Publications by authors named "Andres F Vallejo"

31 Publications

Dual dean entrainment with volume ratio modulation for efficient droplet co-encapsulation: extreme single-cell indexing.

Lab Chip 2021 Jul 9. Epub 2021 Jul 9.

Cancer Sciences, Faculty of Medicine, University of Southampton, UK. and Institute for Life Sciences, University of Southampton, UK.

The future of single cell diversity screens involves ever-larger sample sizes, dictating the need for higher throughput methods with low analytical noise to accurately describe the nature of the cellular system. Current approaches are limited by the Poisson statistic, requiring dilute cell suspensions and associated losses in throughput. In this contribution, we apply Dean entrainment to both cell and bead inputs, defining different volume packets to effect efficient co-encapsulation. Volume ratio scaling was explored to identify optimal conditions. This enabled the co-encapsulation of single cells with reporter beads at rates of ∼1 million cells per hour, while increasing assay signal-to-noise with cell multiplet rates of ∼2.5% and capturing ∼70% of cells. The method, called Pirouette coupling, extends our capacity to investigate biological systems.
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http://dx.doi.org/10.1039/d1lc00292aDOI Listing
July 2021

An IRF1-IRF4 Toggle-Switch Controls Tolerogenic and Immunogenic Transcriptional Programming in Human Langerhans Cells.

Front Immunol 2021 15;12:665312. Epub 2021 Jun 15.

Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, Faculty of Medicine, University of Southampton, Southampton, United Kingdom.

Langerhans cells (LCs) reside in the epidermis as a dense network of immune system sentinels, coordinating both immunogenic and tolerogenic immune responses. To determine molecular switches directing induction of LC immune activation, we performed mathematical modelling of gene regulatory networks identified by single cell RNA sequencing of LCs exposed to TNF-alpha, a key pro-inflammatory signal produced by the skin. Our approach delineated three programmes of LC phenotypic activation (immunogenic, tolerogenic or ambivalent), and confirmed that TNF-alpha enhanced LC immunogenic programming. Through regulon analysis followed by mutual information modelling, we identified IRF1 as the key transcription factor for the regulation of immunogenicity in LCs. Application of a mathematical toggle switch model, coupling IRF1 with tolerance-inducing transcription factors, determined the key set of transcription factors regulating the switch between tolerance and immunogenicity, and correctly predicted LC behaviour in LCs derived from different body sites. Our findings provide a mechanistic explanation of how combinatorial interactions between different transcription factors can coordinate specific transcriptional programmes in human LCs, interpreting the microenvironmental context of the local tissue microenvironments.
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http://dx.doi.org/10.3389/fimmu.2021.665312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8239435PMC
June 2021

Integrated transcriptomic analysis of human tuberculosis granulomas and a biomimetic model identifies therapeutic targets.

J Clin Invest 2021 Aug;131(15)

NIHR Biomedical Research Center, School of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom.

Tuberculosis (TB) is a persistent global pandemic, and standard treatment for it has not changed for 30 years. Mycobacterium tuberculosis (Mtb) has undergone prolonged coevolution with humans, and patients can control Mtb even after extensive infection, demonstrating the fine balance between protective and pathological host responses within infected granulomas. We hypothesized that whole transcriptome analysis of human TB granulomas isolated by laser capture microdissection could identify therapeutic targets, and that comparison with a noninfectious granulomatous disease, sarcoidosis, would identify disease-specific pathological mechanisms. Bioinformatic analysis of RNAseq data identified numerous shared pathways between TB and sarcoidosis lymph nodes, and also specific clusters demonstrating TB results from a dysregulated inflammatory immune response. To translate these insights, we compared 3 primary human cell culture models at the whole transcriptome level and demonstrated that the 3D collagen granuloma model most closely reflected human TB disease. We investigated shared signaling pathways with human disease and identified 12 intracellular enzymes as potential therapeutic targets. Sphingosine kinase 1 inhibition controlled Mtb growth, concurrently reducing intracellular pH in infected monocytes and suppressing inflammatory mediator secretion. Immunohistochemical staining confirmed that sphingosine kinase 1 is expressed in human lung TB granulomas, and therefore represents a host therapeutic target to improve TB outcomes.
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http://dx.doi.org/10.1172/JCI148136DOI Listing
August 2021

Doxycycline host-directed therapy in human pulmonary tuberculosis.

J Clin Invest 2021 Jun 15. Epub 2021 Jun 15.

Department of Medicine, Yong Loo Ling School of Medicine, National University of Singapore, Singapore, Singapore.

Background: Matrix metalloproteinases (MMPs) are implicated as key regulators of tissue destruction in tuberculosis (TB) and may be a target for host-directed therapy. Here, we conducted a Phase 2 randomized, double-blind, placebo-controlled trial investigating doxycycline, a licensed broad spectrum MMP inhibitor, in pulmonary TB patients.

Methods: Thirty pulmonary TB patients were enrolled within 7 days of initiating anti-TB treatment and randomly assigned to receive either doxycycline 100 mg or placebo twice a day for 14 days in addition to standard care.

Results: There were significant changes in the host transcriptome, and suppression of systemic and respiratory markers of tissue destruction with the doxycycline intervention. Whole blood RNA-sequencing demonstrated that doxycycline accelerated restoration of dysregulated gene expression patterns in TB towards normality, with more rapid down-regulation of type I and II interferon and innate immune response genes and concurrent up-regulation of B-cell modules relative to placebo. The effects persisted for 6 weeks after doxycycline was discontinued, concurrent with suppression of plasma MMP-1. In respiratory samples, doxycycline reduced MMP-1, -8, -9, -12 and -13 concentrations, suppressed type I collagen and elastin destruction, and reduced pulmonary cavity volume despite unchanged sputum Mycobacterium tuberculosis loads between the study arms. Two weeks of adjunctive doxycycline with standard anti-TB treatment was well-tolerated, with no serious adverse events related to doxycycline.

