PET-PCR reveals low parasitaemia and submicroscopic malarial infections in Honduran Moskitia.

BACKGROUND: Malaria remains a main parasitic disease of humans. Although the largest number of cases is reported in the African region, there are still endemic foci in the Americas. Central America reported 36,000 malaria cases in 2020, which represents 5.5% of cases in the Americas and 0.015% of cases globally. Most malaria infections in Central America are reported in La Moskitia, shared by Honduras and Nicaragua. In the Honduran Moskitia, less than 800 cases were registered in 2020, considering it an area of low endemicity. In low endemicity settings, the number of submicroscopic and asymptomatic infections tends to increase, leaving many cases undetected and untreated. These reservoirs challenge national malaria elimination programmes. This study aimed to assess the diagnostic performance of Light Microscopy (LM), a nested PCR test and a photoinduced electron transfer polymerase chain reaction (PET-PCR) in a population of febrile patients from La Moskitia. METHODS: A total of 309 febrile participants were recruited using a passive surveillance approach at the Puerto Lempira hospital. Blood samples were analysed by LM, nested PCR, and PET-PCR. Diagnostic performance including sensitivity, specificity, negative and positive predictive values, kappa index, accuracy, and ROC analysis was evaluated. The parasitaemia of the positive samples was quantified by both LM and PET-PCR. RESULTS: The overall prevalence of malaria was 19.1% by LM, 27.8% by nPCR, and 31.1% by PET-PCR. The sensitivity of LM was 67.4% compared to nPCR, and the sensitivity of LM and nPCR was 59.6% and 80.8%, respectively, compared to PET-PCR. LM showed a kappa index of 0.67, with a moderate level of agreement. Forty positive cases by PET-PCR were not detected by LM. CONCLUSIONS: This study demonstrated that LM is unable to detect parasitaemia at low levels and that there is a high degree of submicroscopic infections in the Honduran Moskitia.

Assessment of Plasmodium falciparum anti-malarial drug resistance markers in pfcrt and pfmdr1 genes in isolates from Honduras and Nicaragua, 2018-2021.

BACKGROUND: Central America and the island of Hispaniola have set out to eliminate malaria by 2030. However, since 2014 a notable upturn in the number of cases has been reported in the Mosquitia region shared by Nicaragua and Honduras. In addition, the proportion of Plasmodium falciparum malaria cases has increased significantly relative to vivax malaria. Chloroquine continues to be the first-line drug to treat uncomplicated malaria in the region. The objective of this study was to evaluate the emergence of chloroquine resistant strains of P. falciparum using a genetic approach. Plasmodium vivax populations are not analysed in this study. METHODS: 205 blood samples from patients infected with P. falciparum between 2018 and 2021 were analysed. The pfcrt gene fragment encompassing codons 72-76 was analysed. Likewise, three fragments of the pfmdr1 gene were analysed in 51 samples by nested PCR and sequencing. RESULTS: All samples revealed the CVMNK wild phenotype for the pfcrt gene and the N86, Y184F, S1034C, N1042D, D1246 phenotype for the pfmdr1 gene. CONCLUSIONS: The increase in falciparum malaria cases in Nicaragua and Honduras cannot be attributed to the emergence of chloroquine-resistant mutants. Other possibilities should be investigated further. This is the first study to report the genotype of pfmdr1 for five loci of interest in Central America.

Molecular surveillance of the Plasmodium vivax multidrug resistance 1 gene in Peru between 2006 and 2015.

