Clonal expansion of Plasmodium falciparum and Plasmodium vivax during a malaria outbreak in the Bay Islands of Honduras, 2025

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As transmission declines, malaria increasingly occurs in focal outbreaks, particularly in geographically confined settings such as islands. Molecular surveillance can provide critical insights into parasite population structure during these events. In 2025, an outbreak of malaria occurred in the Bay Islands Department of Honduras. This study aimed to characterize the genetic diversity and clonal structure of Plasmodium falciparum and P. vivax circulating during this outbreak. Methods Malaria cases detected during the outbreak were confirmed by photo-induced electron transfer PCR (PET-PCR). Genetic diversity was assessed using targeted amplification and Sanger sequencing of polymorphic loci, including pfmsp1 and pfmdr1 for P. falciparum , and pvcsp and pvmsp3α for P. vivax. Haplotype diversity and population genetic parameters were estimated using DnaSP, and multilocus haplotypes were analysed to evaluate clonal structure and temporal dynamics during the outbreak. Results A total of 49 malaria cases were analysed, including 18 P. falciparum and 31 P. vivax infections across three municipalities. All P. falciparum isolates exhibited identical haplotypes across both analysed loci, indicating complete genetic homogeneity. In P. vivax , two haplotypes were identified at the pvcsp locus and two at pvmsp3α , with one dominant haplotype accounting for most infections. Cluster B was detected only during the early phase of the outbreak, while cluster A became predominant from epidemiological week 11 onward. Complete concordance between loci was observed across all successfully genotyped samples, and no mixed multilocus genotypes were detected. Conclusions The outbreak was characterized by marked genetic homogeneity in P. falciparum and restricted multilocus diversity in P. vivax , with temporal dominance of a single haplotype, consistent with clonal amplification within a geographically confined, low-transmission setting. These findings suggest that the outbreak likely resulted from local expansion of a limited number of parasite lineages rather than multiple independent introductions. Integrating molecular genotyping into routine surveillance may help distinguish local transmission from importation and strengthen malaria elimination strategies in Honduras and the wider Mesoamerican region. Malaria outbreak Honduras molecular markers Plasmodium vivax Plasmodium falciparum Bay Islands Figures Figure 1 Figure 2 Figure 3 Background Malaria transmission in Honduras has historically been concentrated in the eastern and north atlantic regions of the country, particularly in the department of Gracias a Dios [ 1 ]. However, in recent years, focal transmission has re-emerged in other regions, including the Bay Islands Department (Islas de la Bahía), an insular Caribbean territory with ecological conditions highly receptive to malaria transmission. This epidemiological context is particularly relevant given that countries in Central America and the island of Hispaniola have committed to eliminating malaria by 2030, in alignment with the World Health Organization’s Global Technical Strategy for Malaria 2016–2030 [ 2 ] and regional elimination initiatives. Sustained interruption of transmission in focal areas is therefore critical to achieving these regional targets. After several years of relatively low case numbers, a marked increase in malaria incidence was documented in the Bay Islands beginning in 2024, when 184 cases were reported—representing more than a 100% increase compared to 2022 and 2023, which each recorded only 32 cases. The upward trend continued into 2025 and by the end of that year (epidemiological week 53), a total of 239 confirmed malaria cases were documented in the Bay Islands, of which 209 (87%) were caused by Plasmodium vivax and 30 (13%) by Plasmodium falciparum . The vast majority of cases (95%) were classified as autochthonous, indicating sustained local transmission. Transmission was spatially heterogeneous across the department. The municipality of José Santos Guardiola (JSG) accounted for approximately 65% of all reported cases in 2025 (156/239), all attributable to P. vivax . Within JSG, the locality of The Bight represented the primary hotspot, concentrating more than half of the municipality’s cases (Fig. 1 ). Roatán contributed 26% of cases (61/239), with both P. vivax and P. falciparum infections reported, and Crawfish Rock identified as the most affected locality. In contrast, the municipality of Guanaja experienced a distinct outbreak exclusively due to P. falciparum , with 22 confirmed cases distributed across four localities, primarily Vista Hermosa, where transmission had not been reported for approximately a decade. The outbreak in Guanaja was first detected in epidemiological week 5 of 2025, when two autochthonous P. falciparum cases were identified through passive surveillance. Despite immediate implementation of reactive case detection, indoor residual spraying, supervised treatment, and vector control interventions, additional cases continued to occur through epidemiological week 36, suggesting sustained local transmission. The concurrence of geographically distinct transmission foci— P. vivax predominating in JSG and Roatán, and a localized P. falciparum outbreak in Guanaja—raised concerns regarding focal amplification and potential clonal expansion of parasite populations. In insular settings, restricted gene flow and ecological confinement may amplify founder effects, facilitating rapid expansion of a limited number of parasite lineages once transmission is re-established. In Honduras, progressive reductions in transmission over the past 25 years have been accompanied by a documented contraction in parasite genetic diversity, including evidence of a population bottleneck in P. falciparum along the Honduran–Nicaraguan border [ 3 – 7 ]. Such demographic constriction events reduce effective population size and may facilitate the expansion of a limited number of successful lineages when transmission resurges. In low-transmission and pre-elimination settings, apparent increases in case numbers may therefore reflect not only renewed exposure but also the amplification of genetically homogeneous parasite populations derived from a restricted ancestral pool [ 8 , 9 ]. Conventional epidemiological surveillance alone cannot distinguish between multiple independent introductions and sustained local transmission driven by clonal expansion. Molecular markers provide the necessary resolution to assess parasite population structure, quantify genetic diversity, and detect patterns of homogeneity consistent with recent expansion events [ 10 ]. Based on prior molecular surveillance experience in Honduras, four highly informative loci were selected for this study: pfmsp1 and pfmdr1 for P. falciparum [ 5 , 11 – 13 ], and pvcsp and pvmsp3α for P. vivax [ 5 , 7 , 12 , 13 ]. These markers have previously demonstrated strong discriminatory capacity in the national context, capturing both allelic family structure and SNP-defined haplotypes associated with lineage differentiation. While pfmsp1 and pvmsp3α serve as classical markers of genetic diversity and multiplicity of infection, pfmdr1 provides additional resolution at the haplotypic level relevant to lineage tracking, and pvcsp enables characterization of allelic types and repeat polymorphisms reflective of population structure. Together, these loci allow assessment of whether the 2025 outbreak was characterized by high diversity suggestive of multiple introductions or by reduced diversity indicative of genetic homogeneity and clonal expansion. Understanding whether the observed epidemiological pattern resulted from multiple introductions or from local expansion of a limited number of parasite genotypes is critical for guiding elimination strategies in insular and highly receptive settings. Therefore, this study aimed to investigate the molecular epidemiology of P. falciparum and P. vivax during the 2025 outbreak in the Bay Islands of Honduras, with particular emphasis on distinguishing between multiple introductions and local clonal amplification through temporal and multilocus genetic analysis. Methods Study area and sample collection This study was conducted in the Bay Islands Department of Honduras, including the municipalities of Roatán, JSG, and Guanaja (Fig. 1 ), where a malaria outbreak involving P. falciparum and P. vivax was reported in 2025. A total of 239 confirmed malaria cases were documented in the department. Of these, 49 (20.5%) malaria-positive blood samples collected between January and July 2025 were included for molecular analysis. These samples corresponded to the subset of confirmed cases for which dried blood spots on filter paper were available through routine malaria surveillance activities coordinated by the Honduran Ministry of Health. The samples originated from the three affected municipalities and were used for genetic characterization of circulating parasite populations. No additional selection criteria were applied beyond sample availability. For each case, a unique sample identification code was assigned, and epidemiological information including the municipality where the case was detected, the date of diagnostic reporting, and the infecting Plasmodium species was recorded. Cases were classified as Plasmodium vivax (Pv) or Plasmodium falciparum (Pf) based on molecular diagnosis. The inclusion of samples spanning multiple municipalities and several months allowed the assessment of spatial and temporal patterns of parasite circulation during the outbreak. Finger-prick blood samples were obtained from patients with malaria confirmed by thick blood smear microscopy and/or rapid diagnostic tests (RDTs). Blood was spotted onto Whatman® 3MM filter paper, air-dried, and individually stored in paper envelopes with silica desiccant at room temperature until processing. Epidemiological and basic demographic data, including date of sample collection, municipality of origin, patient age and sex, and Plasmodium species diagnosis, were recorded in a project-specific database. Samples were anonymized prior to molecular analysis. DNA extraction Genomic DNA was extracted from dried blood spots using two 2-mm diameter punches per sample. DNA extraction was performed using the Extracta® DNA Prep for PCR kit (QuantaBio, Beverly, MA, USA), following the manufacturer’s instructions. Extracted DNA was stored at − 20°C until further molecular analysis. Molecular marker amplification Molecular diagnosis of Plasmodium infection and species identification were confirmed using photo-induced electron transfer real-time PCR (PET-PCR) [ 14 – 16 ]. All DNA samples were first screened using a genus-specific PET-PCR assay targeting Plasmodium spp., and samples testing positive were subsequently analyzed with species-specific PET-PCR assays for P. falciparum and P. vivax . Reactions were run in duplicate using probe-based real-time PCR chemistry, with appropriate positive and no-template controls included in each run. To assess parasite genetic diversity and clonal structure, highly polymorphic molecular markers were selected for each species and amplified using conventional or nested PCR approaches. In P. falciparum –positive samples, fragments of the merozoite surface protein 1 ( pfmsp1 ) gene were amplified by nested PCR to discriminate the K1, MAD20, and RO33 allelic families [ 5 , 17 ]. In addition, two segments of the multidrug resistance gene 1 ( pfmdr1 ) encompassing codons 86, 184, 1034, and 1042 were amplified through nested PCR assays designed to generate fragments suitable for downstream sequencing [ 11 ]. For P. vivax , genetic diversity was assessed by amplifying fragments of the circumsporozoite protein ( pvcsp ) [ 5 ] and merozoite surface protein 3 alpha ( pvmsp3a ) genes [ 7 , 18 ]. The pvcsp marker allows discrimination between the major allelic variants VK210 and VK247, which differ in the amino acid repeat motifs within the central repeat region of the gene and are widely used to characterize parasite population structure and geographic distribution. PCR reactions were performed in a final volume of 50 µL containing template DNA and 10 µM primers (Table 1 ). Amplification of the pfmsp1 marker was carried out using the high-fidelity KOD DNA polymerase (Toyobo, Osaka, Japan), whereas all other molecular markers were amplified using 2× Taq DNA polymerase (Promega Corp., Madison, WI, USA), according to the corresponding amplification protocols. Amplification conditions followed previously published and validated protocols for malaria molecular surveillance in Central America [ 5 , 11 – 13 , 19 ]. All PCR runs included positive controls consisting of reference parasite DNA and negative controls (no-template controls) to monitor contamination. PCR products were visualized by agarose gel electrophoresis, purified, and subjected to Sanger sequencing. Raw chromatograms were inspected, edited, and assembled into consensus sequences using Geneious® software v. 2024.0.5. Multiple sequence alignments were generated using ClustalW to identify single nucleotide polymorphisms (SNPs), haplotypes, and sequence variation across samples, and inference of clonal structure. The resulting sequences were deposited in GenBank, and accession numbers were obtained. For the pvcsp marker, nucleotide sequences were translated in silico to amino acid sequences using the correct open reading frame (ORF) corresponding to the circumsporozoite protein coding region. Translation was performed to confirm sequence integrity and to characterize the composition and order of the nine–amino acid repeat motifs within the central repeat region. The resulting polypeptide sequences were examined to identify nonameric repeat patterns typical of VK210-type alleles and to detect amino acid substitutions among haplotypes. Repeat motif organization was compared across isolates to support haplotype classification at the protein level. Table 1 List of primers used for the amplification of molecular markers of Plasmodium vivax and P. falciparum. Target gene Reaction Primer name Primer sequence (5´- 3´) References 18Sr RNA gene PET-PCR for genus Plasmodium Genus forward GGC CTA ACA TGG CTA TGA CG [ 15 , 16 ] Genus reverse 6FAM-agg cgc ata gcg cct gg CTG CCT TCC TTA GAT GTG GTA GCT 18Sr RNA gene PET-PCR for P. falciparum Falciparum forward ACC CCT CGC CTG GTG TTT TT [ 15 ] Falciparum reverse HEX-agg cgc ata gcg cct gg TCG GGC CCC AAA AAT AGG AA 18Sr RNA gene PET-PCR for P. vivax Vivax forward ACT GAC ACT GAT GAT TTA GAA CCC ATT T [ 20 ] Vivax reverse HEX- agg cgc ata gcg cct ggT GGA GAG ATC TTT CCA TCC TAA ACC T AL5632 AGG AAT AAA CAA TAA AGA ACA TAA TCA TAC Pfmsp1 -K1 1st round MI-OF CTA GAA GCT TTA GAA GAT GCA GTA TTG [ 5 , 17 ] MI-OR CTT AAA TAG ATT CTA ATT CAA GTG GAT CA 2nd round K1F AAA TGA AGA AGA AAT TAC TAC AAA AGG TGC K1R GCT TGC ATC AGC TGG AGG GCT TGC ACC AGA pfmdr1 SNPs 86, 184 MDR1-1F TTA AAT GTT TAC CTG CAC AAC ATA GAA AAT T [ 11 ] MDR1-1R CTC CAC AAT AAC TTG CAA CAG TTC TTA MDR1-2F TGT ATG TGC TGT ATT ATC AGGA MDR1-2R CTC TTC TAT AAT GGA CAT GGTA pfmdr1 SNPs 1034, 1046 1042-A GTC GAA AAG ACT ATG AAA CGT AGA [ 11 ] 1042-C CTC