Conclusion: These data demonstrate that adjunctive doxycycline with standard anti-TB treatment suppresses pathological MMPs in pulmonary tuberculosis patients, and suggest that larger studies on adjunctive doxycycline to limit immunopathology in TB are merited.
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http://dx.doi.org/10.1172/JCI141895DOI Listing
June 2021

Peptide: MHC-based DNA vaccination strategy to activate natural killer cells by targeting killer cell immunoglobulin-like receptors.

J Immunother Cancer 2021 May;9(5)

School of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK

Background: Natural killer (NK) cells are increasingly being recognized as agents for cancer immunotherapy. The killer cell immunoglobulin-like receptors (KIRs) are expressed by NK cells and are immunogenetic determinants of the outcome of cancer. In particular, KIR2DS2 is associated with protective responses to several cancers and also direct recognition of cancer targets in vitro. Due to the high homology between activating and inhibitory KIR genes to date, it has been challenging to target individual KIR for therapeutic benefit.

Methods: A novel KIR2DS2-targeting therapeutic peptide:MHC DNA vaccine was designed and used to immunize mice transgenic for KIR genes (KIR-Tg). NK cells were isolated from the livers and spleens of vaccinated mice and then analyzed for activation by flow cytometry, RNA profiling and cytotoxicity assays. In vivo assays of NK cell function using a syngeneic cancer model (B16 melanoma) and an adoptive transfer model for human hepatocellular carcinoma (Huh7) were performed.

Results: Injecting KIR-Tg mice with the vaccine construct activated NK cells in both liver and spleens of mice, with preferential activation of KIR2DS2-positive NK cells. KIR-specific activation was most marked on the CD11b+CD27+ mature subset of NK cells. RNA profiling indicated that the DNA vaccine upregulated genes associated with cellular metabolism and downregulated genes related to histone H3 methylation, which are associated with immune cell maturation and NK cell function. Vaccination led to canonical and cross-reactive peptide:MHC-specific NK cell responses. In vivo, DNA vaccination led to enhanced antitumor responses against B16F10 melanoma cells and also enhanced responses against a tumor model expressing the KIR2DS2 ligand HLA-C*0102.

Conclusion: We show the feasibility of a peptide-based KIR-targeting vaccine strategy to activate NK cells and hence generate functional antitumor responses. This approach does not require detailed knowledge of the tumor peptidomes nor HLA matching with the patient. It therefore offers a novel opportunity for targeting NK cells for cancer immunotherapy.
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http://dx.doi.org/10.1136/jitc-2020-001912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8141441PMC
May 2021

Resolving cellular systems by ultra-sensitive and economical single-cell transcriptome filtering.

iScience 2021 Mar 5;24(3):102147. Epub 2021 Feb 5.

Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, UK.

Single-cell transcriptomics suffer from sensitivity limits that restrict low abundance transcript identification, affects clustering and can hamper downstream analyses. Here, we describe Constellation sequencing (Constellation-Seq), a molecular transcriptome filter that delivers two orders of magnitude sensitivity gains by maximizing read utility while reducing the data sparsity and sequencing costs. The technique reliably measures changes in gene expression and was demonstrated by resolving rare dendritic cell populations from a peripheral blood mononuclear cell sample sample and exploring their biology with extreme resolution. The simple and powerful method is fully compatible with standard scRNA-Seq library preparation protocols and can be used for hypothesis testing, marker validation or investigating pathways.
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http://dx.doi.org/10.1016/j.isci.2021.102147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900351PMC
March 2021

Ileal Transcriptomic Analysis in Paediatric Crohn's Disease Reveals IL17- and NOD-signalling Expression Signatures in Treatment-naïve Patients and Identifies Epithelial Cells Driving Differentially Expressed Genes.

J Crohns Colitis 2021 May;15(5):774-786

Department of Human Genetics and Genomic Medicine, University of Southampton, Southampton, UK.

Background And Aims: Crohn's disease [CD] arises through host-environment interaction. Abnormal gene expression results from disturbed pathway activation or response to bacteria. We aimed to determine activated pathways and driving cell types in paediatric CD.

Methods: We employed contemporary targeted autoimmune RNA sequencing, in parallel to single-cell sequencing, to ileal tissue derived from paediatric CD and controls. Weighted gene co-expression network analysis [WGCNA] was performed and differentially expressed genes [DEGs] were determined. We integrated clinical data to determine co-expression modules associated with outcomes.

Results: In all, 27 treatment-naive CD [TN-CD], 26 established CD patients and 17 controls were included. WGCNA revealed a 31-gene signature characterising TN-CD patients, but not established CD, nor controls. The CSF3R gene is a hub within this module and is key in neutrophil expansion and differentiation. Antimicrobial genes, including S100A12 and the calprotectin subunit S100A9, were significantly upregulated in TN CD compared with controls [p = 2.61 x 10-15 and p = 9.13 x 10-14, respectively] and established CD [both p = 0.0055]. Gene-enrichment analysis confirmed upregulation of the IL17-, NOD- and Oncostatin-M-signalling pathways in TN-CD patients, identified in both WGCNA and DEG analyses. An upregulated gene signature was enriched for transcripts promoting Th17-cell differentiation and correlated with prolonged time to relapse [correlation-coefficient-0.36, p = 0.07]. Single-cell sequencing of TN-CD patients identified specialised epithelial cells driving differential expression of S100A9. Cell groups, determined by single-cell gene expression, demonstrated enrichment of IL17-signalling in monocytes and epithelial cells.

