Simplified Model to Survey Tuberculosis Transmission in Countries Without Systematic Molecular Epidemiology Programs.

Systematic molecular/genomic epidemiology studies for tuberculosis surveillance cannot be implemented in many countries. We selected Panama as a model for an alternative strategy. Mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) analysis revealed a high proportion (50%) of Mycobacterium tuberculosis isolates included in 6 clusters (A-F) in 2 provinces (Panama and Colon). Cluster A corresponded to the Beijing sublineage. Whole-genome sequencing (WGS) differentiated clusters due to active recent transmission, with low single-nucleotide polymorphism-based diversity (cluster C), from clusters involving long-term prevalent strains with higher diversity (clusters A, B). Prospective application in Panama of 3 tailored strain-specific PCRs targeting marker single-nucleotide polymorphisms identified from WGS data revealed that 31.4% of incident cases involved strains A-C and that the Beijing strain was highly represented and restricted mainly to Colon. Rational integration of MIRU-VNTR, WGS, and tailored strain-specific PCRs could be a new model for tuberculosis surveillance in countries without molecular/genomic epidemiology programs.

Exportation of MDR TB to Europe from Setting with Actively Transmitted Persistent Strains in Peru.

We performed a cross-border molecular epidemiology analysis of multidrug-resistant tuberculosis in Peru, Spain, and Italy. This analysis revealed frequent transmission in Peru and exportation of a strain that recreated similar levels of transmission in Europe during 2007-2017. Transnational efforts are needed to control transmission of multidrug-resistant tuberculosis globally.

Whole genome sequencing-based analysis of tuberculosis (TB) in migrants: rapid tools for cross-border surveillance and to distinguish between recent transmission in the host country and new importations.

BackgroundThe analysis of transmission of tuberculosis (TB) is challenging in areas with a large migrant population. Standard genotyping may fail to differentiate transmission within the host country from new importations, which is key from an epidemiological perspective.AimTo propose a new strategy to simplify and optimise cross-border surveillance of tuberculosis and to distinguish between recent transmission in the host country and new importationsMethodsWe selected 10 clusters, defined by 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR), from a population in Spain rich in migrants from eastern Europe, north Africa and west Africa and reanalysed 66 isolates by whole-genome sequencing (WGS). A multiplex-allele-specific PCR was designed to target strain-specific marker single nucleotide polymorphisms (SNPs), identified from WGS data, to optimise the surveillance of the most complex cluster.ResultsIn five of 10 clusters not all isolates showed the short genetic distances expected for recent transmission and revealed a higher number of SNPs, thus suggesting independent importations of prevalent strains in the country of origin. In the most complex cluster, rich in Moroccan cases, a multiplex allele-specific oligonucleotide-PCR (ASO-PCR) targeting the marker SNPs for the transmission subcluster enabled us to prospectively identify new secondary cases. The ASO-PCR-based strategy was transferred and applied in Morocco, demonstrating that the strain was prevalent in the country.ConclusionWe provide a new model for optimising the analysis of cross-border surveillance of TB transmission in the scenario of global migration.

Comparative whole-genome sequence analysis of Mycobacterium tuberculosis isolated from pulmonary tuberculosis and tuberculous lymphadenitis patients in Northwest Ethiopia.