AAA TGA TAA TTT TGC AT 1042-B GAT CCA AGT TTT TTA ATA CA 1042-C CTC AAA TGA TAA TTT TGC AT pvcsp 1st round VCS-OF ATG TAG ATC TGT CCA AGG CCA TAA A [ 5 ] VCS-OR TAA TTG AAT AAT GCT AGG ACT AAC AAT ATG 2nd round VCS-NF GCA GAA CCA AAA AAT CCA CGT GAA AAT AAG VCS-NR CCA ACG GTA GCT CTA ACT TTA TCT AGG TAT pvmsp3α 1st round Pvmsp3a P1 CAG CAG ACA CCA TTT AAG G [ 7 , 18 ] Pvmsp3a P2 CCG TTT GTT GAT TAG TTG C 2nd round Pvmsp3a N1 GAC CAG TGT GAT ACC ATT AAC C Pvmsp - 3a N2 ATA CTG GTT CTT CGT CTT CAG G Genetic diversity and clonal structure analysis Genetic diversity indices were estimated from aligned nucleotide sequences for each molecular marker. Multiple sequence alignments were generated using ClustalW implemented in Geneious® v. 2024.0.5. Estimates of the number of haplotypes (h), haplotype diversity (Hd), number of segregating sites (S), total number of mutations (Eta), average number of nucleotide differences (k), nucleotide diversity (π per site), and Watterson’s estimator (θ per site) were calculated using DnaSP v5 software. Neutrality tests, including Tajima’s D and Fu and Li’s D* and F* statistics, were also computed in DnaSP v5 to assess departures from neutral expectations consistent with demographic expansion or selection. Clonal expansion was operationally defined as the detection of identical haplotypes (100% sequence identity across the analysed fragment) in two or more epidemiologically independent malaria cases, and across all successfully genotyped loci for the corresponding species (Pf: pfmsp1 and pfmdr1 ; Pv: pvcsp and pvmsp3α ). Samples showing evidence of mixed infections, identified by multiple alleles or ambiguous Sanger sequencing chromatograms, were excluded from haplotype-based clustering analyses for the affected marker. Only unambiguous haplotypes were considered for clonal structure analyses. The assessment of multiplicity of infection (MOI) was not a primary objective of this study. Ethical considerations This study used anonymized samples collected as part of routine malaria surveillance activities conducted by the Honduran Ministry of Health. No personal identifiers were included in the molecular analyses. In accordance with national regulations governing public health surveillance activities, individual informed consent was not required for the use of these anonymized samples. The study protocol was reviewed and approved by the appropriate institutional and national ethical review committees, in accordance with national regulations and international ethical guidelines (approval PI-10-2025). Results Demographic and epidemiological characteristics of malaria cases in the Bay Islands A total of 49 malaria-positive cases from the Bay Islands Department were included in the analysis, comprising 20 cases from Roatán, 15 from José Santos Guardiola (JSG), and 14 from Guanaja (Table 2 ). Overall, cases were more frequent among males (34/49; 69.4%), with a comparable sex distribution across municipalities. The median age was 32.8 years (SD ± 18.4), with comparable age distributions among municipalities and a wide age range spanning from 2 to 80 years, indicating involvement of both paediatric and adult populations. Marked differences in Plasmodium species distribution were observed by municipality. Plasmodium vivax infections predominated in Roatán (16/20; 80%) and JSG (15/15; 100%), accounting for 31 cases overall (63.3%). In contrast, P. falciparum infections were concentrated in Guanaja (14/14; 100%), with only four cases detected in Roatán and none in JSG. Microscopy-based parasite counts indicated generally low to moderate parasite densities for P. vivax and low parasite densities for P. falciparum , consistent with a focal outbreak detected in a low-transmission setting (data not shown). Table 2 Demographic and clinical characteristics of malaria cases included in the molecular analysis in the Bay Islands Department, Honduras, 2025. The table summarizes the number of cases by municipality (Roatán, José Santos Guardiola [JSG], and Guanaja), sex distribution, median age with standard deviation (SD), age range, and Plasmodium species identified by molecular diagnosis. Percentages are calculated within each municipality. Variable Roatán JSG Guanaja Total n 20 15 14 49 Sex, n (%) • Male 13 (65%) 10 (66.7%) 11 (78.6%) 34 (69.4%) • Female 7 (35%) 5 (33.3%) 3 (21.4%) 15 (30.6%) Median age (IQR) ± SD 34.6 ± 16.32 33.1 ± 24.01 30.1 ± 15.5 32.8 ± 18.4 Age range (years) 4 to 66 8 to 80 2 to 52 2 to 80 Plasmodium species, n (%) • P. vivax 16 (80%) 15 (100%) 0 (0%) 31 (63.3%) • P. falciparum 4 (20%) 0 (0%) 14 (100%) 18 (36.7%) Molecular confirmation Molecular diagnosis by PET-PCR fully confirmed the results obtained by routine diagnostic methods. All 49 samples were positive by genus-specific PET-PCR, and species identification was concordant in all cases. No mixed-species infections were detected. This complete concordance provided a robust basis for downstream genetic analyses. Genetic diversity of P. falciparum P. falciparum merozoite surface protein 1 ( Pfmsp1 ) All 18 P. falciparum isolates yielded amplification exclusively of the K1 allelic family, producing an amplicon of approximately 200 bp. No MAD20 or RO33 allelic families were detected. Sequence analysis of a 178-bp fragment revealed complete identity among all isolates. Only a single haplotype was identified. One representative sequence was deposited in GenBank (accession PX955271). P. falciparum multidrug resistance gene 1 ( Pfmdr1 ) Two loci of the pfmdr1 gene were analysed. The first segment encompassed codons 86 and 184, while the second segment covered codons 1034 and 1042, all of which are relevant molecular markers associated with antimalarial drug resistance. For the first segment, a 465-base-pair sequence was obtained and found to be identical across all 18 P. falciparum isolates analysed. This sequence corresponded to the N86/184F haplotype. No sequence polymorphisms were observed among the samples. One representative sequence was deposited in GenBank under accession number PX955272. For the second segment, a 285-base-pair sequence was obtained and was likewise identical across all 18 isolates analysed. This sequence corresponded to the 1034C/D1042 haplotype, with no sequence polymorphisms detected among the samples. One representative sequence was deposited in GenBank under accession number PX955273. Overall, a single haplotype (N86/184F/1034C/D1042) was identified for each P. falciparum molecular marker analysed, with no detectable sequence variation among isolates. This pattern is consistent with low genetic diversity and is compatible with a focal outbreak involving limited parasite lineages. Genetic diversity of P. vivax isolates A total of 31 P. vivax isolates were analysed using the pvcsp and pvmsp3α markers. P. vivax circumsporozoite protein (pvcsp) Thirty-one sequences were successfully obtained for pvcsp . Two haplotypes (clusters A and B) were identified. Cluster A predominated (26/31; 83.9%), while cluster B was detected in 5/31 isolates (16.1%). Cluster B was observed during the early phase of transmission (epidemiological weeks 1–5), primarily in Roatán (Table 3 ). From week 6 onward, cluster A became dominant and remained prevalent throughout the study period. No mixed infections were detected. Table 3 Temporal distribution and genotyping results of Plasmodium vivax samples collected in the Bay Islands Department, Honduras, 2025. The table shows the municipality of origin (Roatán and José Santos Guardiola [JSG]), date of collection, corresponding epidemiological week, and allelic clustering results for the pvcsp and pvmsp3α markers. Clusters (A and B) were assigned based on sequence similarity and phylogenetic grouping. n Sample code Municipality Date Epi Week Cluster PvCSP Cluster PvMSP3α 1 42 Roatán January 4 1 B B 2 37 Roatán January 20 4 B B 3 38 Roatán January 20 4 B B 4 39 JSG January 24 4 A A 5 43 Roatán January 27 5 B B 6 45 JSG January 28 5 A A 7 44 Roatán January 29 5 A A 8 46 JSG February 3 6 A A 9 47 JSG February 7 6 A A 10 49 JSG February 9 7 A A 11 55 JSG March 13 11 A A 12 57 Roatán March 20 12 B B 13 56 JSG March 20 12 A A 14 59 JSG March 27 13 A A 15 60 JSG March 31 14 A A 16 72 JSG April 8 15 A A 17 78 Roatán April 9 15 A A 18 73 JSG April 10 15 A A 19 74 JSG April 11 15 A A 20 75 JSG April 14 16 A A 21 76 JSG April 14 16 A A 22 79 Roatán April 15 16 A A 23 80 Roatán April 15 16 A A 24 81 Roatán April 28 18 A A 25 82 Roatán April 29 18 A A 26 93 Roatán May 14 20 A A 27 94 Roatán May 14 20 A A 28 95 Roatán May 14 20 A A 29 107 Roatán June 9 24 A A 30 109 Roatán July 7 28 A A 31 110 JSG July 11 28 A A Sequence analysis of the 657-bp pvcsp alignment identified four segregating sites (S = 4) and two haplotypes (h = 2). Haplotype diversity was low (Hd = 0.2615), and nucleotide diversity was minimal (π = 0.00207), with an average number of nucleotide differences of k = 1.046. Watterson’s estimator per site was θ = 0.00186. Tajima’s D was positive but not statistically significant (D = 0.2685, P > 0.10), and Fu and Li’s D* (1.0269) and F* (0.9305) were likewise non-significant (P > 0.10), suggesting no strong deviation from neutral expectations at this locus. The marked predominance of a single haplotype and the low nucleotide diversity are consistent with restricted allelic variation and temporal structuring during the outbreak period. Representative sequences were deposited in GenBank under accession numbers PZ005775 (cluster A) and PZ005774 (cluster B). The schematic representation of the PvCSP central repeat region (Fig. 2 ) illustrates the organization of the nine–amino acid repeat motifs defining clusters A and B. Both allelic variants shared the canonical repeat structure characteristic of VK210-type sequences, with variation restricted to specific repeat units. The principal difference between clusters was localized to the antepenultimate nonapeptide repeat, where a non-synonymous substitution was identified (red arrow). This amino acid change resulted in a distinct repeat motif in cluster B compared to cluster A, while the remaining repeat blocks were conserved between variants. The limited amino acid divergence and the preservation of the overall repeat architecture further support the close genetic relatedness of the two haplotypes and are consistent with recent divergence from a common ancestral lineage during the outbreak period. P. vivax merozoite protein 3 alpha (pvmsp3α) Analysis of the 761-bp pvmsp3α alignment revealed 37 segregating sites (S = 37) and two haplotypes (h = 2). Despite the presence of multiple polymorphic sites, haplotype diversity was low (Hd = 0.1000), reflecting the predominance of a single haplotype. Nucleotide diversity was π = 0.01101, with an average of k = 3.70 nucleotide differences. Watterson’s estimator per site was θ = 0.03104. Tajima’s D was strongly negative and statistically significant (D = − 2.5603, P < 0.001). Fu and Li’s D* (− 3.9974, P < 0.02) and F* (− 4.1572, P < 0.02) were also significantly negative. This pattern is compatible with recent population expansion from a limited ancestral pool or purifying selection acting on the locus. Despite the nucleotide-level variation and the presence of multiple segregating sites, these polymorphisms were largely restricted to a single dominant haplotype, consistent with recent expansion rather than long-term diversification. Representative sequences were deposited under accession numbers PX955277 (cluster A) and PX955276 (cluster B). Multilocus structure and clonal patterns in P. vivax Combined analysis of pvcsp and pvmsp3α revealed that the predominant multilocus haplotype corresponded to cluster A at both loci (A/A). From epidemiological week 11 onward, nearly all successfully genotyped infections exhibited this multilocus profile across both Roatán and JSG (Fig. 3 ). Cluster B was detected only during the early outbreak phase and did not reappear in later weeks. No evidence of novel haplotypes emerged during the peak transmission period (weeks 12–18) or in the late phase (weeks 20–28). Concordance between pvcsp and pvmsp3α was complete (100%), with identical cluster assignment across both loci in all samples successfully genotyped at both markers. No mixed genotypes were detected. Overall, the outbreak exhibited complete genetic homogeneity in P. falciparum and markedly restricted haplotypic diversity in P. vivax , characterized by temporal replacement of an early cluster B by cluster A and sustained predominance of a single multilocus genotype during peak transmission. Although nucleotide-level variation was detected in pvmsp3α, the overwhelming dominance of one multilocus lineage supports a genetically constrained parasite population structure throughout the outbreak period. Discussion The molecular analyses conducted during the 2025 malaria outbreak in the Bay Islands, Honduras, revealed a strikingly restricted genetic profile among circulating parasites. Plasmodium falciparum isolates exhibited complete haplotypic uniformity across all loci analysed, while P. vivax populations were characterized by only two closely related clusters, with clear temporal structuring and complete inter-locus concordance. Together, these findings indicate markedly reduced allelic diversity and a simplified parasite population structure within a geographically confined setting. In a region committed to malaria elimination by 2030 [ 2 ], such patterns must be interpreted within the broader demographic and epidemiological context of declining transmission and focal persistence. Across Mesoamerica and Hispaniola, sustained reductions in malaria incidence over the past two decades [ 21 ] have led to increasingly focal transmission, with cases concentrated in residual hotspots rather than widespread endemicity. As transmission intensity declines, parasite populations contract, recombination opportunities decrease, and infections become more spatially structured [ 3 , 22 – 25 ]. Under such conditions, small transmission foci may amplify rapidly from a limited ancestral pool, particularly in ecologically receptive areas [ 24 , 26 , 27 ]. Insular transmission systems such as the Bay Islands may be especially vulnerable to these dynamics, as restricted gene flow and ecological confinement can magnify founder effects once transmission is re-established [ 28 ]. Thus, the genetic homogeneity observed during the outbreak is consistent with expectations for malaria resurgence in low-transmission, geographically constrained environments. Importantly, the 2025 outbreak did not emerge on a genetically diverse background. Previous studies in Honduras have consistently documented progressive contraction of parasite genetic diversity during the elimination phase, including reduced multiplicity of infection, dominance of specific haplotypes, and evidence of demographic bottlenecks in P. falciparum populations along the Honduran–Nicaraguan border [ 3 – 7 , 12 , 13 ]. The persistence of homogeneous pfcrt and pfmdr1 profiles, limited allelic family diversity in pfmsp1 , and reduced haplotypic richness in P. vivax markers have collectively indicated shrinking effective parasite population size. In this context, the Bay Islands outbreak appears to represent amplification of a previously restricted parasite reservoir rather than emergence from a highly heterogeneous transmission