Conclusions: Ileal tissue from treatment-naïve paediatric patients is significantly upregulated for genes driving IL17-, NOD- and Oncostatin-M-signalling. This signal is driven by a distinct subset of epithelial cells expressing antimicrobial gene transcripts.
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http://dx.doi.org/10.1093/ecco-jcc/jjaa236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8095388PMC
May 2021

Comprehensive plasma proteomic profiling reveals biomarkers for active tuberculosis.

JCI Insight 2020 09 17;5(18). Epub 2020 Sep 17.

School of Clinical and Experimental Sciences, Faculty of Medicine, and.

BACKGROUNDTuberculosis (TB) kills more people than any other infection, and new diagnostic tests to identify active cases are required. We aimed to discover and verify novel markers for TB in nondepleted plasma.METHODSWe applied an optimized quantitative proteomics discovery methodology based on multidimensional and orthogonal liquid chromatographic separation combined with high-resolution mass spectrometry to study nondepleted plasma of 11 patients with active TB compared with 10 healthy controls. Prioritized candidates were verified in independent UK (n = 118) and South African cohorts (n = 203).RESULTSWe generated the most comprehensive TB plasma proteome to date, profiling 5022 proteins spanning 11 orders-of-magnitude concentration range with diverse biochemical and molecular properties. We analyzed the predominantly low-molecular weight subproteome, identifying 46 proteins with significantly increased and 90 with decreased abundance (peptide FDR ≤ 1%, q ≤ 0.05). Verification was performed for novel candidate biomarkers (CFHR5, ILF2) in 2 independent cohorts. Receiver operating characteristics analyses using a 5-protein panel (CFHR5, LRG1, CRP, LBP, and SAA1) exhibited discriminatory power in distinguishing TB from other respiratory diseases (AUC = 0.81).CONCLUSIONWe report the most comprehensive TB plasma proteome to date, identifying novel markers with verification in 2 independent cohorts, leading to a 5-protein biosignature with potential to improve TB diagnosis. With further development, these biomarkers have potential as a diagnostic triage test.FUNDINGColciencias, Medical Research Council, Innovate UK, NIHR, Academy of Medical Sciences, Program for Advanced Research Capacities for AIDS, Wellcome Centre for Infectious Diseases Research.
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http://dx.doi.org/10.1172/jci.insight.137427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526553PMC
September 2020

Genomic programming of IRF4-expressing human Langerhans cells.

Nat Commun 2020 01 16;11(1):313. Epub 2020 Jan 16.

Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, Faculty of Medicine, University of Southampton, SO16 6YD, Southampton, UK.

Langerhans cells (LC) can prime tolerogenic as well as immunogenic responses in skin, but the genomic states and transcription factors (TF) regulating these context-specific responses are unclear. Bulk and single-cell transcriptional profiling demonstrates that human migratory LCs are robustly programmed for MHC-I and MHC-II antigen presentation. Chromatin analysis reveals enrichment of ETS-IRF and AP1-IRF composite regulatory elements in antigen-presentation genes, coinciding with expression of the TFs, PU.1, IRF4 and BATF3 but not IRF8. Migration of LCs from the epidermis is accompanied by upregulation of IRF4, antigen processing components and co-stimulatory molecules. TNF stimulation augments LC cross-presentation while attenuating IRF4 expression. CRISPR-mediated editing reveals IRF4 to positively regulate the LC activation programme, but repress NF2EL2 and NF-kB pathway genes that promote responsiveness to oxidative stress and inflammatory cytokines. Thus, IRF4-dependent genomic programming of human migratory LCs appears to enable LC maturation while attenuating excessive inflammatory and immunogenic responses in the epidermis.
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http://dx.doi.org/10.1038/s41467-019-14125-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965086PMC
January 2020

Constitutive Activation of Natural Killer Cells in Primary Biliary Cholangitis.

Front Immunol 2019 15;10:2633. Epub 2019 Nov 15.

Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom.

Natural killer (NK) cells are innate immune cells that interface with the adaptive immune system to generate a pro-inflammatory immune environment. Primary Biliary Cholangitis (PBC) is a hepatic autoimmune disorder with extrahepatic associations including systemic sclerosis, Sjogren's syndrome and thyroiditis. Immunogenetic studies have identified polymorphisms of the IL-12/STAT4 pathway as being associated with PBC. As this pathway is important for NK cell function we investigated NK cells in PBC. Circulating NK cells from individuals with PBC were constitutively activated, with higher levels of CD49a and the liver-homing marker, CXCR6, compared to participants with non-autoimmune chronic liver disease and healthy controls. Stimulation with minimal amounts of IL-12 (0.005 ng/ml) led to significant upregulation of CXCR6 ( < 0.005), and enhanced IFNγ production ( < 0.02) on NK cells from PBC patients compared to individuals with non-autoimmune chronic liver disease, indicating dysregulation of the IL-12/STAT4 axis. In RNAseq studies, resting NK cells from PBC patients had a constitutively activated transcriptional profile and upregulation of genes associated with IL-12/STAT4 signaling and metabolic reprogramming. Consistent with these findings, resting NK cells from PBC patients expressed higher levels of pSTAT4 compared to control groups ( < 0.001 vs. healthy controls and < 0.05 vs. liver disease controls). In conclusion NK cells in PBC are sensitive to minute quantities of IL-12 and have a "primed" phenotype. We therefore propose that peripheral priming of NK cells to express tissue-homing markers may contribute to the pathophysiology of PBC.
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http://dx.doi.org/10.3389/fimmu.2019.02633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6874097PMC
November 2020

Malaria systems immunology: Plasmodium vivax induces tolerance during primary infection through dysregulation of neutrophils and dendritic cells.