Background: Tuberculosis (TB), caused by the Mycobacterium tuberculosis complex (MTBC), is a chronic infectious disease with both pulmonary and extrapulmonary forms. This study set out to investigate and compare the genomic diversity and transmission dynamics of Mycobacterium tuberculosis (Mtb) isolates obtained from tuberculous lymphadenitis (TBLN) and pulmonary TB (PTB) cases in Northwest Ethiopia. Methods: A facility-based cross-sectional study was conducted using two groups of samples collected between February 2021 and June 2022 (Group 1) and between June 2020 and June 2022 (Group 2) in Northwest Ethiopia. Deoxyribonucleic acid (DNA) was extracted from 200 heat-inactivated Mtb isolates. Whole-genome sequencing (WGS) was performed from 161 isolates having ≥1 ng DNA/µl using Illumina NovaSeq 6000 technology. Results: From the total 161 isolates sequenced, 146 Mtb isolates were successfully genotyped into three lineages (L) and 18 sub-lineages. The Euro-American (EA, L4) lineage was the prevailing (n = 100; 68.5%) followed by Central Asian (CAS, L3, n = 43; 25.3%) and then L7 (n = 3; 2.05%). The L4.2.2.ETH sub-lineage accounted for 19.9%, while Haarlem estimated at 13.7%. The phylogenetic tree revealed distinct Mtb clusters between PTB and TBLN isolates even though there was no difference at lineages and sub-lineages levels. The clustering rate (CR) and recent transmission index (RTI) for PTB were 30 and 15%, respectively. Similarly, the CR and RTI for TBLN were 31.1 and 18 %, respectively. Conclusion and recommendations: PTB and TBLN isolates showed no Mtb lineages and sub-lineages difference. However, at the threshold of five allelic distances, Mtb isolates obtained from PTB and TBLN form distinct complexes in the phylogenetic tree, which indicates the presence of Mtb genomic variation among the two clinical forms. The high rate of clustering and RTI among TBLN implied that TBLN was likely the result of recent transmission and/or reactivation from short latency. Hence, the high incidence rate of TBLN in the Amhara region could be the result of Mtb genomic diversity and rapid clinical progression from primary infection and/or short latency. To validate this conclusion, a similar community-based study with a large sample size and better sampling technique is highly desirable. Additionally, analysis of genomic variants other than phylogenetic informative regions could give insightful information. Combined analysis of the host and the pathogen genome (GXG) together with environmental (GxGxE) factors could give comprehensive co-evolutionary information.

Mycobacterium tuberculosis Sub-Lineage 4.2.2/SIT149 as Dominant Drug-Resistant Clade in Northwest Ethiopia 2020-2022: In-silico Whole-Genome Sequence Analysis.

Introduction: Drug resistance (DR) in Mycobacterium tuberculosis complex (MTBC) is mainly associated with certain lineages and varies across regions and countries. The Beijing genotype is the leading resistant lineage in Asia and western countries. M. tuberculosis (Mtb) (sub) lineages responsible for most drug resistance in Ethiopia are not well described. Hence, this study aimed to identify the leading drug resistance sub-lineages and characterize first-line anti-tuberculosis drug resistance-associated single nucleotide polymorphisms (SNPs). Methods: A facility-based cross-sectional study was conducted in 2020-2022 among new and presumptive multidrug resistant-TB (MDR-TB) cases in Northwest Ethiopia. Whole-genome sequencing (WGS) was performed on 161 isolates using Illumina NovaSeq 6000 technology. The SNP mutations associated with drug resistance were identified using MtbSeq and TB profiler Bioinformatics softwares. Results: Of the 146 Mtb isolates that were successfully genotyped, 20 (13.7%) harbored one or more resistance-associated SNPs. L4.2.2.ETH was the leading drug-resistant sub-lineage, accounting for 10/20 (50%) of the resistant Mtb. MDR-TB isolates showed extensive mutations against first-line anti-TB drugs. Ser450Leu/(tcg/tTg) for Rifampicin (RIF), Ser315Thr/(agc/aCc) for Isoniazid (INH), Met306Ile/(atg/atA(C)) for Ethambutol (EMB), and Gly69Asp for Streptomycin (STR) were the leading resistance associated mutations which accounted for 56.5%, 89.5%, 47%, and 29.4%, respectively. The presence of both clustered and non-clustered drug resistance (DR) isolates indicated that the epidemics is driven by both new DR development and acquired resistance. Conclusion: The high prevalence of drug-resistant TB due to geographically restricted sub-lineages (L4.2.2.ETH) indicates the ongoing local micro epidemics. The Mtb drug resistance surveillance system must be improved. Further evolutionary analysis of L4.2.2.ETH strain is highly desirable to understand evolutionary forces that leads L4.2.2.ETH in to high level DR and transmissible sub-lineage.