BACKGROUND: The high incidence of Plasmodium vivax infections associated with clinical severity and the emergence of chloroquine (CQ) resistance has posed a challenge to control efforts aimed at eliminating this disease. Despite conflicting evidence regarding the role of mutations of P. vivax multidrug resistance 1 gene (pvmdr1) in drug resistance, this gene can be a tool for molecular surveillance due to its variability and spatial patterns. METHODS: Blood samples were collected from studies conducted between 2006 and 2015 in the Northern and Southern Amazon Basin and the North Coast of Peru. Thick and thin blood smears were prepared for malaria diagnosis by microscopy and PCR was performed for detection of P. vivax monoinfections. The pvmdr1 gene was subsequently sequenced and the genetic data was used for haplotype and diversity analysis. RESULTS: A total of 550 positive P. vivax samples were sequenced; 445 from the Northern Amazon Basin, 48 from the Southern Amazon Basin and 57 from the North Coast. Eight non-synonymous mutations and three synonymous mutations were analysed in 4,395 bp of pvmdr1. Amino acid changes at positions 976F and 1076L were detected in the Northern Amazon Basin (12.8%) and the Southern Amazon Basin (4.2%) with fluctuations in the prevalence of both mutations in the Northern Amazon Basin during the course of the study that seemed to correspond with a malaria control programme implemented in the region. A total of 13 pvmdr1 haplotypes with non-synonymous mutations were estimated in Peru and an overall nucleotide diversity of π = 0.00054. The Northern Amazon Basin was the most diverse region (π = 0.00055) followed by the Southern Amazon and the North Coast (π = 0.00035 and π = 0.00014, respectively). CONCLUSION: This study showed a high variability in the frequencies of the 976F and 1076L polymorphisms in the Northern Amazon Basin between 2006 and 2015. The low and heterogeneous diversity of pvmdr1 found in this study underscores the need for additional research that can elucidate the role of this gene on P. vivax drug resistance as well as in vitro and clinical data that can clarify the extend of CQ resistance in Peru.

A cluster of the first reported Plasmodium ovale spp. infections in Peru occuring among returning UN peace-keepers, a review of epidemiology, prevention and diagnostic challenges in nonendemic regions.

BACKGROUND: Plasmodium ovale curtisi and Plasmodium ovale wallikeri are regarded as less virulent forms of malaria with a geographic distribution including Southeast Asia, Central and West Africa, and is increasingly reported as an infection in returning travellers. A species of malaria that may have delayed or relapsing presentations similar to Plasmodium vivax, the clinical presentation of P. ovale spp. has been described to have prepatent periods of 2 weeks or slightly longer with reports of relapse following primary infection out to 8-9 months. This presentation may be obscured further in the setting of anti-malarial exposure, with report of delayed primary infection out to 4 years. Presented is a cluster of 4 imported P. ovale spp. cases in returning Peruvian military personnel assigned to United Nations peace-keeping operations in the Central African Republic. CASE PRESENTATION: From January to December 2016, Peruvian peace-keepers were deployed in support of United Nations (UN) operations in the Central African Republic (CAR). While serving abroad, Navy, Army, and Air Force members experienced 223 episodes of Plasmodium falciparum malaria following interruption of prophylaxis with mefloquine. Diagnosis was made using rapid diagnostics tests (RDTs) and/or smear with no coinfections identified. Cases of malaria were treated with locally-procured artemether-lumefantrine. Returning to Peru in January 2017, 200 peace-keepers were screened via thick and thin smear while on weekly mefloquine prophylaxis with only 1 showing nucleic acid within red blood cells consistent with Plasmodium spp. and 11 reporting syndromes of ill-defined somatic complaints. Between a period of 5 days to 11 months post return, 4 cases of P. ovale spp. were diagnosed using smear and polymerase chain reaction (PCR) following febrile complaints. All cases were subsequently treated with chloroquine and primaquine, with cure of clinical disease and documented clearance of parasitaemia. CONCLUSION: These patients represent the first imported cases in Peru of this species of malaria as well as highlight the challenges in implementing population level prophylaxis in a deployed environment, and the steps for timely diagnosis and management in a non-endemic region where risk of introduction for local transmission exists.

Whole genome sequencing of Trypanosoma cruzi field isolates reveals extensive genomic variability and complex aneuploidy patterns within TcII DTU.