J Infect 2018 11 22;77(5):440-447. Epub 2018 Sep 22.

Clinical and Experimental Sciences and NIHR Southampton Biomedical Research Centre, Faculty of Medicine, University of Southampton, Southampton General Hospital, LE59, MP813, SO16 6YD, Southampton, UK; Institute for Life Sciences, University of Southampton, SO17 1BJ, UK. Electronic address:

Objectives: To dissect the transcriptional networks underpinning immune cells responses during primary Plasmodium vivax infection of healthy human adults.

Methods: We conducted network co-expression analysis of next-generation RNA sequencing data from whole blood from P. vivax and P. falciparum controlled human malaria infection (CHMI) of healthy naïve and malaria-exposed volunteers. Single cell transcription signatures were used to deconvolute the bulk RNA-Seq data into cell-specific signals.

Results: Initial exposure to P. vivax induced activation of innate immunity, including efficient antigen presentation and complement activation. However, this effect was accompanied by strong immunosuppression mediated by dendritic cells via the induction of Indoleamine 2,3-Dioxygenase 1(IDO1) and Lymphocyte Activation Gene 3 (LAG3). Additionally, P. vivax induced depletion of neutrophil populations associated with down regulation of 3G-protein coupled receptors, CRXCR1, CXCR2 and CSF3R. Accordingly, in malaria-exposed volunteers the inflammatory response was attenuated, with a decreased class II antigen presentation in dendritic cells. While the immunosuppressive signalling was maintained between plasmodium species, response to P. falciparum was significantly more immunogenic.

Conclusions: In silico analyses suggest that primary infection with P. vivax induces potent immunosuppression mediated by dendritic cells, conditioning subsequent anti-malarial immune responses. Targeting immune evasion mechanisms could be an effective alternative for improving vaccine efficacy.
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http://dx.doi.org/10.1016/j.jinf.2018.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6203889PMC
November 2018

Langerhans Cells-Programmed by the Epidermis.

Front Immunol 2017 29;8:1676. Epub 2017 Nov 29.

Systems Immmunology Group, Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, Faculty of Medicine, University of Southampton, Southampton, United Kingdom.

Langerhans cells (LCs) reside in the epidermis as a dense network of immune system sentinels. These cells determine the appropriate adaptive immune response (inflammation or tolerance) by interpreting the microenvironmental context in which they encounter foreign substances. In a normal physiological, "non-dangerous" situation, LCs coordinate a continuous state of immune tolerance, preventing unnecessary and harmful immune activation. Conversely, when they sense a danger signal, for example during infection or when the physical integrity of skin has been compromised as a result of a trauma, they instruct T lymphocytes of the adaptive immune system to mount efficient effector responses. Recent advances investigating the molecular mechanisms underpinning the cross talk between LCs and the epidermal microenvironment reveal its importance for programming LC biology. This review summarizes the novel findings describing LC origin and function through the analysis of the transcriptomic programs and gene regulatory networks (GRNs). Review and meta-analysis of publicly available datasets clearly delineates LCs as distinct from both conventional dendritic cells (DCs) and macrophages, suggesting a primary role for the epidermal microenvironment in programming LC biology. This concept is further supported by the analysis of the effect of epidermal pro-inflammatory signals, regulating key GRNs in human and murine LCs. Applying whole transcriptome analyses and analysis has advanced our understanding of how LCs receive, integrate, and process signals from the steady-state and diseased epidermis. Interestingly, in homeostasis and under immunological stress, the molecular network in LCs remains relatively stable, reflecting a key evolutionary need related to tissue localization. Importantly, to fulfill their key role in orchestrating antiviral adaptive immune responses, LC share specific transcriptomic modules with other DC types able to cross-present antigens to cytotoxic CD8 T cells, pointing to a possible evolutionary convergence mechanism. With the development of more advanced technologies allowing delineation of the molecular networks at the level of chromatin organization, histone modifications, protein translation, and phosphorylation, future "omics" investigations will bring in-depth understanding of the complex molecular mechanisms underpinning human LC biology.
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http://dx.doi.org/10.3389/fimmu.2017.01676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712534PMC
November 2017

Malaria epidemiology in low-endemicity areas of the northern coast of Ecuador: high prevalence of asymptomatic infections.

Malar J 2017 07 26;16(1):300. Epub 2017 Jul 26.

Caucaseco Scientific Research Center, Cali, Colombia.

Background: The recent scale-up in malaria control measures in Latin America has resulted in a significant decrease in the number of reported cases in several countries including Ecuador, where it presented a low malaria incidence in recent years (558 reported cases in 2015) with occasional outbreaks of both Plasmodium falciparum and Plasmodium vivax in the coastal and Amazonian regions. This success in malaria control in recent years has led Ecuador to transition its malaria policy from control to elimination.

Results: This study evaluated the general knowledge, attitude and practices (KAP) about malaria, as well as its prevalence in four communities of an endemic area in northwest Ecuador. A total of 258 interviews to assess KAP in the community indicated that most people in the study area have a basic knowledge about the disease but did not use to contribute to its control. Six hundred and forty-eight blood samples were collected and analysed by thick blood smear and real-time PCR. In addition, the distribution of the infections was mapped in the study communities. Although, no parasites were found by microscopy, by PCR the total malaria prevalence was 7.5% (6.9% P. vivax and 0.6% P. falciparum), much higher than expected and comparable to that reported in endemic areas of neighbouring countries with higher malaria transmission. Serology using ELISA and immunofluorescence indicated 27% respondents for P. vivax and 22% respondents for P. falciparum.