network. The predominance of a single multilocus genotype in P. falciparum and the near-fixation of cluster A in P. vivax are therefore best interpreted as expansion from a constrained ancestral pool. When placed in a global comparative framework, the genetic profile observed in the Bay Islands contrasts sharply with patterns reported from moderate- to high-transmission settings. In sub-Saharan Africa, multiplicity of infection frequently exceeds 2, polyclonal infections may surpass 50%, and high allelic richness in markers such as pfmsp1 reflects sustained recombination and intense transmission [ 29 , 30 ]. Similarly, in parts of Southeast Asia with ongoing but unstable transmission, pvcsp and pvmsp3α loci often display substantial haplotypic diversity and multiple co-circulating genotypes [ 31 , 32 ]. Such scenarios are incompatible with the extreme monomorphism observed in P. falciparum and the limited haplotypic spectrum detected in P. vivax in this study. In contrast, the Bay Islands outbreak closely resembles patterns described in pre-elimination or post-interruption contexts characterized by focal clonal amplification. In Suriname, consecutive P. vivax outbreaks in previously controlled communities were driven by persistence and expansion of a single lineage [ 26 ]. In eastern Panama, epidemic resurgence of P. falciparum was associated with highly related parasite subpopulations and reduced recombination [ 24 ]. Similar clonal signatures have been reported in insular or geographically constrained settings such as Cape Verde, where outbreak isolates shared > 99% SNP similarity [ 27 ], and in southern Thailand, where limited pvcsp diversity was associated with localized transmission [ 33 ]. These observations support the interpretation that reduced transmission intensity and geographic restriction favor inbreeding, linkage disequilibrium, and expansion of a limited number of successful parasite lineages. The epidemiological interpretation of the Bay Islands outbreak is further clarified by the multilocus structure observed. For P. falciparum , complete monomorphism across pfmsp1 and pfmdr1 , coupled with identical haplotypes in all analysed isolates, is compatible with either a single introduction followed by local amplification or resurgence from a persistent low-level reservoir. The absence of allelic family diversity and the uniform drug-resistance haplotype argue against multiple independent introductions during the outbreak period. For P. vivax , the presence of two closely related clusters and the temporal replacement of cluster B by cluster A during early epidemiological weeks suggest limited initial diversity followed by selective or demographic dominance of a single lineage. Complete concordance between pvcsp and pvmsp3α across all successfully genotyped samples further reinforces the inference of restricted recombination and limited parasite mixing. Notably, no novel haplotypes emerged during peak transmission, and no mixed multilocus genotypes were detected, findings that collectively support local amplification rather than repeated importation. Spatially, the predominance of cluster A across both Roatán and José Santos Guardiola and the genetic uniformity of P. falciparum in Guanaja suggest largely independent focal dynamics within the department. The P. falciparum outbreak in Guanaja, restricted to a single multilocus genotype, appears epidemiologically distinct from the P. vivax transmission patterns observed in the other municipalities. This compartmentalization is consistent with structured transmission in geographically discrete settings and parallels observations from Mexico and Panama, where significant genetic differentiation has been documented between adjacent residual foci [ 34 , 35 ]. Together, these findings argue more strongly for focal amplification of limited lineages than for widespread connectivity or multiple contemporaneous introductions. The programmatic implications of these findings are substantial. In elimination settings, conventional surveillance metrics such as case counts and incidence rates cannot distinguish between importation-driven outbreaks and local clonal amplification. Molecular genotyping provides critical resolution to disentangle these scenarios. As emphasized in regional experiences from Costa Rica and other countries [ 36 , 37 ], routine integration of parasite genotyping into surveillance systems enhances outbreak attribution and guides targeted interventions. In insular contexts such as the Bay Islands, where ecological receptivity remains high and vector species such as Anopheles albimanus persist [ 38 ], rapid identification of genetically homogeneous outbreaks may facilitate focused containment before expansion occurs. Conversely, failure to detect early clonal amplification may allow rapid spread from a single introduced or persistent lineage. Overall, the 2025 Bay Islands outbreak illustrates how malaria resurgence in low-transmission settings may be driven by amplification of genetically constrained parasite populations rather than by repeated independent introductions. The observed homogeneity in P. falciparum and the restricted multilocus diversity in P. vivax are consistent with expansion from a reduced ancestral pool in a geographically confined environment. These findings underscore the importance of sustained molecular surveillance during the elimination phase and highlight the vulnerability of insular transmission systems to founder-driven outbreaks. Integrating multilocus genotyping into routine surveillance frameworks will be essential to differentiate local transmission from importation and to support evidence-based elimination strategies in Honduras and across the Mesoamerican region. Conclusions The 2025 malaria outbreak in the Bay Islands of Honduras was characterized by marked genetic homogeneity, with complete monomorphism in Plasmodium falciparum and restricted multilocus diversity in P. vivax , including temporal dominance of a single haplotype. These findings are consistent with clonal amplification within a geographically confined, low-transmission setting rather than with multiple independent parasite introductions. In the context of sustained transmission reduction and prior evidence of demographic contraction in Honduras, the outbreak appears to have emerged from a genetically constrained ancestral pool. Our results highlight the value of multilocus molecular surveillance for distinguishing local amplification from importation in elimination settings and underscore the vulnerability of insular transmission systems to founder-driven outbreaks. Integrating routine genotyping into malaria surveillance frameworks will be essential to support evidence-based decision-making and to safeguard progress toward regional elimination goals by 2030. Limitations This study has several limitations. First, genotyping was based on targeted amplification and Sanger sequencing of selected polymorphic loci rather than whole-genome sequencing, which may limit resolution for detecting very recent micro-divergence events. In addition, Sanger-based approaches primarily capture dominant haplotypes and may underestimate low-frequency subclonal variants in mixed infections. However, no ambiguous chromatograms suggestive of multiclonal infections were observed, and complete multilocus concordance across successfully genotyped samples supports the inference of restricted recombination during the outbreak period. The number of samples analysed represented a subset of confirmed cases and was constrained by sample availability through routine surveillance. Nonetheless, the consistent detection of identical haplotypes across municipalities and over several epidemiological weeks indicates that the observed genetic homogeneity reflects a true biological pattern rather than sampling artefact. Future studies incorporating genome-wide approaches and broader temporal sampling would further refine understanding of parasite population dynamics in insular elimination settings. Abbreviations ACT Artemisinin-based combination therapy bp Base pairs CIG-UNAH Centro de Investigaciones Genéticas – Universidad Nacional Autónoma de Honduras D* Fu and Li’s D* statistic Eta Total number of mutations F* Fu and Li’s F* statistic Hd Haplotype diversity JSG José Santos Guardiola k Average number of nucleotide differences MAD20 Merozoite surface protein 1 allelic family MAD20 MOI Multiplicity of infection ORF Open reading frame PCR Polymerase chain reaction PET-PCR Photo-induced electron transfer polymerase chain reaction Pf Plasmodium falciparum pfcrt Plasmodium falciparum chloroquine resistance transporter gene pfmdr1 Plasmodium falciparum multidrug resistance gene 1 pfmsp1 Plasmodium falciparum merozoite surface protein 1 Pv Plasmodium vivax pvcsp Plasmodium vivax circumsporozoite protein gene pvmsp3α Plasmodium vivax merozoite surface protein 3 alpha gene RDT Rapid diagnostic test S Number of segregating sites SD Standard deviation SNP Single nucleotide polymorphism θ Watterson’s estimator π Nucleotide diversity VK210 Plasmodium vivax circumsporozoite protein VK210 variant VK247 Plasmodium vivax circumsporozoite protein VK247 variant Declarations Ethics approval and consent to participate . This study used anonymized samples collected as part of routine malaria surveillance activities conducted by the Honduran Ministry of Health. No personal identifiers were included in the molecular analyses. In accordance with national regulations governing public health surveillance activities, individual informed consent was not required for the use of these anonymized samples. The study protocol was reviewed and approved by the appropriate institutional and national ethical review committees, in accordance with national regulations and international ethical guidelines (approval PI-10-2025). Consent for publication . Not applicable. Competing interests. One of the authors of this manuscript (H.O.V.) is an employee of the U.S. Government. This work was prepared as part of his official duties. Title 17 U.S.C. § 105 provides that “Copyright protection under this Title is not available for any work of the United States Government”. Title 17 U.S.C. § 101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person’s official duties. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government. Funding. Funding for this study was provided by the Genetic Research Center, CIG-UNAH, and by the Armed Forces Health Surveillance Division (AFHSD), Global Emerging Infections Surveillance (GEIS) Branch (PROMIS ID P0146_25_N6). The funders had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript. The APC was funded by DICIHT-UNAH. Author Contribution GF: Conceptualization, Methodology validation, Formal analysis, Data curation, Visualization, Writing – original draft, Supervision, Project administration. FP, DE, GA, LC: Methodology, Investigation, Writing – review and editing. HOV, GF: Resources, Funding acquisition. LC, GA, AM, DO, MC: Epidemiological surveillance and field follow-up of the malaria outbreak in the Bay Islands. DE, GF: Project administration. All authors read and approved the final manuscript. Acknowledgements. Not applicable. Availability of data and materials. All nucleotide sequences generated in this study have been deposited in GenBank under the accession numbers reported in the Results section. Additional data supporting the findings of this study are available from the corresponding author upon reasonable request. References Subvención Malaria del. 1 de enero del 2024 al 31 de diciembre de 2025 [ https://mcp-honduras.hn/malaria/] World Health organization. Global technical strategy and targets for malaria 2016–2030. Geneva: WHO; 2015. Pinto A, Archaga O, Mejia A, Escober L, Henriquez J, Montoya A, Valdivia HO, Fontecha G. 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Molecular diagnosis of malaria by photo-induced electron transfer fluorogenic primers: PET-PCR. PLoS ONE. 2013;8:e56677. Matamoros G, Escobar D, Pinto A, Serrano D, Ksandrova E, Grimaldi N, Juarez-Fontecha G, Moncada M, Valdivia HO, Fontecha G. PET-PCR reveals low parasitaemia and submicroscopic malarial infections in Honduran Moskitia. Malar J. 2023;22:110. Snounou G, Zhu X, Siripoon N, Jarra W, Thaithong S, Brown KN, Viriyakosol S. Biased distribution of msp1 and msp2 allelic variants in Plasmodium falciparum populations in Thailand. Trans R Soc Trop Med Hyg. 1999;93:369–74. Thanapongpichat S, Khammanee T, Sawangjaroen N, Buncherd H, Tun AW. Genetic Diversity of Plasmodium vivax in Clinical Isolates from Southern Thailand using PvMSP1, PvMSP3 (PvMSP3alpha, PvMSP3beta) Genes and Eight Microsatellite Markers. Korean J Parasitol. 2019;57:469–79. Jovel IT, Mejia RE, Banegas E, Piedade R, Alger J, Fontecha G, Ferreira PE, Veiga MI, Enamorado IG, Bjorkman A, Ursing J. 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Genetic polymorphism of merozoite surface protein 1 and merozoite surface protein 2 in the Vietnam Plasmodium falciparum population. BMC Infect Dis. 2024;24:1216. Obaldia N 3rd, Baro NK, Calzada JE, Santamaria AM, Daniels R, Wong W, Chang HH, Hamilton EJ, Arevalo-Herrera M, Herrera S, et al. Clonal outbreak of Plasmodium falciparum infection in eastern Panama. J Infect Dis. 2015;211:1087–96. Saenz FE, Morton LC, Okoth SA, Valenzuela G, Vera-Arias CA, Velez-Alvarez E, Lucchi NW, Castro LE, Udhayakumar V. Clonal population expansion in an outbreak of Plasmodium falciparum on the northwest coast of Ecuador. Malar J. 2015;13(1):497. Labadie-Bracho MY, Adhin MR. Reconstruction of Plasmodium vivax outbreaks in a low malaria endemic setting utilizing conventional restriction fragment length polymorphism. Int J Mol Epidemiol Genet. 2021;12:9–15. Da Veiga Leal S, Ward D, Campino S, Benavente ED, Ibrahim A, Claret T, Isaias V, Monteiro D, Clark TG, Goncalves L, et al. Drug resistance profile and clonality of Plasmodium falciparum parasites in Cape Verde: the 2017 malaria outbreak. Malar J. 2021;20:172. Huang B, Tuo F, Liang Y, Wu W, Wu G, Huang S, Zhong Q, Su XZ, Zhang H, Li M, et al. Temporal changes in genetic diversity of msp-1, msp-2, and msp-3 in Plasmodium falciparum isolates from Grande Comore Island after introduction of ACT. Malar J. 2018;17:83. Simpson SV, Nundu SS, Arima H, Kaneko O, Mita T, Culleton R, Yamamoto T. The diversity of Plasmodium falciparum isolates from asymptomatic and symptomatic school-age children in Kinshasa Province, Democratic Republic of Congo. Malar J. 2023;22:102. Andika B, Mobegi V, Gathii K, Nyataya J, Maina N, Awinda G, Mutai B, Waitumbi J. Plasmodium falciparum population structure inferred by msp1 amplicon sequencing of parasites collected from febrile patients in Kenya. Malar J. 2023;22:263. Vo TC, Trinh NTM, Le HG, Kang JM, Yoo WG, Quang HH, Na BK. Genetic Diversity of Circumsporozoite Surface Protein of Plasmodium vivax from the Central Highlands. Vietnam Pathogens 2022, 11. Hayat C, Kamil A, Khan A, Sayed A, Akbar K, Afridi SG. Genetic diversity of Plasmodium falciparum and Plasmodium vivax field isolates from the Nowshera district of Pakistan. Malar J. 2024;23:358. Khulmanee T, Thita T, Kritsiriwutinan K, Boonyuen U, Saai A, Inkabjan K, Chakrabarti R, Rathod PK, Krudsood S, Mungthin M, Patrapuvich R. Low Genetic Diversity of Plasmodium vivax Circumsporozoite Surface Protein in Clinical Isolates from Southern Thailand. Trop Med Infect Dis 2024, 9. Santamaria AM, Vasquez V, Rigg C, Samudio F, Moreno D, Romero L, Saldana A, Chaves LF, Calzada JE. Plasmodium vivax Genetic Diversity in Panama: Challenges for Malaria Elimination in Mesoamerica. Pathogens 2021, 10. Gonzalez-Ceron L, Gomez-Perez DJ, Santillan-Valenzuela F, Ovilla-Munoz M, Guzman-Bracho C, Pech-May A, Amores GR, Montoya-Perez A, Villarreal-Trevino C. Population Dynamics of Plasmodium vivax in Mexico Determined by CSP, Pvs25, and SSU 18S rRNA S-Type Polymorphism Analyses. Microorganisms 2025, 13. Chaves LF, Huber JH, Rojas Salas O, Ramirez Rojas M, Romero LM, Gutierrez Alvarado JM, Perkins TA, Prado M, Rodriguez RM. Malaria Elimination in Costa Rica: Changes in Treatment and Mass Drug Administration. Microorganisms 2020, 8. Labadie-Bracho MY, Adhin MR. Advocating for PCR-RFLP as molecular tool within malaria programs in low endemic areas and low resource settings. PLoS Negl Trop Dis. 2023;17:e0011747. Escobar D, Archaga O, Reyes A, Palma A, Larson RT, Vasquez GM, Fontecha G. A Follow-Up to the Geographical Distribution of Anopheles Species in Malaria-Endemic and Non-Endemic Areas of Honduras. Insects 2022, 13. