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Background <p>Understanding the molecular epidemiology of <i>Ascaris</i> is essential for clarifying species boundaries and transmission dynamics within regions where human-animal interfaces may influence parasite dispersal. This study provides a comprehensive multilocus characterization of <i>Ascaris</i> populations in Pakistan using nuclear (partial 5.8&#xa0;S, ITS2, partial 28&#xa0;S) and mitochondrial (<i>cox1</i>) markers.</p> Methods <p>A total of 1675 children were enrolled across six districts of Khyber Pakhtunkhwa, and expelled adult worms were collected following single-dose anthelmintic treatment; genomic DNA from morphologically confirmed specimens was analysed using standard rDNA and <i>cox1</i> sequencing workflows.</p> Results <p>Nuclear rDNA analysis revealed extremely low intraspecific variation among Pakistani <i>Ascaris</i> isolates, with only a few variable sites detected across the partial 5.8&#xa0;S, ITS2, and partial 28&#xa0;S regions. Most sequences (Pk_01, Pk_02, Pk_04, Pk_05) were nearly identical, while Pk_03 and Pk_06 showed slightly higher but still limited divergence, clustering within mixed <i>A. lumbricoides/A. suum</i> lineages. The haplotype network placed Pk_01 in the dominant global ITS2 haplotype, with Pk_02 and Pk_06 only one mutational step away, indicating strong global connectivity. Mitochondrial <i>cox1</i> sequences showed similarly shallow divergence (&lt; 1%), with PK_03 and PK_04 identical and PK_06 exhibiting the greatest but still modest variation. Phylogenetic analysis positioned all Pakistani cox1 haplotypes within a well-supported monophyletic <i>Ascaris</i> clade (bootstrap = 100%) showing intermixing of both species, consistent with incomplete lineage sorting and historical introgression. The <i>cox1</i> haplotype network identified multiple maternal lineages, including globally common types and one divergent Pakistani haplotype (PK_06).</p> Conclusions <p>The intermixing of <i>A. lumbricoides</i>–<i>A. suum</i> sequences supports the concept of a genetically interconnected species complex rather than clearly separated species. The presence of slightly divergent haplotypes further indicates ongoing microevolutionary processes within Pakistani <i>Ascaris</i> populations.</p>

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Multilocus Characterization of Human Ascaris in Pakistan Indicates Ongoing Introgression and Shared Global Haplotypes

  • 期刊论文

  • 发表日期：

    2026年4月21日

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  • Amjad Ullah Khan,
  • Tayyaba Shan,
  • Mashal Khalid,
  • Sabika Firasat,
  • Kiran Afshan

摘要

Background

Understanding the molecular epidemiology of Ascaris is essential for clarifying species boundaries and transmission dynamics within regions where human-animal interfaces may influence parasite dispersal. This study provides a comprehensive multilocus characterization of Ascaris populations in Pakistan using nuclear (partial 5.8 S, ITS2, partial 28 S) and mitochondrial (cox1) markers.

Methods

A total of 1675 children were enrolled across six districts of Khyber Pakhtunkhwa, and expelled adult worms were collected following single-dose anthelmintic treatment; genomic DNA from morphologically confirmed specimens was analysed using standard rDNA and cox1 sequencing workflows.

Results

Nuclear rDNA analysis revealed extremely low intraspecific variation among Pakistani Ascaris isolates, with only a few variable sites detected across the partial 5.8 S, ITS2, and partial 28 S regions. Most sequences (Pk_01, Pk_02, Pk_04, Pk_05) were nearly identical, while Pk_03 and Pk_06 showed slightly higher but still limited divergence, clustering within mixed A. lumbricoides/A. suum lineages. The haplotype network placed Pk_01 in the dominant global ITS2 haplotype, with Pk_02 and Pk_06 only one mutational step away, indicating strong global connectivity. Mitochondrial cox1 sequences showed similarly shallow divergence (< 1%), with PK_03 and PK_04 identical and PK_06 exhibiting the greatest but still modest variation. Phylogenetic analysis positioned all Pakistani cox1 haplotypes within a well-supported monophyletic Ascaris clade (bootstrap = 100%) showing intermixing of both species, consistent with incomplete lineage sorting and historical introgression. The cox1 haplotype network identified multiple maternal lineages, including globally common types and one divergent Pakistani haplotype (PK_06).

Conclusions

The intermixing of A. lumbricoides–A. suum sequences supports the concept of a genetically interconnected species complex rather than clearly separated species. The presence of slightly divergent haplotypes further indicates ongoing microevolutionary processes within Pakistani Ascaris populations.