BACKGROUND: Trypanosoma cruzi, the etiologic agent of Chagas disease, is currently divided into six discrete typing units (DTUs), named TcI-TcVI. TcII is among the major DTUs enrolled in human infections in South America southern cone, where it is associated with severe cardiac and digestive symptoms. Despite the importance of TcII in Chagas disease epidemiology and pathology, so far, no genome-wide comparisons of the mitochondrial and nuclear genomes of TcII field isolates have been performed to track the variability and evolution of this DTU in endemic regions. RESULTS: In the present work, we have sequenced and compared the whole nuclear and mitochondrial genomes of seven TcII strains isolated from chagasic patients from the central and northeastern regions of Minas Gerais, Brazil, revealing an extensive genetic variability within this DTU. A comparison of the phylogeny based on the nuclear or mitochondrial genomes revealed that the majority of branches were shared by both sequences. The subtle divergences in the branches are probably consequence of mitochondrial introgression events between TcII strains. Two T. cruzi strains isolated from patients living in the central region of Minas Gerais, S15 and S162a, were clustered in the nuclear and mitochondrial phylogeny analysis. These two strains were isolated from the other five by the Espinhaço Mountains, a geographic barrier that could have restricted the traffic of insect vectors during T. cruzi evolution in the Minas Gerais state. Finally, the presence of aneuploidies was evaluated, revealing that all seven TcII strains have a different pattern of chromosomal duplication/loss. CONCLUSIONS: Analysis of genomic variability and aneuploidies suggests that there is significant genomic variability within Minas Gerais TcII strains, which could be exploited by the parasite to allow rapid selection of favorable phenotypes. Also, the aneuploidy patterns vary among T. cruzi strains and does not correlate with the nuclear phylogeny, suggesting that chromosomal duplication/loss are recent and frequent events in the parasite evolution.

Gene and Chromosomal Copy Number Variations as an Adaptive Mechanism Towards a Parasitic Lifestyle in Trypanosomatids.

Trypanosomatids are a group of kinetoplastid parasites including some of great public health importance, causing debilitating and life-long lasting diseases that affect more than 24 million people worldwide. Among the trypanosomatids, Trypanosoma cruzi, Trypanosoma brucei and species from the Leishmania genus are the most well studied parasites, due to their high prevalence in human infections. These parasites have an extreme genomic and phenotypic variability, with a massive expansion in the copy number of species-specific multigene families enrolled in host-parasite interactions that mediate cellular invasion and immune evasion processes. As most trypanosomatids are heteroxenous, and therefore their lifecycles involve the transition between different hosts, these parasites have developed several strategies to ensure a rapid adaptation to changing environments. Among these strategies, a rapid shift in the repertoire of expressed genes, genetic variability and genome plasticity are key mechanisms. Trypanosomatid genomes are organized into large directional gene clusters that are transcribed polycistronically, where genes derived from the same polycistron may have very distinct mRNA levels. This particular mode of transcription implies that the control of gene expression operates mainly at post-transcriptional level. In this sense, gene duplications/losses were already associated with changes in mRNA levels in these parasites. Gene duplications also allow the generation of sequence variability, as the newly formed copy can diverge without loss of function of the original copy. Recently, aneuploidies have been shown to occur in several Leishmania species and T. cruzi strains. Although aneuploidies are usually associated with debilitating phenotypes in superior eukaryotes, recent data shows that it could also provide increased fitness in stress conditions and generate drug resistance in unicellular eukaryotes. In this review, we will focus on gene and chromosomal copy number variations and their relevance to the evolution of trypanosomatid parasites.

TipMT: Identification of PCR-based taxon-specific markers.

BACKGROUND: Molecular genetic markers are one of the most informative and widely used genome features in clinical and environmental diagnostic studies. A polymerase chain reaction (PCR)-based molecular marker is very attractive because it is suitable to high throughput automation and confers high specificity. However, the design of taxon-specific primers may be difficult and time consuming due to the need to identify appropriate genomic regions for annealing primers and to evaluate primer specificity. RESULTS: Here, we report the development of a Tool for Identification of Primers for Multiple Taxa (TipMT), which is a web application to search and design primers for genotyping based on genomic data. The tool identifies and targets single sequence repeats (SSR) or orthologous/taxa-specific genes for genotyping using Multiplex PCR. This pipeline was applied to the genomes of four species of Leishmania (L. amazonensis, L. braziliensis, L. infantum and L. major) and validated by PCR using artificial genomic DNA mixtures of the Leishmania species as templates. This experimental validation demonstrates the reliability of TipMT because amplification profiles showed discrimination of genomic DNA samples from Leishmania species. CONCLUSIONS: The TipMT web tool allows for large-scale identification and design of taxon-specific primers and is freely available to the scientific community at http://200.131.37.155/tipMT/ .