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 12 May, 2026 Reviews received at journal 12 May, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers invited by journal 21 Apr, 2026 Editor assigned by journal 08 Apr, 2026 Submission checks completed at journal 08 Apr, 2026 First submitted to journal 07 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9347818","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":628021606,"identity":"55892712-bbd0-4f9f-b406-ceb7c243ce9f","order_by":0,"name":"Lesly Chaver","email":"","orcid":"","institution":"Laboratorio Nacional de Vigilancia de Malaria, Secretaría de Salud de Honduras","correspondingAuthor":false,"prefix":"","firstName":"Lesly","middleName":"","lastName":"Chaver","suffix":""},{"id":628021607,"identity":"c5f9010f-c67a-4c3e-b2fd-fd21a3e9597a","order_by":1,"name":"Gloria Ardón","email":"","orcid":"","institution":"Laboratorio Nacional de Vigilancia de Malaria, Secretaría de Salud de Honduras","correspondingAuthor":false,"prefix":"","firstName":"Gloria","middleName":"","lastName":"Ardón","suffix":""},{"id":628021608,"identity":"587bc403-5903-45cb-8e81-23d5fc3308dc","order_by":2,"name":"Amed Matute","email":"","orcid":"","institution":"Secretaría de Salud de Honduras","correspondingAuthor":false,"prefix":"","firstName":"Amed","middleName":"","lastName":"Matute","suffix":""},{"id":628021609,"identity":"5348e08d-5cf2-4eeb-a73d-781fa1934b66","order_by":3,"name":"Dámaris Ortez","email":"","orcid":"","institution":"Secretaría de Salud de Honduras","correspondingAuthor":false,"prefix":"","firstName":"Dámaris","middleName":"","lastName":"Ortez","suffix":""},{"id":628021610,"identity":"46a95c1f-0266-4859-a03f-17a0686aa0ed","order_by":4,"name":"Mitzi Castro","email":"","orcid":"","institution":"Laboratorio Nacional de Vigilancia de Malaria, Secretaría de Salud de Honduras","correspondingAuthor":false,"prefix":"","firstName":"Mitzi","middleName":"","lastName":"Castro","suffix":""},{"id":628021611,"identity":"01a44756-95c4-4034-a8e6-770dc2f27a28","order_by":5,"name":"Fernando Pérez","email":"","orcid":"","institution":"Universidad Nacional Autónoma de Honduras","correspondingAuthor":false,"prefix":"","firstName":"Fernando","middleName":"","lastName":"Pérez","suffix":""},{"id":628021612,"identity":"3f9bf61e-03a8-4ec3-b9fe-eb4742336999","order_by":6,"name":"Denis Escobar","email":"","orcid":"","institution":"Universidad Nacional Autónoma de Honduras","correspondingAuthor":false,"prefix":"","firstName":"Denis","middleName":"","lastName":"Escobar","suffix":""},{"id":628021613,"identity":"142b264c-e50b-4f6a-a071-cf7c02987b89","order_by":7,"name":"Hugo O. Valdivia","email":"","orcid":"","institution":"U.S. Naval Medical Research Unit SOUTH (NAMRU SOUTH)","correspondingAuthor":false,"prefix":"","firstName":"Hugo","middleName":"O.","lastName":"Valdivia","suffix":""},{"id":628021614,"identity":"b1ba08eb-ed10-4d13-830c-13bfa4b33328","order_by":8,"name":"Gustavo Fontecha","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABJUlEQVRIiWNgGAWjYDAD9gYwdYCBH0gyAzFjAwMPLsWMYMU8B8CMAwySDSRrMThAQIt8RPrzBz/bbBh42HuPP/hQc0fe+Ebuwc8FDDayGw7wHnuARYvhjRzDxt62NAYennOJjTOOPTPcdiMvWXoGQ5rxhgN86QbYtMzIYWzgOXOYwV4ix7CZt+Ew47YbOQbSPAyHEzcc4DGTwKol/WHjH6AWHpCWvw2H7TfPyDH+zcPwH6cWeYkEw2aeCqgWxgag4RI5ZkBbDuDUYsDzxnC2TEUaDw/PGcOZPccOJ88488bMmscg2XjmYRy2tKc/+PjGwEaOh73H4MOPmsO2/e05xrd5Kuxk+473YLflAIRGjwJQUDFjUQ+ypQG7+CgYBaNgFIwCBAAAlFRoFdv0xHEAAAAASUVORK5CYII=","orcid":"","institution":"Universidad Nacional Autónoma de Honduras","correspondingAuthor":true,"prefix":"","firstName":"Gustavo","middleName":"","lastName":"Fontecha","suffix":""}],"badges":[],"createdAt":"2026-04-07 16:39:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9347818/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9347818/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108389885,"identity":"afc59111-69e0-4222-a39c-3e80da11b55c","added_by":"auto","created_at":"2026-05-04 06:52:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2328875,"visible":true,"origin":"","legend":"\u003cp\u003eGeographic distribution of malaria-positive samples in the Bay Islands Department, Honduras. Pie charts represent the proportion of \u003cem\u003eP. vivax\u003c/em\u003eand \u003cem\u003eP. falciparum\u003c/em\u003e infections by municipality (Roatán, José Santos Guardiola (JSG), and Guanaja), with chart size proportional to the total number of samples analysed per municipality. The highlighted localities indicate the three areas reporting the highest number of cases within each municipality: The Bight (JSG), Crawfish Rock (Roatán), and Vista Hermosa (Guanaja). Insets show the location of the Bay Islands within Honduras and the regional position of Honduras in Central America. El mapa de las islas de la Bahia fue tomado de Google Maps. The map of the Bay Islands was obtained from Google Maps.\u003c/p\u003e","description":"","filename":"Figure1Hondurasmap.png","url":"https://assets-eu.researchsquare.com/files/rs-9347818/v1/be9ec40f0734e413251bb11e.png"},{"id":108492492,"identity":"cde23aa5-ce18-4515-aee8-d772f6c79d8c","added_by":"auto","created_at":"2026-05-05 09:57:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":324957,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the nine–amino acid repeat motifs of the two PvCSP allelic variants. Each color represents a distinct repeat motif. Red arrow indicates the site at which a non-synonymous mutation was identified.\u003c/p\u003e","description":"","filename":"Figure2.PvCSP.png","url":"https://assets-eu.researchsquare.com/files/rs-9347818/v1/52264518a0e63956f6fd76f2.png"},{"id":108389887,"identity":"a7d69262-ff9a-42db-9d43-adcdb4051259","added_by":"auto","created_at":"2026-05-04 06:52:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":589750,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal distribution of malaria cases by epidemiological week in the Bay Islands Department, Honduras, 2025. Square plots depict the occurrence of \u003cem\u003ePlasmodium vivax\u003c/em\u003e (Pv) and \u003cem\u003eP. falciparum \u003c/em\u003e(Pf) cases by epidemiological week (x-axis) and parasite species (y-axis), stratified by municipality (Guanaja, José Santos Guardiola, and Roatán). Each square represents one or more malaria cases confirmed by molecular diagnosis, with square size proportional to the number of cases reported per epidemiological week. For \u003cem\u003eP. vivax\u003c/em\u003einfections, the letters A and B shown below the squares indicate the haplotype (cluster A or B) identified based on \u003cem\u003epvcsp\u003c/em\u003e and \u003cem\u003epvmsp3α\u003c/em\u003egenotyping. Weeks without reported cases are shown to illustrate gaps in transmission during the study period.\u003c/p\u003e","description":"","filename":"Figure3.Outbreak.png","url":"https://assets-eu.researchsquare.com/files/rs-9347818/v1/14cf1b1e8233b7bcb15b794a.png"},{"id":108804262,"identity":"cbc14045-ec1f-412a-920d-a506e7522329","added_by":"auto","created_at":"2026-05-08 15:18:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3296879,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9347818/v1/6f19dec8-17e1-4970-a609-1fe4ea720811.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Clonal expansion of Plasmodium falciparum and Plasmodium vivax during a malaria outbreak in the Bay Islands of Honduras, 2025","fulltext":[{"header":"Background","content":"\u003cp\u003eMalaria transmission in Honduras has historically been concentrated in the eastern and north atlantic regions of the country, particularly in the department of Gracias a Dios [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, in recent years, focal transmission has re-emerged in other regions, including the Bay Islands Department (Islas de la Bah\u0026iacute;a), an insular Caribbean territory with ecological conditions highly receptive to malaria transmission. This epidemiological context is particularly relevant given that countries in Central America and the island of Hispaniola have committed to eliminating malaria by 2030, in alignment with the World Health Organization\u0026rsquo;s Global Technical Strategy for Malaria 2016\u0026ndash;2030 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and regional elimination initiatives. Sustained interruption of transmission in focal areas is therefore critical to achieving these regional targets.\u003c/p\u003e \u003cp\u003eAfter several years of relatively low case numbers, a marked increase in malaria incidence was documented in the Bay Islands beginning in 2024, when 184 cases were reported\u0026mdash;representing more than a 100% increase compared to 2022 and 2023, which each recorded only 32 cases. The upward trend continued into 2025 and by the end of that year (epidemiological week 53), a total of 239 confirmed malaria cases were documented in the Bay Islands, of which 209 (87%) were caused by \u003cem\u003ePlasmodium vivax\u003c/em\u003e and 30 (13%) by \u003cem\u003ePlasmodium falciparum\u003c/em\u003e. The vast majority of cases (95%) were classified as autochthonous, indicating sustained local transmission.\u003c/p\u003e \u003cp\u003eTransmission was spatially heterogeneous across the department. The municipality of Jos\u0026eacute; Santos Guardiola (JSG) accounted for approximately 65% of all reported cases in 2025 (156/239), all attributable to \u003cem\u003eP. vivax\u003c/em\u003e. Within JSG, the locality of The Bight represented the primary hotspot, concentrating more than half of the municipality\u0026rsquo;s cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Roat\u0026aacute;n contributed 26% of cases (61/239), with both \u003cem\u003eP. vivax\u003c/em\u003e and \u003cem\u003eP. falciparum\u003c/em\u003e infections reported, and Crawfish Rock identified as the most affected locality. In contrast, the municipality of Guanaja experienced a distinct outbreak exclusively due to \u003cem\u003eP. falciparum\u003c/em\u003e, with 22 confirmed cases distributed across four localities, primarily Vista Hermosa, where transmission had not been reported for approximately a decade. The outbreak in Guanaja was first detected in epidemiological week 5 of 2025, when two autochthonous \u003cem\u003eP. falciparum\u003c/em\u003e cases were identified through passive surveillance. Despite immediate implementation of reactive case detection, indoor residual spraying, supervised treatment, and vector control interventions, additional cases continued to occur through epidemiological week 36, suggesting sustained local transmission.\u003c/p\u003e \u003cp\u003eThe concurrence of geographically distinct transmission foci\u0026mdash;\u003cem\u003eP. vivax\u003c/em\u003e predominating in JSG and Roat\u0026aacute;n, and a localized \u003cem\u003eP. falciparum\u003c/em\u003e outbreak in Guanaja\u0026mdash;raised concerns regarding focal amplification and potential clonal expansion of parasite populations. In insular settings, restricted gene flow and ecological confinement may amplify founder effects, facilitating rapid expansion of a limited number of parasite lineages once transmission is re-established. In Honduras, progressive reductions in transmission over the past 25 years have been accompanied by a documented contraction in parasite genetic diversity, including evidence of a population bottleneck in \u003cem\u003eP. falciparum\u003c/em\u003e along the Honduran\u0026ndash;Nicaraguan border [\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Such demographic constriction events reduce effective population size and may facilitate the expansion of a limited number of successful lineages when transmission resurges.\u003c/p\u003e \u003cp\u003eIn low-transmission and pre-elimination settings, apparent increases in case numbers may therefore reflect not only renewed exposure but also the amplification of genetically homogeneous parasite populations derived from a restricted ancestral pool [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Conventional epidemiological surveillance alone cannot distinguish between multiple independent introductions and sustained local transmission driven by clonal expansion. Molecular markers provide the necessary resolution to assess parasite population structure, quantify genetic diversity, and detect patterns of homogeneity consistent with recent expansion events [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on prior molecular surveillance experience in Honduras, four highly informative loci were selected for this study: \u003cem\u003epfmsp1\u003c/em\u003e and \u003cem\u003epfmdr1\u003c/em\u003e for \u003cem\u003eP. falciparum\u003c/em\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and \u003cem\u003epvcsp\u003c/em\u003e and \u003cem\u003epvmsp3α\u003c/em\u003e for \u003cem\u003eP. vivax\u003c/em\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These markers have previously demonstrated strong discriminatory capacity in the national context, capturing both allelic family structure and SNP-defined haplotypes associated with lineage differentiation. While \u003cem\u003epfmsp1\u003c/em\u003e and \u003cem\u003epvmsp3α\u003c/em\u003e serve as classical markers of genetic diversity and multiplicity of infection, \u003cem\u003epfmdr1\u003c/em\u003e provides additional resolution at the haplotypic level relevant to lineage tracking, and \u003cem\u003epvcsp\u003c/em\u003e enables characterization of allelic types and repeat polymorphisms reflective of population structure. Together, these loci allow assessment of whether the 2025 outbreak was characterized by high diversity suggestive of multiple introductions or by reduced diversity indicative of genetic homogeneity and clonal expansion.