The team, led by Sampreeti Tahbildar and Ashis Das of the Molecular Parasitology and Systems Biology Lab at BITS Pilani, published their findings in Frontiers in Malaria on 11 November 2025. Their approach relied on array-based comparative genomic hybridization, a technique that measures the relative abundance of every segment of a parasite genome by competitively hybridizing fluorescently labeled test and reference DNA onto a dense microarray. Rather than sequencing whole genomes, which can struggle with the repetitive, GC-rich regions that litter malaria parasite chromosomes, the researchers designed a custom 2 × 400K tiling array carrying 418,577 oligonucleotide probes spaced an average of just 56 base pairs apart across the entire P. vivax genome. That density, anchored to the P. vivax Sal-1 reference genome, allowed them to detect copy number changes as small as 100 base pairs and as large as nearly 1.5 megabases.

The clinical material came from patients admitted to S.P. Medical College in Bikaner, in northwestern India, a region where P. vivax remains endemic. Blood samples were drawn after informed consent, processed within fifteen minutes of collection, and confirmed as pure P. vivax infections using microscopy, rapid diagnostic tests, and an 18S rRNA gene-based multiplex PCR that ruled out P. falciparum co-infection. Three isolates came from patients with cerebral malaria, defined by a Glasgow coma scale score below 11, and three came from patients with uncomplicated disease. Genomic DNA from each group was pooled, labeled with distinct fluorescent dyes, and hybridized against each other on the tiling array. CNVs were called using the ADM-2 algorithm, a standard tool in Agilent array-CGH analysis, with stringent filters requiring at least three consecutive probes showing an average absolute log2 ratio of 0.3 or greater, a threshold designed to eliminate single-probe artifacts arising from sequence mismatches.

The results were striking in their breadth. Copy number variations appeared across all 14 chromosomes of the P. vivax genome, affecting a total of 2,138 genes. Altered intervals ranged from roughly 100 base pairs to approximately 1,429 kilobases. The single most extensive CNV spanned about 1,450 kilobases on chromosome 12, covering 316 genes in a continuous stretch of amplification. Chromosome 14 and chromosome 10 also carried large amplified blocks, encompassing 100 and 96 genes respectively. Many of the affected genes encode hypothetical proteins whose functions remain uncharacterized, a reflection of the persistent difficulty in maintaining P. vivax in continuous laboratory culture, which has hampered functional studies of the parasite for decades.

Among the most consequential findings were CNVs in genes linked to antimalarial drug resistance. The team detected amplifications in three genes encoding putative multidrug resistance proteins belonging to ABC transporter families, including PVX_097025, PVX_080100, and PVX_118100. Amplification of the P. falciparum ortholog of PVX_080100 has previously been associated with mefloquine resistance, and increased copy number of the multidrug resistance gene 1, mdr1, has been repeatedly linked to elevated drug tolerance in both P. falciparum and P. vivax. Earlier work by Suwanarusk and colleagues showed that pvmdr1 amplification in Thai P. vivax isolates correlated with higher IC50 values for mefloquine and artesunate, providing functional evidence that copy number changes at this locus translate into measurable drug resistance phenotypes. The current study also identified amplification of a putative chloroquine resistance marker protein on chromosome 12 and deletion of a putative chloroquine resistance transporter on chromosome 1, echoing field observations from the Brazilian Amazon where multi-copy pvcrt-o has been tied to chloroquine treatment failure.

Equally significant were the CNVs detected in genes encoding surface antigens, the molecular interface between parasite and host immune system. All 24 tryptophan-rich antigens, or TRAgs, showing copy number changes were amplified, consistent with the tendency of this gene family to proliferate through duplication and diversification. One amplified TRAg, encoded by PVX_088850, is known to interact with PvMSP7 at the merozoite surface, suggesting a role in stabilizing protein complexes required for red blood cell invasion. The study also found copy number changes in 23 genes encoding serine-repeat antigens, all clustered on chromosome 4, with 19 in deleted regions and four in amplified regions. Amplifications were detected in genes for reticulocyte-binding proteins, or PvRBPs, which mediate invasion of young red blood cells, and in genes for rhoptry proteins, early transcribed membrane proteins, and cytoadherence-linked asexual proteins located on chromosomes 7 and 14. In P. falciparum, CLAG proteins are known to drive cytoadherence, the process by which infected erythrocytes bind to host endothelial cells, and to form nutrient uptake channels at the host cell membrane.