The Leishmania metaphylome: a comprehensive survey of Leishmania protein phylogenetic relationships.

BACKGROUND: Leishmaniasis is a neglected parasitic disease with diverse clinical manifestations and a complex epidemiology. It has been shown that its parasite-related traits vary between species and that they modulate infectivity, pathogenicity, and virulence. However, understanding of the species-specific adaptations responsible for these features and their evolutionary background is limited. To improve our knowledge regarding the parasite biology and adaptation mechanisms of different Leishmania species, we conducted a proteome-wide phylogenomic analysis to gain insights into Leishmania evolution. RESULTS: The analysis of the reconstructed phylomes (totaling 45,918 phylogenies) allowed us to detect genes that are shared in pathogenic Leishmania species, such as calpain-like cysteine peptidases and 3'a2rel-related proteins, or genes that could be associated with visceral or cutaneous development. This analysis also established the phylogenetic relationship of several hypothetical proteins whose roles remain to be characterized. Our findings demonstrated that gene duplication constitutes an important evolutionary force in Leishmania, acting on protein families that mediate host-parasite interactions, such as amastins, GP63 metallopeptidases, cathepsin L-like proteases, and our methods permitted a deeper analysis of their phylogenetic relationships. CONCLUSIONS: Our results highlight the importance of proteome wide phylogenetic analyses to detect adaptation and evolutionary processes in different organisms and underscore the need to characterize the role of expanded and species-specific proteins in the context of Leishmania evolution by providing a framework for the phylogenetic relationships of Leishmania proteins. Phylogenomic data are publicly available for use through PhylomeDB (http://www.phylomedb.org).

Comparative genomic analysis of Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis.

BACKGROUND: The Leishmania (Viannia) braziliensis complex is responsible for most cases of New World tegumentary leishmaniasis. This complex includes two closely related species but with different geographic distribution and disease phenotypes, L. (V.) peruviana and L. (V.) braziliensis. However, the genetic basis of these differences is not well understood and the status of L. (V.) peruviana as distinct species has been questioned by some. Here we sequenced the genomes of two L. (V.) peruviana isolates (LEM1537 and PAB-4377) using Illumina high throughput sequencing and performed comparative analyses against the L. (V.) braziliensis M2904 reference genome. Comparisons were focused on the detection of Single Nucleotide Polymorphisms (SNPs), insertions and deletions (INDELs), aneuploidy and gene copy number variations. RESULTS: We found 94,070 variants shared by both L. (V.) peruviana isolates (144,079 in PAB-4377 and 136,946 in LEM1537) against the L. (V.) braziliensis M2904 reference genome while only 26,853 variants separated both L. (V.) peruviana genomes. Analysis in coding sequences detected 26,750 SNPs and 1,513 indels shared by both L. (V.) peruviana isolates against L. (V.) braziliensis M2904 and revealed two L. (V.) braziliensis pseudogenes that are likely to have coding potential in L. (V.) peruviana. Chromosomal read density and allele frequency profiling showed a heterogeneous pattern of aneuploidy with an overall disomic tendency in both L. (V.) peruviana isolates, in contrast with a trisomic pattern in the L. (V.) braziliensis M2904 reference. Read depth analysis allowed us to detect more than 368 gene expansions and 14 expanded gene arrays in L. (V.) peruviana, and the likely absence of expanded amastin gene arrays. CONCLUSIONS: The greater numbers of interspecific SNP/indel differences between L. (V.) peruviana and L. (V.) braziliensis and the presence of different gene and chromosome copy number variations support the classification of both organisms as closely related but distinct species. The extensive nucleotide polymorphisms and differences in gene and chromosome copy numbers in L. (V.) peruviana suggests the possibility that these may contribute to some of the unique features of its biology, including a lower pathology and lack of mucosal development.

Chromosomal copy number variation reveals differential levels of genomic plasticity in distinct Trypanosoma cruzi strains.

