Revisiting the applicability of minisatellite marker CH1T1M13779 to investigate the genetic diversity of P. vivax in Nuh district, Haryana, India

Abstract

The focal rise of P. vivax cases, while not uncommon, remains a significant challenge in India’s malaria elimination efforts. In 2015, a dramatic surge in P. vivax cases occurred in Primary Health Centre (PHC)- Punhana, Nuh district, Haryana, India. This focal rise was attributed to the development of an irrigation system near Hathangaon village leading to a higher malaria incidence, breeding habitats and vector densities. Using our previously validated minisatellite marker CH1T1M13779, we investigated the genetic diversity of 41 P. vivax infected samples and observed 10 alleles in two villages- Hathangaon and Raipuri, Nuh district, Haryana, India. Following are the key takeaways-a) The minisatellite marker CH1T1M13779 proved effective for genotyping; b) P. vivax preferential infection of children and adolescents making them a vulnerable age group. Despite limitations in sample size and geographic scope, this study underscores the value of using minisatellite markers for cost-effective, large-scale genotyping surveillance to help prevent future outbreaks.

Introduction

India contributes 65.7% of the malaria burden in the South East Asian region; and 43% of this is attributable to Plasmodium vivax [1–3]. Given P. vivax’s high genetic diversity, adaptability to its human host, and propensity for relapse, its control and elimination are critical for India’s overall malaria eradication efforts [4]. In 2022, five Indian states- Odisha, Chhattisgarh, Jharkhand, Madhya Pradesh, and Maharashtra were responsible for 70% of all P. vivax malaria cases [5]. Focal rise in malaria cases are not uncommon, as exemplified by the dramatic ~ 26,000% surge in P. vivax cases at Punhana PHC (Primary Health Center), District Nuh, Haryana, in 2015 [6]. This unprecedented increase could be directly attributed to the newly implemented irrigation system on local malaria transmission dynamics (Fig. 1) [6]. While this finding highlights the significant influence of environmental factors on malaria prevalence via altered vector population dynamics and behaviour, it is likely that other contributing variables exacerbated the unusual surge. These factors might include emergence of new parasite strains from genetic mutations and recombination, possibly imparting increased virulence, drug resistance and new genotypes with altered characteristics [7]. Host susceptibility can be heightened by compromised immune systems due to malnutrition, underlying health conditions, or age [8]. Additionally, the introduction of P. vivax to new areas through population migration or displacement can trigger unusual outbreaks [5].

Fig. 1.

Geographical location of Raipuri and Hathangaon villages, PHC Ujina and Punhana, Nuh district, Haryana, India

Previously, we identified a highly polymorphic minisatellite marker CH1T1M13779, which has the potential to differentiate between relapse and new infections of P. vivax from our malaria clinic at ICMR-National Institute of Malaria Research, Delhi, India [9]. In this report, we sought to validate the applicability of this minisatellite marker CH1T1M13779 in assessing the genetic diversity of P. vivax in Nuh district, Haryana, India; that might have contributed to the dramatic surge in vivax malaria cases in 2015. Various molecular approaches and markers have been reported for genetic diversity and phylogenetic studies in P. vivax population, including PCR-based genotyping using minisatellite markers [10] and PCR-RFLP of Pvmsp3 [11, 12]. While sequencing-based genotyping or Next-generation sequencing (NGS) technologies for the Pvmsp3-β gene offer high resolution, they can be expensive and require specialized laboratory infrastructure [10, 13]. In contrast, exploring a highly polymorphic minisatellite marker such as CH1T1M13779 provides a simple, robust and cost effective approach for studying genetic diversity in P. vivax populations [9]. While this preliminary investigation sought to determine the potential causes of the unusual surge in P. vivax cases, the high genetic diversity observed through the minisatellite marker CH1T1M13779 highlighted its applicability in such studies and resource-limited settings.

Methods

Sample collection

In September 2015, an unusual surge of malaria cases occurred in the villages of Raipuri (PHC Ujina) and Hathangaon (PHC Punhana) of district Mewat, Haryana, India (Fig. 1). A total 101 blood samples were collected from these villages, with 34 from Raipuri and 67 from Hathangaon. Ethical approval for blood sample collection was obtained by the Institutional Ethics Committee (ECR/NIMR/2013/102) of the ICMR-National Institute of Malaria Research, New Delhi, India. Written informed consent was acquired from all participants, including legal guardians for minors. Thick and thin blood smears were prepared for microscopic examination and 2–3 drops of blood was collected on Whatman 3MM paper for molecular studies.