Perhaps the most intriguing signal came from the vir gene family, a large subtelomeric multigene superfamily unique to P. vivax. The analysis identified CNVs in 117 unique vir genes, with 84 located in amplified regions, 10 in deleted regions, and 23 showing both gains and losses. Vir genes have been proposed to mediate cytoadherence of infected red blood cells to host endothelial cells, a process that can obstruct microvasculature and contribute to tissue damage, and their extensive sequence diversity is thought to facilitate antigenic variation, allowing the parasite to stay one step ahead of host immunity. Amplification of vir genes in cerebral malaria isolates could, in principle, enhance both immune evasion and the adhesive phenotypes implicated in severe disease, though the authors are careful to note that this remains a hypothesis requiring functional validation.

Beyond coding regions, the study detected CNVs in intergenic stretches of the P. vivax genome, a finding that may carry regulatory implications. Intergenic regions harbor promoters, enhancers, and insulators that govern the expression of neighboring genes, and copy number changes in these non-coding sequences can alter the accessibility of regulatory elements to transcription factors or disrupt long-range chromatin interactions. In P. falciparum, intergenic regions are known to give rise to regulatory long non-coding RNAs that influence gene expression, sexual development, and parasite transmission. In humans, non-coding CNVs have been shown to modify gene regulation and phenotypic outcomes. By analogy, intergenic CNVs in P. vivax could reshape the regulatory landscape of genes encoding surface antigens, drug resistance proteins, and stress-related factors, though the authors emphasize that experimental studies are needed to confirm any such role.

The study also documented copy number changes in genes encoding components of the ubiquitin-proteasome system, with 20 genes from the ubiquitin machinery and 9 from the proteasomal degradation pathway showing CNVs. The proteasome serves as the primary engine of protein degradation in Plasmodium, and ubiquitylation levels rise during the transition from intracellular schizont to extracellular merozoite stages. Ubiquitin modification has also been associated with altered parasite susceptibility to multiple antimalarials, making these CNVs potentially relevant to treatment outcomes. Additional amplifications were detected in 12 genes encoding PHIST proteins, which in P. falciparum participate in host cell remodeling and have been differentially expressed in cerebral and pregnancy-associated malaria, as well as in approximately 80 genes encoding ribosomal complex proteins and in the gene for plasmepsin IV, an aspartic protease involved in hemoglobin digestion within the parasite food vacuole.

The authors are candid about the limitations of their work. All samples came from a single location, Bikaner, and were collected between 2007 and 2008, which may constrain the generalizability of the observed CNV patterns. The small sample size, three cerebral malaria isolates and three uncomplicated controls, reflects the practical difficulty of recruiting severe P. vivax cases, which remain comparatively rare relative to uncomplicated infections. The pooled design provides a group-level overview rather than individual-level resolution, and the authors explicitly state that they cannot conclude whether the observed CNVs contribute directly to disease severity. What they can say is that these copy number changes were present in cerebral malaria isolates when compared with uncomplicated cases, and that they cluster in genes and regions with plausible roles in immune evasion, drug resistance, cytoadherence, and host-pathogen interaction. To their knowledge, no previous whole-genome CNV analysis has been performed on P. vivax cerebral malaria isolates, making this a foundational dataset for future investigations. The microarray data have been deposited in the NCBI Gene Expression Omnibus under accession number GSE288219, allowing other groups to interrogate the findings independently. As P. vivax continues to shed its reputation as a benign parasite, studies like this one point toward a future where genomic surveillance of copy number variation could identify biomarkers for severe disease and inform more effective strategies for malaria control and treatment.

Subject of Research: Copy number variation analysis of Plasmodium vivax isolates from cerebral and uncomplicated malaria cases in India

Article Title: Genomic dynamics of clinical Plasmodium vivax: comparative genomic hybridization in severe malaria cases

Article References: Tahbildar, S., Boopathi, P. A., Kochar, S. K., Kochar, D. K., Aiyaz, M., Mugasimangalam, R. C., Rao, S. N., & Das, A. (2025). Genomic dynamics of clinical Plasmodium vivax: comparative genomic hybridization in severe malaria cases. Frontiers in Malaria, 3, Article 1667330. https://doi.org/10.3389/fmala.2025.1667330

Image Credits: AI Generated

DOI: 10.3389/fmala.2025.1667330

Keywords: Plasmodium vivax, copy number variation, cerebral malaria, comparative genomic hybridization, drug resistance, surface antigens, vir genes, intergenic regions, India, malaria severity, microarray, genomics