\u003c/p\u003e \u003cp\u003eUnderstanding whether the observed epidemiological pattern resulted from multiple introductions or from local expansion of a limited number of parasite genotypes is critical for guiding elimination strategies in insular and highly receptive settings. Therefore, this study aimed to investigate the molecular epidemiology of \u003cem\u003eP. falciparum\u003c/em\u003e and \u003cem\u003eP. vivax\u003c/em\u003e during the 2025 outbreak in the Bay Islands of Honduras, with particular emphasis on distinguishing between multiple introductions and local clonal amplification through temporal and multilocus genetic analysis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area and sample collection\u003c/h2\u003e \u003cp\u003eThis study was conducted in the Bay Islands Department of Honduras, including the municipalities of Roat\u0026aacute;n, JSG, and Guanaja (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), where a malaria outbreak involving \u003cem\u003eP. falciparum\u003c/em\u003e and \u003cem\u003eP. vivax\u003c/em\u003e was reported in 2025. A total of 239 confirmed malaria cases were documented in the department. Of these, 49 (20.5%) malaria-positive blood samples collected between January and July 2025 were included for molecular analysis. These samples corresponded to the subset of confirmed cases for which dried blood spots on filter paper were available through routine malaria surveillance activities coordinated by the Honduran Ministry of Health. The samples originated from the three affected municipalities and were used for genetic characterization of circulating parasite populations. No additional selection criteria were applied beyond sample availability.\u003c/p\u003e \u003cp\u003eFor each case, a unique sample identification code was assigned, and epidemiological information including the municipality where the case was detected, the date of diagnostic reporting, and the infecting \u003cem\u003ePlasmodium\u003c/em\u003e species was recorded. Cases were classified as \u003cem\u003ePlasmodium vivax\u003c/em\u003e (Pv) or \u003cem\u003ePlasmodium falciparum\u003c/em\u003e (Pf) based on molecular diagnosis. The inclusion of samples spanning multiple municipalities and several months allowed the assessment of spatial and temporal patterns of parasite circulation during the outbreak.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinger-prick blood samples were obtained from patients with malaria confirmed by thick blood smear microscopy and/or rapid diagnostic tests (RDTs). Blood was spotted onto Whatman\u0026reg; 3MM filter paper, air-dried, and individually stored in paper envelopes with silica desiccant at room temperature until processing. Epidemiological and basic demographic data, including date of sample collection, municipality of origin, patient age and sex, and \u003cem\u003ePlasmodium\u003c/em\u003e species diagnosis, were recorded in a project-specific database. Samples were anonymized prior to molecular analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA extraction\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from dried blood spots using two 2-mm diameter punches per sample. DNA extraction was performed using the Extracta\u0026reg; DNA Prep for PCR kit (QuantaBio, Beverly, MA, USA), following the manufacturer\u0026rsquo;s instructions. Extracted DNA was stored at \u0026minus;\u0026thinsp;20\u0026deg;C until further molecular analysis.\u003c/p\u003e\n\u003ch3\u003eMolecular marker amplification\u003c/h3\u003e\n\u003cp\u003eMolecular diagnosis of \u003cem\u003ePlasmodium\u003c/em\u003e infection and species identification were confirmed using photo-induced electron transfer real-time PCR (PET-PCR) [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. All DNA samples were first screened using a genus-specific PET-PCR assay targeting \u003cem\u003ePlasmodium\u003c/em\u003e spp., and samples testing positive were subsequently analyzed with species-specific PET-PCR assays for \u003cem\u003eP. falciparum\u003c/em\u003e and \u003cem\u003eP. vivax\u003c/em\u003e. Reactions were run in duplicate using probe-based real-time PCR chemistry, with appropriate positive and no-template controls included in each run.\u003c/p\u003e \u003cp\u003eTo assess parasite genetic diversity and clonal structure, highly polymorphic molecular markers were selected for each species and amplified using conventional or nested PCR approaches. In \u003cem\u003eP. falciparum\u003c/em\u003e\u0026ndash;positive samples, fragments of the merozoite surface protein 1 (\u003cem\u003epfmsp1\u003c/em\u003e) gene were amplified by nested PCR to discriminate the K1, MAD20, and RO33 allelic families [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In addition, two segments of the multidrug resistance gene 1 (\u003cem\u003epfmdr1\u003c/em\u003e) encompassing codons 86, 184, 1034, and 1042 were amplified through nested PCR assays designed to generate fragments suitable for downstream sequencing [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. For \u003cem\u003eP. vivax\u003c/em\u003e, genetic diversity was assessed by amplifying fragments of the circumsporozoite protein (\u003cem\u003epvcsp\u003c/em\u003e) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and merozoite surface protein 3 alpha (\u003cem\u003epvmsp3a\u003c/em\u003e) genes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The \u003cem\u003epvcsp\u003c/em\u003e marker allows discrimination between the major allelic variants VK210 and VK247, which differ in the amino acid repeat motifs within the central repeat region of the gene and are widely used to characterize parasite population structure and geographic distribution.\u003c/p\u003e \u003cp\u003ePCR reactions were performed in a final volume of 50 \u0026micro;L containing template DNA and 10 \u0026micro;M primers (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Amplification of the \u003cem\u003epfmsp1\u003c/em\u003e marker was carried out using the high-fidelity KOD DNA polymerase (Toyobo, Osaka, Japan), whereas all other molecular markers were amplified using 2\u0026times; Taq DNA polymerase (Promega Corp., Madison, WI, USA), according to the corresponding amplification protocols. Amplification conditions followed previously published and validated protocols for malaria molecular surveillance in Central America [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. All PCR runs included positive controls consisting of reference parasite DNA and negative controls (no-template controls) to monitor contamination.\u003c/p\u003e \u003cp\u003ePCR products were visualized by agarose gel electrophoresis, purified, and subjected to Sanger sequencing. Raw chromatograms were inspected, edited, and assembled into consensus sequences using Geneious\u0026reg; software v. 2024.0.5. Multiple sequence alignments were generated using ClustalW to identify single nucleotide polymorphisms (SNPs), haplotypes, and sequence variation across samples, and inference of clonal structure. The resulting sequences were deposited in GenBank, and accession numbers were obtained.\u003c/p\u003e \u003cp\u003eFor the \u003cem\u003epvcsp\u003c/em\u003e marker, nucleotide sequences were translated in silico to amino acid sequences using the correct open reading frame (ORF) corresponding to the circumsporozoite protein coding region. Translation was performed to confirm sequence integrity and to characterize the composition and order of the nine\u0026ndash;amino acid repeat motifs within the central repeat region. The resulting polypeptide sequences were examined to identify nonameric repeat patterns typical of VK210-type alleles and to detect amino acid substitutions among haplotypes. Repeat motif organization was compared across isolates to support haplotype classification at the protein level.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of primers used for the amplification of molecular markers of \u003cem\u003ePlasmodium vivax\u003c/em\u003e and \u003cem\u003eP. falciparum.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget gene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReaction\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrimer name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePrimer sequence (5\u0026acute;- 3\u0026acute;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReferences\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18Sr RNA gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePET-PCR for genus \u003cem\u003ePlasmodium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGenus forward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGGC CTA ACA TGG CTA TGA CG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGenus reverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6FAM-agg cgc ata gcg cct gg CTG CCT TCC TTA GAT GTG GTA GCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18Sr RNA gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePET-PCR for \u003cem\u003eP. falciparum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFalciparum forward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eACC CCT CGC CTG GTG TTT TT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFalciparum reverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHEX-agg cgc ata gcg cct gg TCG GGC CCC AAA AAT AGG AA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18Sr RNA gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePET-PCR for \u003cem\u003eP. vivax\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVivax forward\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eACT GAC ACT GAT GAT TTA GAA CCC ATT T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVivax reverse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHEX- agg cgc ata gcg cct ggT GGA GAG ATC TTT CCA TCC TAA ACC T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAL5632\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAGG AAT AAA CAA TAA AGA ACA TAA TCA TAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePfmsp1\u003c/em\u003e-K1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1st round\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMI-OF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCTA GAA GCT TTA GAA GAT GCA GTA TTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMI-OR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCTT AAA TAG ATT CTA ATT CAA GTG GAT CA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2nd round\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK1F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAAA TGA AGA AGA AAT TAC TAC AAA AGG TGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK1R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGCT TGC ATC AGC TGG AGG GCT TGC ACC AGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003epfmdr1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSNPs 86, 184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMDR1-1F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTTA AAT GTT TAC CTG CAC AAC ATA GAA AAT T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMDR1-1R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCTC CAC AAT AAC TTG CAA CAG TTC TTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMDR1-2F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTGT ATG TGC TGT ATT ATC AGGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMDR1-2R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCTC TTC TAT AAT GGA CAT GGTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003epfmdr1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSNPs 1034, 1046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1042-A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGTC GAA AAG ACT ATG AAA CGT AGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1042-C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCTC AAA TGA TAA TTT TGC AT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1042-B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGAT CCA AGT TTT TTA ATA CA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1042-C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCTC AAA TGA TAA TTT TGC AT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003epvcsp\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1st round\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVCS-OF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eATG TAG ATC TGT CCA AGG CCA TAA A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVCS-OR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTAA TTG AAT AAT GCT AGG ACT AAC AAT ATG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2nd round\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVCS-NF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGCA GAA CCA AAA AAT CCA CGT GAA AAT AAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVCS-NR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCCA ACG GTA GCT CTA ACT TTA TCT AGG TAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003epvmsp3α\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1st round\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePvmsp3a P1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCAG CAG ACA CCA TTT AAG G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePvmsp3a P2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCCG TTT GTT GAT TAG TTG C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2nd round\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePvmsp3a N1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGAC CAG TGT GAT ACC ATT AAC C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePvmsp\u003cb\u003e-\u003c/b\u003e3a N2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eATA CTG GTT CTT CGT CTT CAG G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eGenetic diversity and clonal structure analysis\u003c/h3\u003e\n\u003cp\u003eGenetic diversity indices were estimated from aligned nucleotide sequences for each molecular marker. Multiple sequence alignments were generated using ClustalW implemented in Geneious\u0026reg; v. 2024.0.5. Estimates of the number of haplotypes (h), haplotype diversity (Hd), number of segregating sites (S), total number of mutations (Eta), average number of nucleotide differences (k), nucleotide diversity (π per site), and Watterson\u0026rsquo;s estimator (θ per site) were calculated using DnaSP v5 software. Neutrality tests, including Tajima\u0026rsquo;s D and Fu and Li\u0026rsquo;s D* and F* statistics, were also computed in DnaSP v5 to assess departures from neutral expectations consistent with demographic expansion or selection.\u003c/p\u003e \u003cp\u003eClonal expansion was operationally defined as the detection of identical haplotypes (100% sequence identity across the analysed fragment) in two or more epidemiologically independent malaria cases, and across all successfully genotyped loci for the corresponding species (Pf: \u003cem\u003epfmsp1\u003c/em\u003e and \u003cem\u003epfmdr1\u003c/em\u003e; Pv: \u003cem\u003epvcsp\u003c/em\u003e and \u003cem\u003epvmsp3α\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eSamples showing evidence of mixed infections, identified by multiple alleles or ambiguous Sanger sequencing chromatograms, were excluded from haplotype-based clustering analyses for the affected marker. Only unambiguous haplotypes were considered for clonal structure analyses. The assessment of multiplicity of infection (MOI) was not a primary objective of this study.\u003c/p\u003e\n\u003ch3\u003eEthical considerations\u003c/h3\u003e\n\u003cp\u003eThis study used anonymized samples collected as part of routine malaria surveillance activities conducted by the Honduran Ministry of Health. No personal identifiers were included in the molecular analyses. In accordance with national regulations governing public health surveillance activities, individual informed consent was not required for the use of these anonymized samples. The study protocol was reviewed and approved by the appropriate institutional and national ethical review committees, in accordance with national regulations and international ethical guidelines (approval PI-10-2025).