BACKGROUND: Trypanosoma cruzi, the etiologic agent of Chagas disease, is currently divided into six discrete typing units (DTUs), named TcI-TcVI. CL Brener, the reference strain of the T. cruzi genome project, is a hybrid with a genome assembled into 41 putative chromosomes. Gene copy number variation (CNV) is well documented as an important mechanism to enhance gene expression and variability in T. cruzi. Chromosomal CNV (CCNV) is another level of gene CNV in which whole blocks of genes are expanded simultaneously. Although the T. cruzi karyotype is not well defined, several studies have demonstrated a significant variation in the size and content of chromosomes between different T. cruzi strains. Despite these studies, the extent of diversity in CCNV among T. cruzi strains based on a read depth coverage analysis has not been determined. RESULTS: We identify the CCNV in T. cruzi strains from the TcI, TcII and TcIII DTUs, by analyzing the depth coverage of short reads from these strains using the 41 CL Brener chromosomes as reference. This study led to the identification of a broader extent of CCNV in T. cruzi than was previously speculated. The TcI DTU strains have very few aneuploidies, while the strains from TcII and TcIII DTUs present a high degree of chromosomal expansions. Chromosome 31, which is the only chromosome that is supernumerary in all six T. cruzi samples evaluated in this study, is enriched with genes related to glycosylation pathways, highlighting the importance of glycosylation to parasite survival. CONCLUSIONS: Increased gene copy number due to chromosome amplification may contribute to alterations in gene expression, which represents a strategy that may be crucial for parasites that mainly depend on post-transcriptional mechanisms to control gene expression.

Asymptomatic Malaria Reservoirs in Honduras: A Challenge for Elimination.

BACKGROUND: Efforts on a global scale for combating malaria have achieved substantial progress over the past twenty years. Two Central American nations have accomplished their goal of eliminating malaria: El Salvador and Belize. Honduras has decreased the incidence of malaria and now reports fewer than 4000 malaria cases annually, aspiring to reach elimination by 2030. To accomplish this goal, it is essential to assess the existing strategies employed for malaria control and to address the task of incorporating novel intervention strategies to identify asymptomatic reservoirs. METHODS: A survey for detecting asymptomatic cases was carried out in the community of Kaukira, in Gracias a Dios, Honduras, focusing on malaria transmission during 2023. Asymptomatic community members were recruited as participants, malaria screening was performed through a rapid diagnostic test in situ, and a blood sample was collected on filter paper. Highly sensitive molecular assays based on photo-induced electron transfer PCR (PET-PCR) were performed to detect the two species of Plasmodium circulating in Honduras: Plasmodium vivax and Plasmodium falciparum. In addition, the identification of the parasite species was verified by amplifying three genetic markers (Pvmsp3α, Pvmsp3ß, and Pfmsp1). RESULTS: A total of 138 participants were recruited, mostly adult women. All individuals tested negative on the rapid diagnostic test. Positive results for malaria were detected by PET-PCR in 17 samples (12.3%). Most samples (12 out of 17) were amplified with a Ct value between 37 and 42, indicating very low parasitemias. Out of the 17 samples, 16 of them also showed amplification in the species assays. There were nine cases of P. falciparum infections and seven cases of P. vivax infections that were further confirmed by nested PCR (nPCR) of Pvmsp3 and Pfmsp1. Parasitemias ranged from 100 p/µL to less than 0.25 p/µL. One sample showed mixed infection. CONCLUSIONS: The existence of asymptomatic malaria reservoirs in Honduras can contribute to disease transmission and pose a challenge that may hinder elimination efforts, requiring public health authorities to modify surveillance strategies to identify the disease and treat this population accordingly.

Comparison of prevalence estimates of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum determined by conventional PCR and multiplex qPCR and implications for surveillance and monitoring.

