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Define SNP thresholds for delineation of tuberculosis transmissions using whole-genome sequencing.

ABSTRACT: For facilitating tuberculosis (TB) control, we used a whole-genome sequencing (WGS)-based approach to delineate transmission networks in a country with an intermediate burden of TB. A cluster was defined as Mycobacterium tuberculosis isolates with identical genotypes, and an outbreak was defined as clustered cases with epidemiological links (epi-links). To refine a cluster predefined using space oligonucleotide typing and mycobacterial interspersed repetitive unit variable tandem repeat typing, we analyzed one pansusceptible TB (C1) and three multidrug-resistant (MDR)-TB (C2-C4) clusters from different scenarios. Pansusceptible TB cluster (C1) consisting of 28 cases had ≤5 single nucleotide polymorphisms (SNPs) difference between their isolates. C1 was a definite outbreak, with cases attending the same junior high school in 2012. Three MDR-TB clusters (C2-C4) with distinct genotypes were identified, each consisting of 12-22 cases. Some of the cases had either ≤5 or ≤15 SNPs difference with clear or probable epi-links. Of note, even though WGS could effectively assist TB contact tracing, we still observed missing epi-links in some cases within the same cluster. Our results showed that thresholds of ≤5 and ≤15 SNPs difference between isolates were used to categorize definite and probable TB transmission, respectively. Furthermore, a higher SNP threshold might be required to define an MDR-TB outbreak. WGS still needs to be combined with classical epidemiological methods for improving outbreak investigations. Importantly, different SNP thresholds have to be applied to define outbreaks.

Importance

TB is a chronic disease. Depending on host factors and TB burden, clusters of cases may continue to increase for several years. Conventional genotyping methods overestimate TB transmission, hampering precise detection of outbreaks and comprehensive surveillance. WGS can be used to obtain SNP information of M. tuberculosis to improve discriminative limitations of conventional methods and to strengthen delineation of transmission networks. It is important to define the country-specific SNP thresholds for investigation of transmission. This study demonstrated the use of thresholds of ≤5 and ≤15 SNPs difference between isolates to categorize definite and probable transmission, respectively. Different SNP thresholds should be applied while a higher cutoff was required to define an MDR-TB outbreak. The utilization of SNP thresholds proves to be crucial for guiding public health interventions, eliminating the need for unnecessary public health actions, and potentially uncovering undisclosed TB transmissions.

SUBMITTER: Xiao Y-X

PROVIDER: S-EPMC11302064 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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Define SNP thresholds for delineation of tuberculosis transmissions using whole-genome sequencing.

Xiao Yu-Xin Y-X Chan Tai-Hua T-H Liu Kuang-Hung K-H Jou Ruwen R

Microbiology spectrum 20240625 8

For facilitating tuberculosis (TB) control, we used a whole-genome sequencing (WGS)-based approach to delineate transmission networks in a country with an intermediate burden of TB. A cluster was defined as <i>Mycobacterium tuberculosis</i> isolates with identical genotypes, and an outbreak was defined as clustered cases with epidemiological links (epi-links). To refine a cluster predefined using space oligonucleotide typing and mycobacterial interspersed repetitive unit variable tandem repeat t ...[more]

PMID: 38916321

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Project description:BACKGROUND:Although the Illumina 1 G Genome Analyzer generates billions of base pairs of sequence data, challenges arise in sequence selection due to the varying sequence quality. Therefore, in the framework of the International Porcine SNP Chip Consortium, this pilot study aimed to evaluate the impact of the quality level of the sequenced bases on mapping quality and identification of true SNPs on a large scale. RESULTS:DNA pooled from five animals from a commercial boar line was digested with DraI; 150-250-bp fragments were isolated and end-sequenced using the Illumina 1 G Genome Analyzer, yielding 70,348,064 sequences 36-bp long. Rules were developed to select sequences, which were then aligned to unique positions in a reference genome. Sequences were selected based on quality, and three thresholds of sequence quality (SQ) were compared. The highest threshold of SQ allowed identification of a larger number of SNPs (17,489), distributed widely across the pig genome. In total, 3,142 SNPs were validated with a success rate of 96%. The correlation between estimated minor allele frequency (MAF) and genotyped MAF was moderate, and SNPs were highly polymorphic in other pig breeds. Lowering the SQ threshold and maintaining the same criteria for SNP identification resulted in the discovery of fewer SNPs (16,768), of which 259 were not identified using higher SQ levels. Validation of SNPs found exclusively in the lower SQ threshold had a success rate of 94% and a low correlation between estimated MAF and genotyped MAF. Base change analysis suggested that the rate of transitions in the pig genome is likely to be similar to that observed in humans. Chromosome X showed reduced nucleotide diversity relative to autosomes, as observed for other species. CONCLUSION:Large numbers of SNPs can be identified reliably by creating strict rules for sequence selection, which simultaneously decreases sequence ambiguity. Selection of sequences using a higher SQ threshold leads to more reliable identification of SNPs. Lower SQ thresholds can be used to guarantee sufficient sequence coverage, resulting in high success rate but less reliable MAF estimation. Nucleotide diversity varies between porcine chromosomes, with the X chromosome showing less variation as observed in other species.

Project description:Cambodia has one of the highest tuberculosis (TB) incidence rates in the WHO Western Pacific region. Remarkably though, the prevalence of multidrug-resistant TB (MDR-TB) remains low. We explored the genetic diversity of Mycobacterium tuberculosis (MTB) circulating in this unique setting using whole-genome sequencing (WGS). From October 2017 until January 2018, we collected one hundred sputum specimens from consenting adults older than 21 years of age, newly diagnosed with bacteriologically confirmed TB in 3 districts of Phnom Penh and Takeo provinces of Cambodia before they commence on their TB treatment, where eighty MTB isolates were successfully cultured and sequenced. Majority of the isolates belonged to Lineage 1 (Indo-Oceanic) (69/80, 86.25%), followed by Lineage 2 (East Asian) (10/80, 12.5%) and Lineage 4 (Euro-American) (1/80, 1.25%). Phenotypic resistance to both streptomycin and isoniazid was found in 3 isolates (3/80, 3.75%), while mono-resistance to streptomycin and isoniazid was identical at 2.5% (N = 2 each). None of the isolates tested was resistant to either rifampicin or ethambutol. The specificities of genotypic prediction for resistance to all drugs tested were 100%, while the sensitivities of genotypic resistance predictions to isoniazid and streptomycin were lower at 40% (2/5) and 80% (4/5) respectively. We identified 8 clusters each comprising of two to five individuals all residing in the Takeo province, making up half (28/56, 50%) of all individuals sampled in the province, indicating the presence of multiple ongoing transmission events. All clustered isolates were of Lineage 1 and none are resistant to any of the drugs tested. This study while demonstrating the relevance and utility of WGS in predicting drug resistance and inference of disease transmission, highlights the need to increase the representation of genotype-phenotype TB data from low and middle income countries in Asia and Africa to improve the accuracies for prediction of drug resistance.

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Define SNP thresholds for delineation of tuberculosis transmissions using whole-genome sequencing.

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Define SNP thresholds for delineation of tuberculosis transmissions using whole-genome sequencing.

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