Conclusions: Results suggest that despite a great malaria reduction in Ecuador, transition from control to elimination would demand further improvement in malaria diagnostics, including active case detection to identify and treat parasite asymptomatic carriers, as well as community participation in its elimination.
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http://dx.doi.org/10.1186/s12936-017-1947-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5530496PMC
July 2017

Characterizing the malaria rural-to-urban transmission interface: The importance of reactive case detection.

PLoS Negl Trop Dis 2017 Jul 17;11(7):e0005780. Epub 2017 Jul 17.

Caucaseco Scientific Research Center, Cali, Colombia.

Background: Reported urban malaria cases are increasing in Latin America, however, evidence of such trend remains insufficient. Here, we propose an integrated approach that allows characterizing malaria transmission at the rural-to-urban interface by combining epidemiological, entomological, and parasite genotyping methods.

Methods/principal Findings: A descriptive study that combines active (ACD), passive (PCD), and reactive (RCD) case detection was performed in urban and peri-urban neighborhoods of Quibdó, Colombia. Heads of households were interviewed and epidemiological surveys were conducted to assess malaria prevalence and identify potential risk factors. Sixteen primary cases, eight by ACD and eight by PCD were recruited for RCD. Using the RCD strategy, prevalence of 1% by microscopy (6/604) and 9% by quantitative polymerase chain reaction (qPCR) (52/604) were found. A total of 73 houses and 289 volunteers were screened leading to 41 secondary cases, all of them in peri-urban settings (14% prevalence). Most secondary cases were genetically distinct from primary cases indicating that there were independent occurrences. Plasmodium vivax was the predominant species (76.3%, 71/93), most of them being asymptomatic (46/71). Urban and peri-urban neighborhoods had significant sociodemographic differences. Twenty-four potential breeding sites were identified, all in peri-urban areas. The predominant vectors for 1,305 adults were Anopheles nuneztovari (56,2%) and An. Darlingi (42,5%). One An. nuneztovari specimen was confirmed naturally infected with P. falciparum by ELISA.

Conclusions: This study found no evidence supporting the existence of urban malaria transmission in Quibdó. RCD strategy was more efficient for identifying malaria cases than ACD alone in areas where malaria transmission is variable and unstable. Incorporating parasite genotyping allows discovering hidden patterns of malaria transmission that cannot be detected otherwise. We propose to use the term "focal case" for those primary cases that lead to discovery of secondary but genetically unrelated malaria cases indicating undetected malaria transmission.
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http://dx.doi.org/10.1371/journal.pntd.0005780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5531679PMC
July 2017

Protective Efficacy of Plasmodium vivax Radiation-Attenuated Sporozoites in Colombian Volunteers: A Randomized Controlled Trial.

PLoS Negl Trop Dis 2016 Oct 19;10(10):e0005070. Epub 2016 Oct 19.

Malaria Vaccine and Drug Development Center (MVDC), Cali, Colombia.

Background: Immunizing human volunteers by mosquito bite with radiation-attenuated Plasmodium falciparum sporozoites (RAS) results in high-level protection against infection. Only two volunteers have been similarly immunized with P. vivax (Pv) RAS, and both were protected. A phase 2 controlled clinical trial was conducted to assess the safety and protective efficacy of PvRAS immunization.

Methodology/principal Findings: A randomized, single-blinded trial was conducted. Duffy positive (Fy+; Pv susceptible) individuals were enrolled: 14 received bites from irradiated (150 ± 10 cGy) Pv-infected Anopheles mosquitoes (RAS) and 7 from non-irradiated non-infected mosquitoes (Ctl). An additional group of seven Fy- (Pv refractory) volunteers was immunized with bites from non-irradiated Pv-infected mosquitoes. A total of seven immunizations were carried out at mean intervals of nine weeks. Eight weeks after last immunization, a controlled human malaria infection (CHMI) with non-irradiated Pv-infected mosquitoes was performed. Nineteen volunteers completed seven immunizations (12 RAS, 2 Ctl, and 5 Fy-) and received a CHMI. Five of 12 (42%) RAS volunteers were protected (receiving a median of 434 infective bites) compared with 0/2 Ctl. None of the Fy- volunteers developed infection by the seventh immunization or after CHMI. All non-protected volunteers developed symptoms 8-13 days after CHMI with a mean pre-patent period of 12.8 days. No serious adverse events related to the immunizations were observed. Specific IgG1 anti-PvCS response was associated with protection.

Conclusion: Immunization with PvRAS was safe, immunogenic, and induced sterile immunity in 42% of the Fy+ volunteers. Moreover, Fy- volunteers were refractory to Pv malaria.

Trial Registration: Identifier: NCT01082341.
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http://dx.doi.org/10.1371/journal.pntd.0005070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5070852PMC
October 2016

Malaria elimination challenges in Mesoamerica: evidence of submicroscopic malaria reservoirs in Guatemala.

Malar J 2016 08 30;15(1):441. Epub 2016 Aug 30.

Malaria Vaccine and Drug Development Center (MVDC), Cali, Colombia.

Background: Even though malaria incidence has decreased substantially in Guatemala since 2000, Guatemala remains one of the countries with the highest malaria transmission in Mesoamerica. Guatemala is committed to eliminating malaria as part of the initiative 'Elimination of Malaria in Mesoamerica and the Island of Hispaniola' (EMMIE); however, it is still in the control phase. During the past decade, the government strengthened malaria control activities including mass distribution of long-lasting insecticide-impregnated bed nets, early diagnosis and prompt treatment. This study aimed to determine the prevalence of malaria, including gametocytes, in three areas of Guatemala using active case detection (ACD) and quantitative polymerase chain reaction (qPCR).

