population in Lao PDR. First of all, a high proportion of orphan and unknown M. tuberculosis isolates (18.3%) was detected in our sample, probably because of the lack of previous genetic data. Indeed, in countries where many genetic studies have been already performed, the proportion of orphan isolates is lower, for instance 9.5% in Vietnam , and 8.2% in China . Conversely, the proportion of isolates belonging to minor families (T, H, CAS, LAM, and MANU) was lower in Lao PDR than in Myanmar and Vietnam (7.9% vs 15 and 23%, respectively) [8, 10]. Moreover, only one isolate belonged to the CAS family, which is totally absent in Cambodia and Vietnam [9, 10]. This result is in agreement with the reported low prevalence of CAS isolates in Southeast Asia, differently from South-Central Asia (56.5% in Pakistan, 26% in India) [25, 26].
Our findings indicate that the M. tuberculosis population in Lao PDR is mainly composed of strains belonging to the EAI (76.7%) and Beijing (14.4%) families, similarly to neighboring countries but in different proportions. Indeed, in Cambodia and Myanmar, the EAI family is predominant (60 and 48.4% respectively), but the Beijing family also is highly prevalent (30, and 31.9%) [8, 9]. In Vietnam, the Beijing and EAI families represent 38.5%/each of the M. tuberculosis population (Beijing isolates were found particularly in urban areas with high population density, such as Hanoi and Ho Chi Minh) . Conversely, in China, the Beijing family represents 74.1% of the M. tuberculosis population and was detected in all studied provinces, whereas only 0.03% of isolates belongs to the EAI family (only in Fujian province) . The low proportion of Beijing isolates found in our study could be explained by the low population density (27 people per km2) in Lao PDR and the fact that 67% of the Lao population live in rural areas . Moreover, the distribution of the M. tuberculosis families was heterogeneous in the different provinces of Lao PDR. EAI family isolates were from all over the country, whereas Beijing isolates came mainly from the northern and central provinces (see Fig. 1). In most of the biggest provinces (Luang Prabang, Vientiane Capital, Savannakhet), isolates belonged to different M. tuberculosis families, except in Champasack province where all isolates were identified as EAI (Fig. 1). Concerning the EAI subfamilies, the proportion of EAI5 was two times higher in Lao PDR (69.0%) than in Cambodia (28.8%) and in Vietnam (30.6%). On the other hand, EAI4-VNM, which was mainly identified in Vietnam (65.9%), was less frequent (4.5%) and found only in the central provinces. These data suggest that EAI5 is the most ancient M. tuberculosis family circulating in Lao PDR. The long history of social-economic exchange with neighboring countries has undoubtedly favored the spread of specific genotypes in the country. The “4th Population and Housing Census” (PHC) of 2015 estimated the global number of migrants at 42,000 . Most of them came from Thailand (37%), Vietnam (26%), China (23%), Myanmar (6%) and Cambodia (1%). Currently, Vientiane Capital hosts the largest proportion of migrants, and this could explain the high diversity of M. tuberculosis families (n = 5) observed in this province compared with most of the other provinces (0 to 4 families) (Fig. 1). Migrants from China and Myanmar live mostly in northern provinces, those from Thailand are mainly in the central part of the country, and migrants from Vietnam are found in the center and in Attapeu province in the South . The number of migrants from Cambodia (1%) is very low compared with those from other neighboring countries and they are distributed all over the country. These data could partly explain the distribution of the Beijing and EAI4-VNM subfamilies in Lao PDR and raise the question of the risk of a progressive invasion by Beijing strains, as previously observed in Vietnam .
Genetic diversity and transmission of M. tuberculosis families in Lao PDR
To explore the genetic diversity of M. tuberculosis population in Lao PDR, 202 isolates were characterized by spoligotyping and MIRU-VNTR typing. The results revealed 178 genotypes, a result similar to the one reported for Cambodia (91 patterns in 105 isolates) and higher than that for Vietnam (153 genotypes for 221 isolates) [9, 10]. As expected, the EAI family was more diverse than the Beijing family (138 genotypes for 155 isolates vs 23 genotypes for 29 isolates). The 19 clusters grouped 43 isolates that belonged only to the three main families (EAI, Beijing and T). The overall clustering rate was 11.9%, reflecting a non-negligible level of recent transmission compared with high TB burden countries, such as Vietnam (16.3%)  and China (18.4%) . Moreover, the Beijing family clustering rate was higher than the clustering rates of the other families (20.7% for Beijing vs 11.0% for EAI vs 9.1% for T), suggesting a potential association of the Beijing family in recent transmission cases, as demonstrated in many studies [10, 29,30,31]. Nevertheless, it is worth noting that the combination of 24 Loci MIRU-VNTR and spoligotyping can lack discrimination (only the whole genome sequencing can give us the real genotype of each isolate) making possible that some clusters include slightly different genotypes. This lack of discrimination can lead to a global overestimated clustering rate in our study. However, the large difference observed between the families (20.7% for Beijing vs 11.0% for EAI vs 9.1% for T) supports the hypothesis that Beijing, as demonstrated in many studies, might be associated with recent transmission than the other families in Laos. EAI isolate predominance, higher diversity and lower clustering rate compared with the Beijing family reinforce the hypothesis that the EAI family (specifically the EAI5 sub-family) is the more ancient M. tuberculosis family in Lao PDR. Most isolates in clusters (16 of the 19 clusters, and 37 of the 43 clustered isolates) were geographically linked, reflecting the occurrence of recent transmissions. Clusters were mainly observed in the northern and southern provinces, and mostly in rural area. Surprisingly, no cluster was observed in the capital city. This could be explained by the global low population density in cities and the higher patients’ recruitment in rural areas than in urban areas in our study.
Epidemiological consideration and drug resistant TB
The proportion of the two main families was significantly different in function of the age group, region of origin and drug-resistant status. The proportion of isolates belonging to the EAI family was higher in the 35–64 age group, as observed in Cambodia, Vietnam and Myanmar, reflecting the endemic circulation of EAI in this part of the world. On the other hand, in Lao PDR the proportion of Beijing isolates in the 15–34 and 35–64 age groups was similar, whereas in Vietnam the proportion of Beijing isolates decreases with age .
Finally, despite the low prevalence of drug resistance in Lao PDR, the Beijing family was more represented among drug-resistant isolates, as previously reported in Cambodia, Vietnam, and China [9, 10, 32]. The Beijing isolates in clusters were geographically linked and one of the three Beijing clusters included drug-resistant isolates (see Fig. 2 and Additional file 2: Table S1). These findings underline the risk of Beijing strain expansion in Lao PDR and consequently the increasing risk of primary drug resistance in recent transmission.