Assessment of closely related Mycobacterium tuberculosis variants with different transmission success and in vitro infection dynamics.

Whole genome sequencing (WGS) is able to differentiate closely related Mycobacterium tuberculosis variants within the same transmission cluster. Our aim was to evaluate if this higher discriminatory power may help identify and characterize more actively transmitted variants and understand the factors behind their success. We selected a robust MIRU-VNTR-defined cluster from Almería, Spain (22 cases throughout 2003-2019). WGS allowed discriminating, within the same epidemiological setting, between a successfully transmitted variant and seven closely related variants that did not lead to secondary cases, or were involved in self-limiting transmission (one single secondary case). Intramacrophagic growth of representative variants was evaluated in an in vitro infection model using U937 cells. Intramacrophage multiplication ratios (CFUs at Day 4/CFUs at Day 0) were higher for the actively transmitted variant (range 5.3-10.7) than for the unsuccessfully transmitted closely related variants (1.5-3.95). Two SNPs, mapping at the DNA binding domain of DnaA and at kdpD, were found to be specific of the successful variant.

Integrative transnational analysis to dissect tuberculosis transmission events along the migratory route from Africa to Europe.

BACKGROUND: Growing international migration has increased the complexity of tuberculosis transmission patterns. Italy's decision to close its borders in 2018 made of Spain the new European porte entrée for migration from the Horn of Africa (HA). In one of the first rescues of migrants from this region at the end of 2018, tuberculosis was diagnosed in eight subjects, mainly unaccompanied minors. METHODS: Mycobacterium tuberculosis isolates from these recently arrived migrants were analysed by Mycobacterial Interspersed Repetitive-Unit/Variable-Number of Tandem Repeat (MIRU-VNTR) and subsequent whole genome sequencing (WGS) analysis. Data were compared with those from collections from other European countries receiving migrants from the HA and a strain-specific PCR was applied for a fast searching of common strains. Infections in a cellular model were performed to assess strain virulence. RESULTS: MIRU-VNTR analysis allowed identifying an epidemiological cluster involving three of the eight cases from Somalia (0 single-nucleotide polymorphisms between isolates, HA cluster). Following detailed interviews revealed that two of these cases had shared the same migratory route in most of the trip and had spent a long time at a detention camp in Libya. To confirm potential en route transmission for the three cases, we searched the same strain in collections from other European countries receiving migrants from the HA. MIRU-VNTR, WGS and a strain-specific PCR for the HA strain were applied. The same strain was identified in 12 cases from Eritrea diagnosed soon after their arrival in 2018 to the Netherlands, Belgium and Italy. Intracellular replication rate of the strain did not reveal abnormal virulence. CONCLUSIONS: Our study suggests a potential en route transmission of a pan-susceptible strain, which caused at least 15 tuberculosis cases in Somalian and Eritrean migrants diagnosed in four different European countries.

Screening of inmates transferred to Spain reveals a Peruvian prison as a reservoir of persistent Mycobacterium tuberculosis MDR strains and mixed infections.

It is relevant to evaluate MDR-tuberculosis in prisons and its impact on the global epidemiology of this disease. However, systematic molecular epidemiology programs in prisons are lacking. A health-screening program performed on arrival for inmates transferred from Peruvian prisons to Spain led to the diagnosis of five MDR-TB cases from one of the biggest prisons in Latin America. They grouped into two MIRU-VNTR-clusters (Callao-1 and Callao-2), suggesting a reservoir of two prevalent MDR strains. A high-rate of overexposure was deduced because one of the five cases was coinfected by a pansusceptible strain. Callao-1 strain was also identified in 2018 in a community case in Spain who had been in the same Peruvian prison in 2002-5. A strain-specific-PCR tailored from WGS data was implemented in Peru, allowing the confirmation that these strains were currently responsible for the majority of the MDR cases in that prison, including a new mixed infection.