Methods: Cross-sectional surveys were conducted in three departments with varying transmission intensities: Escuintla, Alta Verapaz and Zacapa. Blood samples from 706 volunteers were screened for malaria using microscopy and qPCR which was also used to determine the prevalence of gametocytes among infected individuals. Results were collected and analysed using REDCap and R Project, respectively.

Results: Malaria was diagnosed by microscopy in only 2.8 % (4/141) of the volunteers from Escuintla. By contrast, qPCR detected a prevalence of 7.1 % (10/141) in the same volunteers, 8.4 % (36/429) in Alta Verapaz, and 5.9 % (8/136) in Zacapa. Overall, 7.6 % (54/706) of the screened individuals were positive, with an average parasitaemia level of 40.2 parasites/μL (range 1-1133 parasites/μL) and 27.8 % carried mature gametocytes. Fifty-seven percent (31/54) of qPCR positive volunteers were asymptomatic and out of the 42.6 % of symptomatic individuals, only one had a positive microscopy result.

Conclusions: This study found a considerable number of asymptomatic P. vivax infections that were mostly submicroscopic, of which, approximately one-quarter harboured mature gametocytes. This pattern is likely to contribute to maintaining transmission across the region. Robust surveillance systems, molecular diagnostic tests and tailored malaria detection activities for each endemic site may prove to be imperative in accelerating malaria elimination in Guatemala and possibly across all of Mesoamerica.
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http://dx.doi.org/10.1186/s12936-016-1500-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006524PMC
August 2016

Optimization of a Membrane Feeding Assay for Plasmodium vivax Infection in Anopheles albimanus.

PLoS Negl Trop Dis 2016 06 29;10(6):e0004807. Epub 2016 Jun 29.

Caucaseco Scientific Research Center, Cali, Cali, Valle de Cauca, Colombia.

Introduction: Individuals exposed to malaria infections for a long time develop immune responses capable of blocking Plasmodium transmission to mosquito vectors, potentially limiting parasite spreading in nature. Development of a malaria TB vaccine requires a better understanding of the mechanisms and main effectors responsible for transmission blocking (TB) responses. The lack of an in vitro culture system for Plasmodium vivax has been an important drawback for development of a standardized method to assess TB responses to this parasite. This study evaluated host, vector, and parasite factors that may influence Anopheles mosquito infection in order to develop an efficient and reliable assay to assess the TB immunity.

Methods/principal Findings: A total of 94 P. vivax infected patients were enrolled as parasite donors or subjects of direct mosquito feeding in two malaria endemic regions of Colombia (Tierralta, and Buenaventura). Parasite infectiousness was assessed by membrane feeding assay or direct feeding assay using laboratory reared Anopheles mosquitoes. Infection was measured by qPCR and by microscopically examining mosquito midguts at day 7 for the presence of oocysts. Best infectivity was attained in four day old mosquitoes fed at a density of 100 mosquitos/cage. Membrane feeding assays produced statistically significant better infections than direct feeding assays in parasite donors; cytokine profiles showed increased IFN-γ, TNF and IL-1 levels in non-infectious individuals. Mosquito infections and parasite maturation were more reliably assessed by PCR compared to microscopy.

Conclusions: We evaluated mosquito, parasite and host factors that may affect the outcome of parasite transmission as measured by artificial membrane feeding assays. Results have led us to conclude that: 1) optimal mosquito infectivity occurs with mosquitoes four days after emergence at a cage density of 100; 2) mosquito infectivity is best quantified by PCR as it may be underestimated by microscopy; 3) host cellular immune response did not appear to significantly affect mosquito infectivity; and 4) no statistically significant difference was observed in transmission between mosquitoes directly feeding on humans and artificial membrane feeding assays.
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http://dx.doi.org/10.1371/journal.pntd.0004807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4927173PMC
June 2016

Population genomics studies identify signatures of global dispersal and drug resistance in Plasmodium vivax.

Nat Genet 2016 08 27;48(8):953-8. Epub 2016 Jun 27.

Instituto de Medicine Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru.

Plasmodium vivax is a major public health burden, responsible for the majority of malaria infections outside Africa. We explored the impact of demographic history and selective pressures on the P. vivax genome by sequencing 182 clinical isolates sampled from 11 countries across the globe, using hybrid selection to overcome human DNA contamination. We confirmed previous reports of high genomic diversity in P. vivax relative to the more virulent Plasmodium falciparum species; regional populations of P. vivax exhibited greater diversity than the global P. falciparum population, indicating a large and/or stable population. Signals of natural selection suggest that P. vivax is evolving in response to antimalarial drugs and is adapting to regional differences in the human host and the mosquito vector. These findings underline the variable epidemiology of this parasite species and highlight the breadth of approaches that may be required to eliminate P. vivax globally.
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http://dx.doi.org/10.1038/ng.3588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347536PMC
August 2016

Global genetic diversity of the Plasmodium vivax transmission-blocking vaccine candidate Pvs48/45.

Malar J 2016 Apr 12;15:202. Epub 2016 Apr 12.

Caucaseco Scientific Research Center, Cali, Colombia.

Background: Plasmodium vivax 48/45 protein is expressed on the surface of gametocytes/gametes and plays a key role in gamete fusion during fertilization. This protein was recently expressed in Escherichia coli host as a recombinant product that was highly immunogenic in mice and monkeys and induced antibodies with high transmission-blocking activity, suggesting its potential as a P. vivax transmission-blocking vaccine candidate. To determine sequence polymorphism of natural parasite isolates and its potential influence on the protein structure, all pvs48/45 sequences reported in databases from around the world as well as those from low-transmission settings of Latin America were compared.