Genotyping of P. vivax by minisatellite marker CH1T1M13779

DNA was extracted from three punches of dried blood spots using QIAamp DNA Mini kit, Cat. No. 51306 kit (QIAGEN, Germany) according to manufacturer’s instructions. Briefly, the samples were lysed with ATL buffer followed by addition of AL buffer and absolute ethanol. The lysate was transferred to a QIAamp spin column and washed with AW1 and AW2 buffers. DNA was eluted in 100 µl of TE buffer and stored at -20°C. Nested PCR was performed to amplify gene segments of ssRNA (small subunit ribosomal RNA), using previously described methods by Snounou et al. 1993 [14]. The first round (Nest 1) consisted of a 20 µl reaction containing Plasmodium genus-specific primers (0.2 µM) (BioServe Biotechnologies, Pvt. Ltd. India), 10 µl of GoTaq Green Master Mix (Promega, USA) and 3 µl of gnomic DNA [14]. The second round (Nest 2) used 1 µl of first round amplification, 0.2 µM of each forward and reverse primers and 10 µl of GoTaq Green Master Mix (Promega, USA) in a 20 µl reaction mix [14]. Amplified PCR products were analysed on 1.5% of agarose gel. Genotyping of P. vivax was performed using the minisatellite marker CH1T1M13779 in a 20 µl reaction mix containing 10 pmol of each primer, [Fw-5’GCATCATAATGGGTAAAG-3’ and Rv- 5’-TTGTTGCAGTGATTGAGA-3’], 10 µl of GoTaq Green Master Mix (Promega, USA), 2 µl of P. vivax genomic DNA. Thermal cycling conditions comprised of initial denaturation at 94 °C for 4 min, followed by 40 cycles at 94 °C for 30 s, 46 °C for 1 min, 72 °C for 1.30 min and final extension 72 °C for 10 min. The genomic DNA of P. falciparum (3D7) sample was also checked with minisatellite marker CH1T1M13779 [15]. Amplified products were analysed on 2% of MetaPhor^® agarose gel and visualized in a gel documentation system (Zenith Pvt. Ltd., India).

Results and Discussion

Demographic analysis and infection status of P. vivax suspected samples

A total of 101 samples were collected for this study from two villages (Hathangaon (n = 67) and Raipuri (n = 34)) and all the samples were subjected for diagnosis of P. vivax by microscopy and nested PCR. Both microscopic and nested PCR examination identified 41(40.59%) samples as positive for P. vivax infection (Table 1). Interestingly, Hathangaon 35(52.2%) presented three times more prevalence of malaria cases compared to Raipuri 6(17.6%). While male and female malaria cases were comparable in Hathangaon village, strikingly, the female prevalence was double the male cases in Raipuri village. In both villages, the age-wise analysis showed that children and adolescents (1–19 years) were the predominant age group affected (Table 1). Our investigation of unusual surge of P. vivax cases from PHC Punhana, Nuh district, Haryana has unearthed crucial evidence that underscores the imperative for expanded genotyping surveillance to combat future outbreaks. However, comprehensive evaluation across diverse geographical regions is also essential to validate its widespread applicability.

Table 1.

Village-wise demographic distribution of total no. of samples collected and positive samples along with age and gender-wise distribution

S. No.	Total samples n = 101		Microscopy n = 41		Nested PCR n = 41
Hathangaon (HG)	67(66.3)		35 (52.2)		35 (52.2)
	M	30(44.7)	M	15(42.8)	M	15(42.8)
	F	37(55.2)	F	20(57.1)	F	20(57.1)
	1–19	48(71.6)	1–19	30(85.7)	1–19	30(85.7)
	> 20	19(28.3)	> 20	5(14.2)	> 20	5(14.2)
Raipuri (RP)	34(33.6)		6 (17.6)		6 (17.6)
	M	14(41.1)	M	2(33.3)	M	2(33.3)
	F	20(58.8)	F	4(66.6)	F	4(66.6)
	1–19	23(67.6)	1–19	5(83.3)	1–19	5(83.3)
	> 20	11(32.3)	> 20	1(16.6)	> 20	1(16.6)

(_) parenthesis represents the percentage; M: males; F: females; 1–19: children and adolescents; >20: adults