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDemographic and epidemiological characteristics of malaria cases in the Bay Islands\u003c/h2\u003e \u003cp\u003eA total of 49 malaria-positive cases from the Bay Islands Department were included in the analysis, comprising 20 cases from Roat\u0026aacute;n, 15 from Jos\u0026eacute; Santos Guardiola (JSG), and 14 from Guanaja (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Overall, cases were more frequent among males (34/49; 69.4%), with a comparable sex distribution across municipalities. The median age was 32.8 years (SD\u0026thinsp;\u0026plusmn;\u0026thinsp;18.4), with comparable age distributions among municipalities and a wide age range spanning from 2 to 80 years, indicating involvement of both paediatric and adult populations.\u003c/p\u003e \u003cp\u003eMarked differences in \u003cem\u003ePlasmodium\u003c/em\u003e species distribution were observed by municipality. \u003cem\u003ePlasmodium vivax\u003c/em\u003e infections predominated in Roat\u0026aacute;n (16/20; 80%) and JSG (15/15; 100%), accounting for 31 cases overall (63.3%). In contrast, \u003cem\u003eP. falciparum\u003c/em\u003e infections were concentrated in Guanaja (14/14; 100%), with only four cases detected in Roat\u0026aacute;n and none in JSG. Microscopy-based parasite counts indicated generally low to moderate parasite densities for \u003cem\u003eP. vivax\u003c/em\u003e and low parasite densities for \u003cem\u003eP. falciparum\u003c/em\u003e, consistent with a focal outbreak detected in a low-transmission setting (data not shown).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic and clinical characteristics of malaria cases included in the molecular analysis in the Bay Islands Department, Honduras, 2025. The table summarizes the number of cases by municipality (Roat\u0026aacute;n, Jos\u0026eacute; Santos Guardiola [JSG], and Guanaja), sex distribution, median age with standard deviation (SD), age range, and \u003cem\u003ePlasmodium\u003c/em\u003e species identified by molecular diagnosis. Percentages are calculated within each municipality.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJSG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGuanaja\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026bull; Male\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13 (65%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (66.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11 (78.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34 (69.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026bull; Female\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (35%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (33.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (21.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15 (30.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedian age (IQR)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34.6\u0026thinsp;\u0026plusmn;\u0026thinsp;16.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.1\u0026thinsp;\u0026plusmn;\u0026thinsp;24.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.1\u0026thinsp;\u0026plusmn;\u0026thinsp;15.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32.8\u0026thinsp;\u0026plusmn;\u0026thinsp;18.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge range (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 to 66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 to 80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 to 52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 to 80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePlasmodium\u003c/em\u003e\u0026nbsp;species, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026bull;\u0026nbsp;\u003cem\u003eP. vivax\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16 (80%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31 (63.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026bull;\u0026nbsp;\u003cem\u003eP. falciparum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 (36.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular confirmation\u003c/h3\u003e\n\u003cp\u003eMolecular diagnosis by PET-PCR fully confirmed the results obtained by routine diagnostic methods. All 49 samples were positive by genus-specific PET-PCR, and species identification was concordant in all cases. No mixed-species infections were detected. This complete concordance provided a robust basis for downstream genetic analyses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic diversity of\u003c/b\u003e \u003cb\u003eP. falciparum\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. falciparum\u003c/em\u003e merozoite surface protein 1 (\u003cem\u003ePfmsp1\u003c/em\u003e)\u003c/p\u003e \u003cp\u003eAll 18 \u003cem\u003eP. falciparum\u003c/em\u003e isolates yielded amplification exclusively of the K1 allelic family, producing an amplicon of approximately 200 bp. No MAD20 or RO33 allelic families were detected. Sequence analysis of a 178-bp fragment revealed complete identity among all isolates. Only a single haplotype was identified. One representative sequence was deposited in GenBank (accession PX955271).\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. falciparum\u003c/em\u003e multidrug resistance gene 1 (\u003cem\u003ePfmdr1\u003c/em\u003e)\u003c/p\u003e \u003cp\u003eTwo loci of the \u003cem\u003epfmdr1\u003c/em\u003e gene were analysed. The first segment encompassed codons 86 and 184, while the second segment covered codons 1034 and 1042, all of which are relevant molecular markers associated with antimalarial drug resistance. For the first segment, a 465-base-pair sequence was obtained and found to be identical across all 18 \u003cem\u003eP. falciparum\u003c/em\u003e isolates analysed. This sequence corresponded to the N86/184F haplotype. No sequence polymorphisms were observed among the samples. One representative sequence was deposited in GenBank under accession number PX955272.\u003c/p\u003e \u003cp\u003eFor the second segment, a 285-base-pair sequence was obtained and was likewise identical across all 18 isolates analysed. This sequence corresponded to the 1034C/D1042 haplotype, with no sequence polymorphisms detected among the samples. One representative sequence was deposited in GenBank under accession number PX955273.\u003c/p\u003e \u003cp\u003eOverall, a single haplotype (N86/184F/1034C/D1042) was identified for each \u003cem\u003eP. falciparum\u003c/em\u003e molecular marker analysed, with no detectable sequence variation among isolates. This pattern is consistent with low genetic diversity and is compatible with a focal outbreak involving limited parasite lineages.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic diversity of\u003c/b\u003e \u003cb\u003eP. vivax\u003c/b\u003e \u003cb\u003eisolates\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 31 \u003cem\u003eP. vivax\u003c/em\u003e isolates were analysed using the \u003cem\u003epvcsp\u003c/em\u003e and \u003cem\u003epvmsp3α\u003c/em\u003e markers.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003eP. vivax\u003c/em\u003e circumsporozoite protein \u003cem\u003e(pvcsp)\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThirty-one sequences were successfully obtained for \u003cem\u003epvcsp\u003c/em\u003e. Two haplotypes (clusters A and B) were identified. Cluster A predominated (26/31; 83.9%), while cluster B was detected in 5/31 isolates (16.1%). Cluster B was observed during the early phase of transmission (epidemiological weeks 1\u0026ndash;5), primarily in Roat\u0026aacute;n (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). From week 6 onward, cluster A became dominant and remained prevalent throughout the study period. No mixed infections were detected.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTemporal distribution and genotyping results of \u003cem\u003ePlasmodium vivax\u003c/em\u003e samples collected in the Bay Islands Department, Honduras, 2025. The table shows the municipality of origin (Roat\u0026aacute;n and Jos\u0026eacute; Santos Guardiola [JSG]), date of collection, corresponding epidemiological week, and allelic clustering results for the pvcsp and pvmsp3α markers. Clusters (A and B) were assigned based on sequence similarity and phylogenetic grouping.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample code\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMunicipality\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEpi Week\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCluster PvCSP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCluster PvMSP3α\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJanuary 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJanuary 20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJanuary 20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJSG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJanuary 24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJanuary 27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJSG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJanuary 28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJanuary 29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJSG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFebruary 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJSG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFebruary 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJSG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFebruary 9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJSG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMarch 13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMarch 20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJSG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMarch 20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJSG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMarch 27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJSG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMarch 31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJSG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eApril 8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eApril 9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJSG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eApril 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJSG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eApril 11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJSG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eApril 14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJSG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eApril 14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eApril 15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eApril 15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eApril 28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eApril 29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMay 14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMay 14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMay 14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJune 9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoat\u0026aacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJuly 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJSG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJuly 11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSequence analysis of the 657-bp \u003cem\u003epvcsp\u003c/em\u003e alignment identified four segregating sites (S\u0026thinsp;=\u0026thinsp;4) and two haplotypes (h\u0026thinsp;=\u0026thinsp;2). Haplotype diversity was low (Hd\u0026thinsp;=\u0026thinsp;0.2615), and nucleotide diversity was minimal (π\u0026thinsp;=\u0026thinsp;0.00207), with an average number of nucleotide differences of k\u0026thinsp;=\u0026thinsp;1.046. Watterson\u0026rsquo;s estimator per site was θ\u0026thinsp;=\u0026thinsp;0.00186. Tajima\u0026rsquo;s D was positive but not statistically significant (D\u0026thinsp;=\u0026thinsp;0.2685, P\u0026thinsp;\u0026gt;\u0026thinsp;0.10), and Fu and Li\u0026rsquo;s D* (1.0269) and F* (0.9305) were likewise non-significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.10), suggesting no strong deviation from neutral expectations at this locus. The marked predominance of a single haplotype and the low nucleotide diversity are consistent with restricted allelic variation and temporal structuring during the outbreak period. Representative sequences were deposited in GenBank under accession numbers PZ005775 (cluster A) and PZ005774 (cluster B).\u003c/p\u003e \u003cp\u003eThe schematic representation of the PvCSP central repeat region (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) illustrates the organization of the nine\u0026ndash;amino acid repeat motifs defining clusters A and B. Both allelic variants shared the canonical repeat structure characteristic of VK210-type sequences, with variation restricted to specific repeat units. The principal difference between clusters was localized to the antepenultimate nonapeptide repeat, where a non-synonymous substitution was identified (red arrow). This amino acid change resulted in a distinct repeat motif in cluster B compared to cluster A, while the remaining repeat blocks were conserved between variants. The limited amino acid divergence and the preservation of the overall repeat architecture further support the close genetic relatedness of the two haplotypes and are consistent with recent divergence from a common ancestral lineage during the outbreak period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003eP. vivax\u003c/em\u003e merozoite protein 3 alpha \u003cem\u003e(pvmsp3α)\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eAnalysis of the 761-bp \u003cem\u003epvmsp3α\u003c/em\u003e alignment revealed 37 segregating sites (S\u0026thinsp;=\u0026thinsp;37) and two haplotypes (h\u0026thinsp;=\u0026thinsp;2). Despite the presence of multiple polymorphic sites, haplotype diversity was low (Hd\u0026thinsp;=\u0026thinsp;0.1000), reflecting the predominance of a single haplotype. Nucleotide diversity was π\u0026thinsp;=\u0026thinsp;0.01101, with an average of k\u0026thinsp;=\u0026thinsp;3.70 nucleotide differences. Watterson\u0026rsquo;s estimator per site was θ\u0026thinsp;=\u0026thinsp;0.03104. Tajima\u0026rsquo;s D was strongly negative and statistically significant (D\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;2.5603, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Fu and Li\u0026rsquo;s D* (\u0026minus;\u0026thinsp;3.9974, P\u0026thinsp;\u0026lt;\u0026thinsp;0.02) and F* (\u0026minus;\u0026thinsp;4.1572, P\u0026thinsp;\u0026lt;\u0026thinsp;0.02) were also significantly negative. This pattern is compatible with recent population expansion from a limited ancestral pool or purifying selection acting on the locus.\u003c/p\u003e \u003cp\u003eDespite the nucleotide-level variation and the presence of multiple segregating sites, these polymorphisms were largely restricted to a single dominant haplotype, consistent with recent expansion rather than long-term diversification. Representative sequences were deposited under accession numbers PX955277 (cluster A) and PX955276 (cluster B).