OBJECTIVES: The accuracy of malaria rapid diagnostic tests is threatened by Plasmodium falciparum with pfhrp2/3 deletions. This study compares gene deletion prevalence determined by multiplex real time polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR) using existing samples with clonality previously determined by microsatellite genotyping. METHODS: Multiplex qPCR was used to estimate prevalence of pfhrp2/3 deletions in three sets of previously collected patient samples from Eritrea and Peru. The qPCR was validated by multiplex digital polymerase chain reaction. Sample classification was compared with cPCR, and receiver operating characteristic curve analysis was used to determine the optimal ΔCq threshold that aligned the results of the two assays. RESULTS: qPCR classified 75% (637 of 849) of samples as single, and 212 as mixed-pfhrp2/3 genotypes, with a positive association between clonality and proportion of mixed-pfhrp2/3 genotype samples. The sample classification agreement between cPCR and qPCR was 75.1% (95% confidence interval [CI] 68.6-80.7%) and 47.8% (95% CI 38.9-56.9%) for monoclonal and polyclonal infections. The qPCR prevalence estimates of pfhrp2/3 deletions showed almost perfect (κ = 0.804, 95% CI 0.714-0.895) and substantial agreement (κ = 0.717, 95% CI 0.562-0.872) with cPCR for Peru and 2016 Eritrean samples, respectively. For 2019 Eritrean samples, the prevalence of double pfhrp2/3 deletions was approximately two-fold higher using qPCR. The optimal threshold for matching the assay results was ΔCq = 3. CONCLUSIONS: Multiplex qPCR and cPCR produce comparable estimates of gene deletion prevalence when monoclonal infections dominate; however, qPCR provides higher estimates where multi-clonal infections are common.

Sampling is decisive to determination of Leishmania (Viannia) species.

BACKGROUND: Accuracy of molecular tools for the identification of parasites that cause human cutaneous leishmaniasis (CL) could largely depend on the sampling method. Non-invasive or less-invasive sampling methods such as filter paper imprints and cotton swabs are preferred over punch biopsies and lancet scrapings for detection methods of Leishmania based on polymerase chain reaction (PCR) because they are painless, simple, and inexpensive, and of benefit to military and civilian patients to ensure timely treatment. However, different types of samples can generate false negatives and there is a clear need to demonstrate which sample is more proper for molecular assays. METHODOLOGY: Here, we compared the sensitivity of molecular identification of different Leishmania (Viannia) species from Peru, using three types of sampling: punch biopsy, filter paper imprint and lancet scraping. Different composite reference standards and latent class models allowed to evaluate the accuracy of the molecular tools. Additionally, a quantitative PCR assessed variations in the results and parasite load in each type of sample. PRINCIPAL FINDINGS: Different composite reference standards and latent class models determined higher sensitivity when lancet scrapings were used for sampling in the identification and determination of Leishmania (Viannia) species through PCR-based assays. This was consistent for genus identification through kinetoplastid DNA-PCR and for the determination of species using FRET probes-based Nested Real-Time PCR. Lack of species identification in some samples correlated with the low intensity of the PCR electrophoretic band, which reflects the low parasite load in samples. CONCLUSIONS: The type of clinical sample can directly influence the detection and identification of Leishmania (Viannia) species. Here, we demonstrated that lancet scraping samples consistently allowed the identification of more leishmaniasis cases compared to filter paper imprints or biopsies. This procedure is inexpensive, painless, and easy to implement at the point of care and avoids the need for anesthesia, surgery, and hospitalization and therefore could be used in resource limited settings for both military and civilian populations.

High-Throughput Genotyping of Plasmodium vivax in the Peruvian Amazon via Molecular Inversion Probes.

Plasmodium vivax transmission occurs throughout the tropics and is an emerging threat in areas of Plasmodium falciparum decline, causing relapse infections that complicate treatment and control. Targeted sequencing for P. falciparum has been widely deployed to detect population structure and the geographic spread of antimalarial and diagnostic resistance. However, there are fewer such tools for P. vivax . Leveraging global variation data, we designed four molecular inversion probe (MIP) genotyping panels targeting geographically differentiating SNPs, neutral SNPs, putative antimalarial resistance genes, and vaccine candidate genes. We deployed these MIP panels on 866 infections from the Peruvian Amazon and identified transmission networks with clonality (IBD>0.99), copy number variation in Pvdbp and multiple Pvrbps , fixation of putative antimalarial resistance, and balancing selection in 13 vaccine candidate genes. Our MIP panels are the broadest genotyping panel currently available and are poised for successful deployment in other regions of P. vivax transmission.

Genomic surveillance of malaria parasites in an indigenous community in the Peruvian Amazon.