Methods: Plasmodium vivax parasite isolates from malaria-endemic regions of Colombia, Brazil and Honduras (n = 60) were used to sequence the Pvs48/45 gene, and compared to those previously reported to GenBank and PlasmoDB (n = 222). Pvs48/45 gene haplotypes were analysed to determine the functional significance of genetic variation in protein structure and vaccine potential.

Results: Nine non-synonymous substitutions (E35K, Y196H, H211N, K250N, D335Y, E353Q, A376T, K390T, K418R) and three synonymous substitutions (I73, T149, C156) that define seven different haplotypes were found among the 282 isolates from nine countries when compared with the Sal I reference sequence. Nucleotide diversity (π) was 0.00173 for worldwide samples (range 0.00033-0.00216), resulting in relatively high diversity in Myanmar and Colombia, and low diversity in Mexico, Peru and South Korea. The two most frequent substitutions (E353Q: 41.9 %, K250N: 39.5 %) were predicted to be located in antigenic regions without affecting putative B cell epitopes or the tertiary protein structure.

Conclusions: There is limited sequence polymorphism in pvs48/45 with noted geographical clustering among Asian and American isolates. The low genetic diversity of the protein does not influence the predicted antigenicity or protein structure and, therefore, supports its further development as transmission-blocking vaccine candidate.
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http://dx.doi.org/10.1186/s12936-016-1263-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4828788PMC
April 2016

Consistent prevalence of asymptomatic infections in malaria endemic populations in Colombia over time.

Malar J 2016 Feb 6;15:70. Epub 2016 Feb 6.

Clinical Trials Unit, Malaria Vaccine and Drug Development Center, Cali, Colombia.

Background: Malaria control programmes rely on confirmation of parasite presence in patients' blood prior to treatment administration. Plasmodium parasites are detected mostly by microscopy or rapid diagnostic test (RDT). Although these methods contribute significantly to malaria control/elimination, they are not suitable for detecting the significant proportion of asymptomatic subjects harbouring low levels of parasitaemia, which endure untreated as potential reservoirs for transmission. Malaria prevalence was assessed in endemic regions of Colombia over a 4-year follow-up.

Methods: A series of cross-sectional surveys were conducted between 2011 and 2014 in low to moderate malaria transmission sentinel sites (SS) of Tumaco, Buenaventura and Tierralta municipalities of Colombia. A census was performed and a random sample of houses was selected from each SS prior to each survey. Inhabitants were asked to answer a questionnaire on clinical, epidemiological and demographic aspects, and to provide a blood sample for malaria diagnosis using microscopy and quantitative real time polymerase chain reaction (qPCR).

Results: A total of 3059 blood samples were obtained from all SS, 58.5 % of which were from women and displayed a malaria prevalence ranging from 4 % (95 % CI 3-5 %) to 10 % (95 % CI 8-12 %) in the 4 years' study period. Almost all malaria cases (n = 220, 97 %) were sub-microscopic and only detectable by qPCR; 90 % of the cases were asymptomatic at the time of blood collection. While Buenaventura and Tierralta had a decreasing tendency during the follow-up, Tumaco had a rise in 2013 and then a decrease in 2014. Plasmodium vivax accounted for the majority (66-100 %) of cases in Tierralta and Buenaventura and for 25-50 % of the cases in Tumaco.

Conclusions: This study demonstrates an important prevalence of asymptomatic malaria cases not detectable by microscopy, which therefore remain untreated representing a parasite pool for malaria transmission. This demands the introduction of alternative strategies for diagnosis and treatment, especially for areas of low transmission to reduce it to appropriate levels for malaria pre-elimination efforts to start.
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http://dx.doi.org/10.1186/s12936-016-1124-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4744459PMC
February 2016

Plasmodium vivax gametocyte infectivity in sub-microscopic infections.

Malar J 2016 Jan 28;15:48. Epub 2016 Jan 28.

Malaria Vaccine and Drug Development Centre (MVDC), Cali, Colombia.

Background: The use of molecular techniques has put in the spotlight the existence of a large mass of malaria sub-microscopic infections among apparently healthy populations. These sub-microscopic infections are considered an important pool for maintained malaria transmission.

Methods: In order to assess the appearance of Plasmodium vivax gametocytes in circulation, gametocyte density and the parasite infectivity to Anopheles mosquitoes, a study was designed to compare three groups of volunteers either experimentally infected with P. vivax sporozoites (early infections; n = 16) or naturally infected patients (acute malaria, n = 16 and asymptomatic, n = 14). In order to determine gametocyte stage, a quantitative reverse transcriptase PCR (RT-qPCR) assay targeting two sexual stage-specific molecular markers was used. Parasite infectivity was assessed by membrane feeding assays (MFA).

Results: In early infections P. vivax gametocytes could be detected starting at day 7 without giving rise to infected mosquitoes during 13 days of follow-up. Asymptomatic carriers, with presumably long-lasting infections, presented the highest proportion of mature gametocytes and were as infective as acute patients.

Conclusions: This study shows the potential role of P. vivax asymptomatic carriers in malaria transmission should be considered when new policies are envisioned to redirect malaria control strategies towards targeting asymptomatic infections as a tool for malaria elimination.
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http://dx.doi.org/10.1186/s12936-016-1104-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4730736PMC
January 2016

Multiplicity of Infection and Disease Severity in Plasmodium vivax.

PLoS Negl Trop Dis 2016 Jan 11;10(1):e0004355. Epub 2016 Jan 11.

Institute for Genomics and Evolutionary Medicine (igem), Temple University, Philadelphia, Pennsylvania, United States of America.