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMultilocus structure and clonal patterns in\u003c/b\u003e \u003cb\u003eP. vivax\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCombined analysis of \u003cem\u003epvcsp\u003c/em\u003e and \u003cem\u003epvmsp3α\u003c/em\u003e revealed that the predominant multilocus haplotype corresponded to cluster A at both loci (A/A). From epidemiological week 11 onward, nearly all successfully genotyped infections exhibited this multilocus profile across both Roat\u0026aacute;n and JSG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCluster B was detected only during the early outbreak phase and did not reappear in later weeks. No evidence of novel haplotypes emerged during the peak transmission period (weeks 12\u0026ndash;18) or in the late phase (weeks 20\u0026ndash;28). Concordance between \u003cem\u003epvcsp\u003c/em\u003e and \u003cem\u003epvmsp3α\u003c/em\u003e was complete (100%), with identical cluster assignment across both loci in all samples successfully genotyped at both markers. No mixed genotypes were detected.\u003c/p\u003e \u003cp\u003eOverall, the outbreak exhibited complete genetic homogeneity in \u003cem\u003eP. falciparum\u003c/em\u003e and markedly restricted haplotypic diversity in \u003cem\u003eP. vivax\u003c/em\u003e, characterized by temporal replacement of an early cluster B by cluster A and sustained predominance of a single multilocus genotype during peak transmission. Although nucleotide-level variation was detected in pvmsp3α, the overwhelming dominance of one multilocus lineage supports a genetically constrained parasite population structure throughout the outbreak period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe molecular analyses conducted during the 2025 malaria outbreak in the Bay Islands, Honduras, revealed a strikingly restricted genetic profile among circulating parasites. \u003cem\u003ePlasmodium falciparum\u003c/em\u003e isolates exhibited complete haplotypic uniformity across all loci analysed, while \u003cem\u003eP. vivax\u003c/em\u003e populations were characterized by only two closely related clusters, with clear temporal structuring and complete inter-locus concordance. Together, these findings indicate markedly reduced allelic diversity and a simplified parasite population structure within a geographically confined setting. In a region committed to malaria elimination by 2030 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], such patterns must be interpreted within the broader demographic and epidemiological context of declining transmission and focal persistence.\u003c/p\u003e \u003cp\u003eAcross Mesoamerica and Hispaniola, sustained reductions in malaria incidence over the past two decades [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] have led to increasingly focal transmission, with cases concentrated in residual hotspots rather than widespread endemicity. As transmission intensity declines, parasite populations contract, recombination opportunities decrease, and infections become more spatially structured [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Under such conditions, small transmission foci may amplify rapidly from a limited ancestral pool, particularly in ecologically receptive areas [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Insular transmission systems such as the Bay Islands may be especially vulnerable to these dynamics, as restricted gene flow and ecological confinement can magnify founder effects once transmission is re-established [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Thus, the genetic homogeneity observed during the outbreak is consistent with expectations for malaria resurgence in low-transmission, geographically constrained environments.\u003c/p\u003e \u003cp\u003eImportantly, the 2025 outbreak did not emerge on a genetically diverse background. Previous studies in Honduras have consistently documented progressive contraction of parasite genetic diversity during the elimination phase, including reduced multiplicity of infection, dominance of specific haplotypes, and evidence of demographic bottlenecks in \u003cem\u003eP. falciparum\u003c/em\u003e populations along the Honduran\u0026ndash;Nicaraguan border [\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The persistence of homogeneous \u003cem\u003epfcrt\u003c/em\u003e and \u003cem\u003epfmdr1\u003c/em\u003e profiles, limited allelic family diversity in \u003cem\u003epfmsp1\u003c/em\u003e, and reduced haplotypic richness in \u003cem\u003eP. vivax\u003c/em\u003e markers have collectively indicated shrinking effective parasite population size. In this context, the Bay Islands outbreak appears to represent amplification of a previously restricted parasite reservoir rather than emergence from a highly heterogeneous transmission network. The predominance of a single multilocus genotype in \u003cem\u003eP. falciparum\u003c/em\u003e and the near-fixation of cluster A in \u003cem\u003eP. vivax\u003c/em\u003e are therefore best interpreted as expansion from a constrained ancestral pool.\u003c/p\u003e \u003cp\u003eWhen placed in a global comparative framework, the genetic profile observed in the Bay Islands contrasts sharply with patterns reported from moderate- to high-transmission settings. In sub-Saharan Africa, multiplicity of infection frequently exceeds 2, polyclonal infections may surpass 50%, and high allelic richness in markers such as \u003cem\u003epfmsp1\u003c/em\u003e reflects sustained recombination and intense transmission [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Similarly, in parts of Southeast Asia with ongoing but unstable transmission, \u003cem\u003epvcsp\u003c/em\u003e and \u003cem\u003epvmsp3α\u003c/em\u003e loci often display substantial haplotypic diversity and multiple co-circulating genotypes [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Such scenarios are incompatible with the extreme monomorphism observed in \u003cem\u003eP. falciparum\u003c/em\u003e and the limited haplotypic spectrum detected in \u003cem\u003eP. vivax\u003c/em\u003e in this study.\u003c/p\u003e \u003cp\u003eIn contrast, the Bay Islands outbreak closely resembles patterns described in pre-elimination or post-interruption contexts characterized by focal clonal amplification. In Suriname, consecutive \u003cem\u003eP. vivax\u003c/em\u003e outbreaks in previously controlled communities were driven by persistence and expansion of a single lineage [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In eastern Panama, epidemic resurgence of \u003cem\u003eP. falciparum\u003c/em\u003e was associated with highly related parasite subpopulations and reduced recombination [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Similar clonal signatures have been reported in insular or geographically constrained settings such as Cape Verde, where outbreak isolates shared\u0026thinsp;\u0026gt;\u0026thinsp;99% SNP similarity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and in southern Thailand, where limited \u003cem\u003epvcsp\u003c/em\u003e diversity was associated with localized transmission [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These observations support the interpretation that reduced transmission intensity and geographic restriction favor inbreeding, linkage disequilibrium, and expansion of a limited number of successful parasite lineages.\u003c/p\u003e \u003cp\u003eThe epidemiological interpretation of the Bay Islands outbreak is further clarified by the multilocus structure observed. For \u003cem\u003eP. falciparum\u003c/em\u003e, complete monomorphism across \u003cem\u003epfmsp1\u003c/em\u003e and \u003cem\u003epfmdr1\u003c/em\u003e, coupled with identical haplotypes in all analysed isolates, is compatible with either a single introduction followed by local amplification or resurgence from a persistent low-level reservoir. The absence of allelic family diversity and the uniform drug-resistance haplotype argue against multiple independent introductions during the outbreak period. For \u003cem\u003eP. vivax\u003c/em\u003e, the presence of two closely related clusters and the temporal replacement of cluster B by cluster A during early epidemiological weeks suggest limited initial diversity followed by selective or demographic dominance of a single lineage. Complete concordance between \u003cem\u003epvcsp\u003c/em\u003e and \u003cem\u003epvmsp3α\u003c/em\u003e across all successfully genotyped samples further reinforces the inference of restricted recombination and limited parasite mixing. Notably, no novel haplotypes emerged during peak transmission, and no mixed multilocus genotypes were detected, findings that collectively support local amplification rather than repeated importation.\u003c/p\u003e \u003cp\u003eSpatially, the predominance of cluster A across both Roat\u0026aacute;n and Jos\u0026eacute; Santos Guardiola and the genetic uniformity of \u003cem\u003eP. falciparum\u003c/em\u003e in Guanaja suggest largely independent focal dynamics within the department. The \u003cem\u003eP. falciparum\u003c/em\u003e outbreak in Guanaja, restricted to a single multilocus genotype, appears epidemiologically distinct from the \u003cem\u003eP. vivax\u003c/em\u003e transmission patterns observed in the other municipalities. This compartmentalization is consistent with structured transmission in geographically discrete settings and parallels observations from Mexico and Panama, where significant genetic differentiation has been documented between adjacent residual foci [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Together, these findings argue more strongly for focal amplification of limited lineages than for widespread connectivity or multiple contemporaneous introductions.\u003c/p\u003e \u003cp\u003eThe programmatic implications of these findings are substantial. In elimination settings, conventional surveillance metrics such as case counts and incidence rates cannot distinguish between importation-driven outbreaks and local clonal amplification. Molecular genotyping provides critical resolution to disentangle these scenarios. As emphasized in regional experiences from Costa Rica and other countries [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], routine integration of parasite genotyping into surveillance systems enhances outbreak attribution and guides targeted interventions. In insular contexts such as the Bay Islands, where ecological receptivity remains high and vector species such as \u003cem\u003eAnopheles albimanus\u003c/em\u003e persist [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], rapid identification of genetically homogeneous outbreaks may facilitate focused containment before expansion occurs. Conversely, failure to detect early clonal amplification may allow rapid spread from a single introduced or persistent lineage.\u003c/p\u003e \u003cp\u003eOverall, the 2025 Bay Islands outbreak illustrates how malaria resurgence in low-transmission settings may be driven by amplification of genetically constrained parasite populations rather than by repeated independent introductions. The observed homogeneity in \u003cem\u003eP. falciparum\u003c/em\u003e and the restricted multilocus diversity in \u003cem\u003eP. vivax\u003c/em\u003e are consistent with expansion from a reduced ancestral pool in a geographically confined environment. These findings underscore the importance of sustained molecular surveillance during the elimination phase and highlight the vulnerability of insular transmission systems to founder-driven outbreaks. Integrating multilocus genotyping into routine surveillance frameworks will be essential to differentiate local transmission from importation and to support evidence-based elimination strategies in Honduras and across the Mesoamerican region.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe 2025 malaria outbreak in the Bay Islands of Honduras was characterized by marked genetic homogeneity, with complete monomorphism in \u003cem\u003ePlasmodium falciparum\u003c/em\u003e and restricted multilocus diversity in \u003cem\u003eP. vivax\u003c/em\u003e, including temporal dominance of a single haplotype. These findings are consistent with clonal amplification within a geographically confined, low-transmission setting rather than with multiple independent parasite introductions. In the context of sustained transmission reduction and prior evidence of demographic contraction in Honduras, the outbreak appears to have emerged from a genetically constrained ancestral pool. Our results highlight the value of multilocus molecular surveillance for distinguishing local amplification from importation in elimination settings and underscore the vulnerability of insular transmission systems to founder-driven outbreaks. Integrating routine genotyping into malaria surveillance frameworks will be essential to support evidence-based decision-making and to safeguard progress toward regional elimination goals by 2030.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eThis study has several limitations. First, genotyping was based on targeted amplification and Sanger sequencing of selected polymorphic loci rather than whole-genome sequencing, which may limit resolution for detecting very recent micro-divergence events. In addition, Sanger-based approaches primarily capture dominant haplotypes and may underestimate low-frequency subclonal variants in mixed infections. However, no ambiguous chromatograms suggestive of multiclonal infections were observed, and complete multilocus concordance across successfully genotyped samples supports the inference of restricted recombination during the outbreak period. The number of samples analysed represented a subset of confirmed cases and was constrained by sample availability through routine surveillance. Nonetheless, the consistent detection of identical haplotypes across municipalities and over several epidemiological weeks indicates that the observed genetic homogeneity reflects a true biological pattern rather than sampling artefact. Future studies incorporating genome-wide approaches and broader temporal sampling would further refine understanding of parasite population dynamics in insular elimination settings.