Allelic variations using minisatellite marker CH1T1M13779

Fourty one (41) samples positive for P. vivax infection were subjected to molecular genotyping by minisatellite marker CH1T1M13779. A total of 10 different allelic variants were observed, as shown by the electrophoretic migration of genotyped products at different sizes and one negative control and one P. falciparum 3D7 sample did not amplify using the minisatellite marker CH1T1M13779 (Fig. 2). The sizes (bp) and percentage allele frequencies are demonstrated in Fig. 3. The allelic variants observed varied in size and frequency. For instance, alleles [260, 300 & 320 bp] in 8(20%) samples each; alleles [230 & 245 bp] in 4(9.75%) each; alleles [160, 280 & 400 bp] in 1(2%) each and allele 250 bp in 5(12%) samples were observed. Interestingly, one case of mixed alleles [260 and 230 bp] was found in one patient sample. Remarkably, the most common allelic variants were mostly represented in children and adolescents. Village-wise analysis revealed 4 allelic variants from 6 P. vivax samples in Raipuri village; while in Hathangaon village, a total 8 allelic variants were observed from 35 P. vivax samples (Fig. 3). While sequencing-based methods (e.g., NGS of Pvmsp3-β) offer comprehensive genetic analysis, their cost and technical requirements limit their widespread application [10, 13]. In contrast, PCR-based genotyping using minisatellite markers is a simpler, more affordable approach for assessing P. vivax genetic diversity [15]. Understanding P. vivax’s genetic diversity, influenced by mutations, varied genotypes, and recombination, is crucial for effective malaria control and treatment. Multiple P. vivax strains, characterized by distinct relapse patterns, have been identified worldwide (e.g., Chesson, St. Elizabeth, Netherlands, North Korean, Sal-1, Madagascar, McCoy, North Indian) [16–18]. This high genetic diversity, likely resulting from inbreeding among diverse P. vivax populations [19, 20], contributes to the parasite’s adaptability to various environments and efficient transmission. Consequently, studying P. vivax genetic diversity, especially in endemic regions, is essential. The presence of multiple P. vivax strains suggests frequent transmission by different infected mosquitoes or potential polyclonal infections. This genetic diversity can facilitate immune evasion, drug resistance, and adaptation to the host and vector, hindering vaccine development [17, 21]. Our findings corroborate previous research linking high P. vivax endemicity with increased genetic diversity [22]. The identified conditions, including a favourable environment for mosquito breeding and high transmission rates in September 2015, likely facilitated the spread of these diverse P. vivax strains.

Fig. 2.

PCR amplification of P. vivax samples by minisatellite marker CH1T1M13779. The samples showing base size polymorphisms on metaphor agarose gel (M: 20 bp DNA ladder marker, Sample: Lane 1 to 18, lane 15 showing 2 alleles in a single patient, lane 10 P. falciparum)

Fig. 3.

Village-wise distribution of the minisatellite marker CH1T1M13779 alleles. The bars in blue colour represents the alleles in Hathangaon village and grey colour bars represent the alleles from Raipuri village

As reported by Thapar et al. [6], the irrigation canal near Hathangaon village created a breeding ground for mosquitoes, leading to a significant increase in malaria cases. This emphasizes the indispensable role of water management in preventing mosquito-borne diseases. Man-made ecological disruptions, accelerated by infrastructure projects, have led to a proliferation of mosquito breeding sites across India. This, combined with climate change, poses a significant challenge to malaria control. To prevent outbreaks, particularly in areas with declining malaria immunity, proactive surveillance of breeding habitats near development zones is imperative. While adults often develop immunity to P. vivax malaria, children and adolescents remain highly vulnerable both in India and globally. This heightened risk is a complex interplay of biological and behavioural factors. Children and adolescents are more likely to spend time outdoors, with less caution about mosquito bites. Their developing immune systems are less equipped to combat the parasite, creating ideal conditions for its propagation. Severe complications, recurrent infections, and anaemia with long-term developmental consequences are common challenges faced by this age group. The lack of sensitive diagnostic tests specifically designed for children and appropriate paediatric drug formulations further hinder effective prevention and treatment, perpetuating the malaria cycle [23].

Conclusion

The crucial findings from our investigation were: (a) The minisatellite marker CH1T1M13779 emerged as a powerful tool for large-scale genotyping surveillance of P. vivax, essential for effectively understanding the future outbreaks; (b) Hathangaon village’s disproportionately high vivax malaria cases correlated with a significantly increased diversity of parasite strains [8] compared to Raipuri village [4] and (c) children and adolescents constitute a highly vulnerable population for P. vivax infection, with a striking 85% infection rate, aligning with previous reports of a high P. vivax burden in this age group in India [15, 24–29]. Our findings revealed a complex interplay of environmental, epidemiological, and genetic factors contributing to this outbreak. While increased breeding habitats and vector densities due to the irrigation canal likely exacerbated the situation, our genotyping analysis identified the preferential infection of children and adolescents, the presence of multiple P. vivax strains, and potential recombination as the primary drivers of the rapid case increase. We recognize that due to site-specific and seasonal focal transmission, only a small sample size could be analyzed, thereby limiting the implication in a larger population. However, these findings highlight the critical role of genetic factors in understanding and addressing malaria outbreaks, as well as the applicability of the inexpensive minisatellite marker CH1T1M13779 particularly in regions with atypical epidemiological patterns.

Author contributions

Conceptualization: RD Sample collection: RD, DS, VKT Data generation and analysis: RD, KV, JK, YG, AA, RH, SC Manuscript writing and review: RD, KV, KCP. All authors approved the contents of the manuscript.

Funding

The author Ram Das (RD) received the fund (No. ICMR/REC NO ECD/ad-hoc/38) for the research from the Indian Council of Medical Research (ICMR), India.

Data availability

The dataset(s) supporting the conclusions of this article are available upon suitable request made to the corresponding author.

Declarations

Ethical approval

Ethical approval for blood sample collection was obtained by the Institutional Ethics Committee (ECR/NIMR/2013/102) of the ICMR-National Institute of Malaria Research, New Delhi, India. Written informed consent was acquired from all participants, including legal guardians for minors.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the author(s) used Gemini from Google in order to improve the language presentation. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

Competing interests

The authors declare no competing interests.