Direct genotyping of Mycobacterium tuberculosis from Xpert® MTB/RIF remnants.

Genotyping of Mycobacterium tuberculosis (MTB) isolates has markedly improved our knowledge of its transmission dynamics. MIRU-VNTR is considered the reference molecular tool for MTB fingerprinting. However, the dependence of this technique on cultured isolates means that we lack molecular epidemiology data from many settings where culture facilities have not been implemented. Efforts have been made to adapt the MIRU-VNTR procedure to direct analysis of clinical specimens, although implementation of these efforts has not proven successful. The large-scale roll-out of Xpert MTB/RIF (Xpert) technology, which is now in almost every TB-endemic country, including many where MTB is not cultured, provides us with a new opportunity to explore whether MTB genotyping could be performed from the remnants of the Xpert cartridge. We ran a pilot study in Mozambique in which the remnants of 24 positive Xpert assays for detection of MTB were used as template material for the 15-locus or the more discriminatory 24-locus MIRU-VNTR technique. MTB fingerprinting was possible in specimens with a high bacterial burden, according to the Xpert load categories, and within the first week after Xpert was performed. Given the wide availability, simple processing, and rapid reporting of results with Xpert, our findings suggest that MIRU-VNTR-based fingerprinting from remnants of Xpert could play a major role in extending MTB molecular epidemiology studies to settings where information on the transmission dynamics of this pathogen is lacking.

A deletion hampering appropriate typing of Mycobacterium africanum.

Molecular epidemiology analysis of tuberculosis transmission is based mostly on the application of MIRU-VNTR. In certain isolates a complete 24-loci genotype is not obtained and these incompletely genotyped isolates can not be used in the definition of clusters. In a population-based molecular epidemiology study performed in Almería, Southeast Spain, a context with a high proportion of immigrants, we found that an 88-bp deletion in isolates of Mycobacterium africanum Lineage 5 hampers MIRU-VNTR analysis. A more extensive analysis revealed that this deletion was shared by all the Lineage 5 isolates in certain countries of origin of immigrants, such as Equatorial Guinea, and is likely present in several other African countries and also in the USA. A procedure is proposed to enable epidemiological analysis of these isolates.

Optimizing and accelerating the assignation of lineages in Mycobacterium tuberculosis using novel alternative single-tube assays.

The assignation of lineages in Mycobacterium tuberculosis (MTB) provides valuable information for evolutionary and phylogeographic studies and makes for more accurate knowledge of the distribution of this pathogen worldwide. Differences in virulence have also been found for certain lineages. MTB isolates were initially assigned to lineages based on data obtained from genotyping techniques, such as spoligotyping or MIRU-VNTR analysis, some of which are more suitable for molecular epidemiology studies. However, since these methods are subject to a certain degree of homoplasy, other criteria have been chosen to assign lineages. These are based on targeting robust and specific SNPs for each lineage. Here, we propose two newly designed multiplex targeting methods-both of which are single-tube tests-to optimize the assignation of the six main lineages in MTB. The first method is based on ASO-PCR and offers an inexpensive and easy-to-implement assay for laboratories with limited resources. The other, which is based on SNaPshot, enables more refined standardized assignation of lineages for laboratories with better resources. Both methods performed well when assigning lineages from cultured isolates from a control panel, a test panel, and a problem panel from an unrelated population, Mexico, which included isolates in which standard genotyping was not able to classify lineages. Both tests were also able to assign lineages from stored isolates, without the need for subculture or purification of DNA, and even directly from clinical specimens with a medium-high bacilli burden. Our assays could broaden the contexts where information on lineages can be acquired, thus enabling us to quickly update data from retrospective collections and to merge data with those obtained at the time of diagnosis of a new TB case.