Hard-to-reach communities represent Peru's main challenge for malaria elimination, but information about transmission in these areas is scarce. Here, we assessed Plasmodium vivax (Pv) and P. falciparum (Pf) transmission dynamics, resistance markers, and Pf hrp2/3 deletions in Nueva Jerusalén (NJ), a remote, indigenous community in the Peruvian Amazon with high population mobility. We collected samples from November 2019 to May 2020 by active (ACD) and passive case detection (PCD) in NJ. Parasites were identified with microscopy and PCR. Then, we analyzed a representative set of positive-PCR samples (Pv = 68, Pf = 58) using highly-multiplexed deep sequencing assays (AmpliSeq) and compared NJ parasites with ones from other remote Peruvian areas using population genetics indexes. The ACD intervention did not reduce malaria cases in the short term, and persistent malaria transmission was observed (at least one Pv infection was detected in 96% of the study days). In Nueva Jerusalen, the Pv population had modest genetic diversity (He = 0.27). Pf population had lower diversity (He = 0.08) and presented temporal clustering, one of these clusters linked to an outbreak in February 2020. Moreover, Pv and Pf parasites from NJ exhibited variable levels of differentiation (Pv Fst = 0.07-0.52 and Pf Fst = 0.11-0.58) with parasites from other remote areas. No artemisin resistance mutations but chloroquine (57%) and sulfadoxine-pyrimethamine (35-67%) were detected in NJ's Pf parasites. Moreover, pfhrp2/3 gene deletions were common (32-50% of parasites with one or both genes deleted). The persistent Pv transmission and the detection of a Pf outbreak with parasites genetically distinct from the local ones highlight the need for tailored interventions focusing on mobility patterns and imported infections in remote areas to eliminate malaria in the Peruvian Amazon.

Development of species-specific multiplex PCR for Leishmania identification.

Leishmaniasis is a diverse group of clinical diseases caused by protozoan parasites of the Leishmania genus. Species-specific identification of Leishmania spp. is challenging due to the high number of different pathogenic species that sometimes co-circulate in the same foci, hampering efforts to effectively control the disease. Multiplex PCR is an attractive alternative for rapid differentiation of Leishmania species with high sensitivity and specificity. We aimed to generate a panel of primers optimized for a multiplex PCR assay capable of identifying different Leishmania species in a single reaction. Species-specific primers were designed based on genomic data using the TipMT tooL. Potential non-specific amplifications of other trypanosomatids as well as human, dog, and sandfly hosts were first evaluated in silico using the Primer-Blast tooL. Species-specific primers for Leishmania amazonensis, Leishmania braziliensis, Leishmania donovani, Leishmania infantum, Leishmania mexicana and for the Leishmania guyanensis complex were tested in vitro. The primers have a limit of detection ranging from 1 to 0.01 ng of parasite gDNA using the same annealing temperature of 66 °C. The primers were specific for their targets when tested against 13 species of Leishmania, six trypanosomatids, and Babesia sp., and to detect the target species in a prepared pool with gDNA of six pathogenic Leishmania species. The designed primers were optimized for multiplex PCR, enabling species-specific identification of all five Leishmania species and one species complex. This new primer set could allow for efficient, fast, and reliable identification of Leishmania parasites.

Genomic surveillance of malaria parasites in an indigenous community in the Peruvian Amazon.

Hard-to-reach communities represent Peru's main challenge for malaria elimination, but information about transmission in these areas is scarce. Here, we assessed Plasmodium vivax (Pv) and P. falciparum (Pf) transmission dynamics, resistance markers, and Pf hrp2/3 deletions in Nueva Jerusalén (NJ), a remote, indigenous community in the Peruvian Amazon with high population mobility. We collected samples from November 2019 to May 2020 by active (ACD) and passive case detection (PCD) in NJ. Parasites were identified with microscopy and PCR. Then, we analyzed a representative set of positive-PCR samples (Pv = 68, Pf = 58) using highly-multiplexed deep sequencing assays (AmpliSeq) and compared NJ parasites with ones from other remote Peruvian areas using population genetics indexes. The ACD intervention did not reduce malaria cases in the short term, and persistent malaria transmission was observed (at least one Pv infection was detected in 96% of the study days). In Nueva Jerusalen, the Pv population had modest genetic diversity (He = 0.27). Pf population had lower diversity (He = 0.08) and presented temporal clustering, one of these clusters linked to an outbreak in February 2020. Moreover, Pv and Pf parasites from NJ exhibited variable levels of differentiation (Pv Fst = -0.52 & Pf Fst = 0.11-0.58) with parasites from other remote areas. No artemisin resistance mutations but chloroquine (57%) and sulfadoxine-pyrimethamine (35-67%) were detected in NJ's Pf parasites. Moreover, pfhrp2/3 gene deletions were common (32-50% of parasites with one or both genes deleted). The persistent Pv transmission and the detection of a Pf outbreak with parasites genetically distinct from the local ones highlight the need for tailored interventions focusing on mobility patterns and imported infections in remote areas to eliminate malaria in the Peruvian Amazon.