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eACT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eArtemisinin-based combination therapy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ebp\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBase pairs\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCIG-UNAH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCentro de Investigaciones Gen\u0026eacute;ticas \u0026ndash; Universidad Nacional Aut\u0026oacute;noma de Honduras\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eD*\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFu and Li\u0026rsquo;s D* statistic\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEta\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTotal number of mutations\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eF*\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFu and Li\u0026rsquo;s F* statistic\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHd\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHaplotype diversity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJSG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eJos\u0026eacute; Santos Guardiola\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ek\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAverage number of nucleotide differences\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMAD20\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMerozoite surface protein 1 allelic family MAD20\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMOI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMultiplicity of infection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eORF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOpen reading frame\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePET-PCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhoto-induced electron transfer polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePf\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003ePlasmodium falciparum\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003epfcrt\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003ePlasmodium falciparum\u003c/em\u003e chloroquine resistance transporter gene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003epfmdr1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003ePlasmodium falciparum\u003c/em\u003e multidrug resistance gene 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003epfmsp1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003ePlasmodium falciparum\u003c/em\u003e merozoite surface protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePv\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003ePlasmodium vivax\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003epvcsp\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003ePlasmodium vivax\u003c/em\u003e circumsporozoite protein gene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003epvmsp3α\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003ePlasmodium vivax\u003c/em\u003e merozoite surface protein 3 alpha gene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRDT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRapid diagnostic test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNumber of segregating sites\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSNP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSingle nucleotide polymorphism\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eθ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWatterson\u0026rsquo;s estimator\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eπ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNucleotide diversity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVK210\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003ePlasmodium vivax\u003c/em\u003e circumsporozoite protein VK210 variant\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVK247\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003ePlasmodium vivax\u003c/em\u003e circumsporozoite protein VK247 variant\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003e \u003cb\u003eEthics approval and consent to participate\u003c/b\u003e.\u003c/strong\u003e \u003cp\u003eThis study used anonymized samples collected as part of routine malaria surveillance activities conducted by the Honduran Ministry of Health. No personal identifiers were included in the molecular analyses. In accordance with national regulations governing public health surveillance activities, individual informed consent was not required for the use of these anonymized samples. The study protocol was reviewed and approved by the appropriate institutional and national ethical review committees, in accordance with national regulations and international ethical guidelines (approval PI-10-2025).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e \u003cb\u003eConsent for publication\u003c/b\u003e.\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests.\u003c/h2\u003e \u003cp\u003eOne of the authors of this manuscript (H.O.V.) is an employee of the U.S. Government. This work was prepared as part of his official duties. Title 17 U.S.C. \u0026sect;\u0026nbsp;105 provides that \u0026ldquo;Copyright protection under this Title is not available for any work of the United States Government\u0026rdquo;. Title 17 U.S.C. \u0026sect;\u0026nbsp;101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person\u0026rsquo;s official duties. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding.\u003c/h2\u003e \u003cp\u003eFunding for this study was provided by the Genetic Research Center, CIG-UNAH, and by the Armed Forces Health Surveillance Division (AFHSD), Global Emerging Infections Surveillance (GEIS) Branch (PROMIS ID P0146_25_N6). The funders had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript. The APC was funded by DICIHT-UNAH.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eGF: Conceptualization, Methodology validation, Formal analysis, Data curation, Visualization, Writing \u0026ndash; original draft, Supervision, Project administration. FP, DE, GA, LC: Methodology, Investigation, Writing \u0026ndash; review and editing. HOV, GF: Resources, Funding acquisition. LC, GA, AM, DO, MC: Epidemiological surveillance and field follow-up of the malaria outbreak in the Bay Islands. DE, GF: Project administration. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements.\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials.\u003c/h2\u003e \u003cp\u003eAll nucleotide sequences generated in this study have been deposited in GenBank under the accession numbers reported in the Results section. Additional data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSubvenci\u0026oacute;n Malaria del. 1 de enero del 2024 al 31 de diciembre de 2025 [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mcp-honduras.hn/malaria/]\u003c/span\u003e\u003cspan address=\"https://mcp-honduras.hn/malaria/]\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Health organization. Global technical strategy and targets for malaria 2016\u0026ndash;2030. Geneva: WHO; 2015.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinto A, Archaga O, Mejia A, Escober L, Henriquez J, Montoya A, Valdivia HO, Fontecha G. Evidence of a Recent Bottleneck in Plasmodium falciparum Populations on the Honduran-Nicaraguan Border. 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Geneva: WHO; 2024. p. 316.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGriffing S, Syphard L, Sridaran S, McCollum AM, Mixson-Hayden T, Vinayak S, Villegas L, Barnwell JW, Escalante AA. Udhayakumar V: pfmdr1 amplification and fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum in Venezuela. Antimicrob Agents Chemother. 2010;54:1572\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVo TC, Le HG, Kang JM, Trinh NTM, Quang HH, Na BK. Genetic polymorphism of merozoite surface protein 1 and merozoite surface protein 2 in the Vietnam Plasmodium falciparum population. BMC Infect Dis. 2024;24:1216.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObaldia N 3rd, Baro NK, Calzada JE, Santamaria AM, Daniels R, Wong W, Chang HH, Hamilton EJ, Arevalo-Herrera M, Herrera S, et al. Clonal outbreak of Plasmodium falciparum infection in eastern Panama. J Infect Dis. 2015;211:1087\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaenz FE, Morton LC, Okoth SA, Valenzuela G, Vera-Arias CA, Velez-Alvarez E, Lucchi NW, Castro LE, Udhayakumar V. Clonal population expansion in an outbreak of Plasmodium falciparum on the northwest coast of Ecuador. Malar J. 2015;13(1):497.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLabadie-Bracho MY, Adhin MR. Reconstruction of Plasmodium vivax outbreaks in a low malaria endemic setting utilizing conventional restriction fragment length polymorphism. Int J Mol Epidemiol Genet. 2021;12:9\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDa Veiga Leal S, Ward D, Campino S, Benavente ED, Ibrahim A, Claret T, Isaias V, Monteiro D, Clark TG, Goncalves L, et al. Drug resistance profile and clonality of Plasmodium falciparum parasites in Cape Verde: the 2017 malaria outbreak. Malar J. 2021;20:172.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang B, Tuo F, Liang Y, Wu W, Wu G, Huang S, Zhong Q, Su XZ, Zhang H, Li M, et al. Temporal changes in genetic diversity of msp-1, msp-2, and msp-3 in Plasmodium falciparum isolates from Grande Comore Island after introduction of ACT. Malar J. 2018;17:83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimpson SV, Nundu SS, Arima H, Kaneko O, Mita T, Culleton R, Yamamoto T. The diversity of Plasmodium falciparum isolates from asymptomatic and symptomatic school-age children in Kinshasa Province, Democratic Republic of Congo. Malar J. 2023;22:102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndika B, Mobegi V, Gathii K, Nyataya J, Maina N, Awinda G, Mutai B, Waitumbi J. Plasmodium falciparum population structure inferred by msp1 amplicon sequencing of parasites collected from febrile patients in Kenya. 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Trop Med Infect Dis 2024, 9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantamaria AM, Vasquez V, Rigg C, Samudio F, Moreno D, Romero L, Saldana A, Chaves LF, Calzada JE. Plasmodium vivax Genetic Diversity in Panama: Challenges for Malaria Elimination in Mesoamerica. Pathogens 2021, 10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonzalez-Ceron L, Gomez-Perez DJ, Santillan-Valenzuela F, Ovilla-Munoz M, Guzman-Bracho C, Pech-May A, Amores GR, Montoya-Perez A, Villarreal-Trevino C. Population Dynamics of Plasmodium vivax in Mexico Determined by CSP, Pvs25, and SSU 18S rRNA S-Type Polymorphism Analyses. Microorganisms 2025, 13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChaves LF, Huber JH, Rojas Salas O, Ramirez Rojas M, Romero LM, Gutierrez Alvarado JM, Perkins TA, Prado M, Rodriguez RM. Malaria Elimination in Costa Rica: Changes in Treatment and Mass Drug Administration. \u003cem\u003eMicroorganisms\u003c/em\u003e 2020, 8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLabadie-Bracho MY, Adhin MR. Advocating for PCR-RFLP as molecular tool within malaria programs in low endemic areas and low resource settings. PLoS Negl Trop Dis. 2023;17:e0011747.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEscobar D, Archaga O, Reyes A, Palma A, Larson RT, Vasquez GM, Fontecha G. A Follow-Up to the Geographical Distribution of Anopheles Species in Malaria-Endemic and Non-Endemic Areas of Honduras. Insects 2022, 13.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Malaria, outbreak, Honduras, molecular markers, Plasmodium vivax, Plasmodium falciparum, Bay Islands","lastPublishedDoi":"10.21203/rs.3.rs-9347818/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9347818/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntroduction\u003c/p\u003e \u003cp\u003eHonduras is progressing toward malaria elimination as part of the Mesoamerican regional initiative targeting elimination by 2030. As transmission declines, malaria increasingly occurs in focal outbreaks, particularly in geographically confined settings such as islands. Molecular surveillance can provide critical insights into parasite population structure during these events. In 2025, an outbreak of malaria occurred in the Bay Islands Department of Honduras. This study aimed to characterize the genetic diversity and clonal structure of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e and \u003cem\u003eP. vivax\u003c/em\u003e circulating during this outbreak.\u003c/p\u003e \u003cp\u003eMethods\u003c/p\u003e \u003cp\u003eMalaria cases detected during the outbreak were confirmed by photo-induced electron transfer PCR (PET-PCR). Genetic diversity was assessed using targeted amplification and Sanger sequencing of polymorphic loci, including \u003cem\u003epfmsp1\u003c/em\u003e and \u003cem\u003epfmdr1\u003c/em\u003e for \u003cem\u003eP. falciparum\u003c/em\u003e, and \u003cem\u003epvcsp\u003c/em\u003e and \u003cem\u003epvmsp3α\u003c/em\u003e for \u003cem\u003eP. vivax.\u003c/em\u003e Haplotype diversity and population genetic parameters were estimated using DnaSP, and multilocus haplotypes were analysed to evaluate clonal structure and temporal dynamics during the outbreak.\u003c/p\u003e \u003cp\u003eResults\u003c/p\u003e \u003cp\u003eA total of 49 malaria cases were analysed, including 18 \u003cem\u003eP. falciparum\u003c/em\u003e and 31 \u003cem\u003eP. vivax\u003c/em\u003e infections across three municipalities. All \u003cem\u003eP. falciparum\u003c/em\u003e isolates exhibited identical haplotypes across both analysed loci, indicating complete genetic homogeneity. In \u003cem\u003eP. vivax\u003c/em\u003e, two haplotypes were identified at the \u003cem\u003epvcsp\u003c/em\u003e locus and two at \u003cem\u003epvmsp3α\u003c/em\u003e, with one dominant haplotype accounting for most infections. Cluster B was detected only during the early phase of the outbreak, while cluster A became predominant from epidemiological week 11 onward. Complete concordance between loci was observed across all successfully genotyped samples, and no mixed multilocus genotypes were detected.\u003c/p\u003e \u003cp\u003eConclusions\u003c/p\u003e \u003cp\u003eThe outbreak was characterized by marked genetic homogeneity in \u003cem\u003eP. falciparum\u003c/em\u003e and restricted multilocus diversity in \u003cem\u003eP. vivax\u003c/em\u003e, with temporal dominance of a single haplotype, consistent with clonal amplification within a geographically confined, low-transmission setting. These findings suggest that the outbreak likely resulted from local expansion of a limited number of parasite lineages rather than multiple independent introductions. Integrating molecular genotyping into routine surveillance may help distinguish local transmission from importation and strengthen malaria elimination strategies in Honduras and the wider Mesoamerican region.\u003c/p\u003e","manuscriptTitle":"Clonal expansion of Plasmodium falciparum and Plasmodium vivax during a malaria outbreak in the Bay Islands of Honduras, 2025","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 06:52:40","doi":"10.21203/rs.3.rs-9347818/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-12T19:27:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T18:50:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62421518325853228525977526797282922692","date":"2026-04-22T23:05:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-21T14:59:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-08T05:01:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-08T05:00:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Malaria Journal","date":"2026-04-07T16:25:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"481ee8bf-0585-4f8e-8bca-d7222f95f49a","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-12T19:27:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T18:50:12+00:00","index":14,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T15:53:35+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 06:52:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9347818","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9347818","identity":"rs-9347818","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}