Parasitological and molecular detection of Trypanosoma evansi in a dog from Tocache, San Martin, Peru.

This study presents the first case report of canine trypanosomiasis caused by Trypanosoma evansi in Peru. The case was admitted to a veterinary clinic in the Peruvian Amazon region of San Martin with severe clinical symptomatology which resulted in the dog's death. Microscopy screening showed the presence of trypomastigotes in blood and bone marrow and postmortem histopathology found damage at the cardiac, lung, kidney and spleen levels. Collected specimens were tested by nested-PCR which were positive for Trypanosoma spp., but negative for T. cruzi. High-throughput sequencing determined that the infecting species was closely related to T. equiperdom/evansi and subsequent phylogenetic analysis confirmed that the sample was related to T. evansi. The presence of T. evansi in the area highlights the need for increased surveillance to assess the impact of surra in the region and to develop measures to prevent socioeconomic damage resulting from infections in domestic and farm animals as well as prevent zoonotic transmission.

Correction: Diversity, distribution and natural Leishmania infection of sand flies from communities along the Interoceanic Highway in the Southeastern Peruvian Amazon.

[This corrects the article DOI: 10.1371/journal.pntd.0009000.].

Malaria Molecular Surveillance in the Peruvian Amazon with a Novel Highly Multiplexed Plasmodium falciparum AmpliSeq Assay.

Molecular surveillance for malaria has great potential to support national malaria control programs (NMCPs). To bridge the gap between research and implementation, several applications (use cases) have been identified to align research, technology development, and public health efforts. For implementation at NMCPs, there is an urgent need for feasible and cost-effective tools. We designed a new highly multiplexed deep sequencing assay (Pf AmpliSeq), which is compatible with benchtop sequencers, that allows high-accuracy sequencing with higher coverage and lower cost than whole-genome sequencing (WGS), targeting genomic regions of interest. The novelty of the assay is its high number of targets multiplexed into one easy workflow, combining population genetic markers with 13 nearly full-length resistance genes, which is applicable for many different use cases. We provide the first proof of principle for hrp2 and hrp3 deletion detection using amplicon sequencing. Initial sequence data processing can be performed automatically, and subsequent variant analysis requires minimal bioinformatic skills using any tabulated data analysis program. The assay was validated using a retrospective sample collection (n = 254) from the Peruvian Amazon between 2003 and 2018. By combining phenotypic markers and a within-country 28-single-nucleotide-polymorphism (SNP) barcode, we were able to distinguish different lineages with multiple resistance haplotypes (in dhfr, dhps, crt and mdr1) and hrp2 and hrp3 deletions, which have been increasing in recent years. We found no evidence to suggest the emergence of artemisinin (ART) resistance in Peru. These findings indicate a parasite population that is under drug pressure but is susceptible to current antimalarials and demonstrate the added value of a highly multiplexed molecular tool to inform malaria strategies and surveillance systems. IMPORTANCE While the power of next-generation sequencing technologies to inform and guide malaria control programs has become broadly recognized, the integration of genomic data for operational incorporation into malaria surveillance remains a challenge in most countries where malaria is endemic. The main obstacles include limited infrastructure, limited access to high-throughput sequencing facilities, and the need for local capacity to run an in-country analysis of genomes at a large-enough scale to be informative for surveillance. In addition, there is a lack of standardized laboratory protocols and automated analysis pipelines to generate reproducible and timely results useful for relevant stakeholders. With our standardized laboratory and bioinformatic workflow, malaria genetic surveillance data can be readily generated by surveillance researchers and malaria control programs in countries of endemicity, increasing ownership and ensuring timely results for informed decision- and policy-making.