Background: Multiplicity of infection (MOI) refers to the average number of distinct parasite genotypes concurrently infecting a patient. Although several studies have reported on MOI and the frequency of multiclonal infections in Plasmodium falciparum, there is limited data on Plasmodium vivax. Here, MOI and the frequency of multiclonal infections were studied in areas from South America where P. vivax and P. falciparum can be compared.

Methodology/principal Findings: As part of a passive surveillance study, 1,328 positive malaria patients were recruited between 2011 and 2013 in low transmission areas from Colombia. Of those, there were only 38 P. vivax and 24 P. falciparum clinically complicated cases scattered throughout the time of the study. Samples from uncomplicated cases were matched in time and location with the complicated cases in order to compare the circulating genotypes for these two categories. A total of 92 P. vivax and 57 P. falciparum uncomplicated cases were randomly subsampled. All samples were genotyped by using neutral microsatellites. Plasmodium vivax showed more multiclonal infections (47.7%) than P. falciparum (14.8%). Population genetics and haplotype network analyses did not detect differences in the circulating genotypes between complicated and uncomplicated cases in each parasite. However, a Fisher exact test yielded a significant association between having multiclonal P. vivax infections and complicated malaria. No association was found for P. falciparum infections.

Conclusion: The association between multiclonal infections and disease severity in P. vivax is consistent with previous observations made in rodent malaria. The contrasting pattern between P. vivax and P. falciparum could be explained, at least in part, by the fact that P. vivax infections have lineages that were more distantly related among them than in the case of the P. falciparum multiclonal infections. Future research should address the possible role that acquired immunity and exposure may have on multiclonal infections and their association with disease severity.
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http://dx.doi.org/10.1371/journal.pntd.0004355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709143PMC
January 2016

Whole Genome Sequencing of Field Isolates Reveals Extensive Genetic Diversity in Plasmodium vivax from Colombia.

PLoS Negl Trop Dis 2015 Dec 28;9(12):e0004252. Epub 2015 Dec 28.

The Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America.

Plasmodium vivax is the most prevalent malarial species in South America and exerts a substantial burden on the populations it affects. The control and eventual elimination of P. vivax are global health priorities. Genomic research contributes to this objective by improving our understanding of the biology of P. vivax and through the development of new genetic markers that can be used to monitor efforts to reduce malaria transmission. Here we analyze whole-genome data from eight field samples from a region in Cordóba, Colombia where malaria is endemic. We find considerable genetic diversity within this population, a result that contrasts with earlier studies suggesting that P. vivax had limited diversity in the Americas. We also identify a selective sweep around a substitution known to confer resistance to sulphadoxine-pyrimethamine (SP). This is the first observation of a selective sweep for SP resistance in this species. These results indicate that P. vivax has been exposed to SP pressure even when the drug is not in use as a first line treatment for patients afflicted by this parasite. We identify multiple non-synonymous substitutions in three other genes known to be involved with drug resistance in Plasmodium species. Finally, we found extensive microsatellite polymorphisms. Using this information we developed 18 polymorphic and easy to score microsatellite loci that can be used in epidemiological investigations in South America.
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http://dx.doi.org/10.1371/journal.pntd.0004252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692395PMC
December 2015

Malaria Molecular Epidemiology: Lessons from the International Centers of Excellence for Malaria Research Network.

Am J Trop Med Hyg 2015 Sep 10;93(3 Suppl):79-86. Epub 2015 Aug 10.

Molecular epidemiology leverages genetic information to study the risk factors that affect the frequency and distribution of malaria cases. This article describes molecular epidemiologic investigations currently being carried out by the International Centers of Excellence for Malaria Research (ICEMR) network in a variety of malaria-endemic settings. First, we discuss various novel approaches to understand malaria incidence and gametocytemia, focusing on Plasmodium falciparum and Plasmodium vivax. Second, we describe and compare different parasite genotyping methods commonly used in malaria epidemiology and population genetics. Finally, we discuss potential applications of molecular epidemiological tools and methods toward malaria control and elimination efforts.
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http://dx.doi.org/10.4269/ajtmh.15-0005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574277PMC
September 2015

High prevalence of sub-microscopic infections in Colombia.

Malar J 2015 May 15;14:201. Epub 2015 May 15.

Caucaseco Scientific Research Center, Cali, Colombia.

Background: Malaria transmission in Latin America is typically characterized as hypo-endemic and unstable with ~170 million inhabitants at risk of malaria infection. Although Colombia has witnessed an important decrease in malaria transmission, the disease remains a public health problem with an estimated ~10 million people currently living in areas with malaria risk and ~61,000 cases reported in 2012. This study aimed to establish the malaria prevalence in three endemic regions of Colombia to aid in designing new interventions for malaria elimination.

Methods: A cross-sectional survey was conducted in three regions of Colombia with different malaria epidemiological profiles: Tierralta (Ta), Tumaco (Tu) and Buenaventura (Bv). The Annual Parasite Index (API) was 10.7, 6.9 and 3.1, respectively. Participants were asked to respond to a sociodemographic questionnaire and then were bled to determine the Duffy genotype and the prevalence of malaria infection by microscopy and quantitative real-time PCR (qPCR).

Results: The study was conducted between October 2011 and January 2012. Eight sentinel sites with 1,169 subjects from 267 households were included. The overall prevalence of sub-microscopic infections measured by thick blood smear (TBS) was 0.3% (n=4) whereas by qPCR it was 9.7% (n=113), with a greater proportion (13%) in 40-50 years old individuals. Furthermore, different regions displayed different prevalence of sub-microscopic infections: Bv 12%, Ta 15%, and Tu 4%. From these 113 samples (qPCR), 74% were positive for P. vivax and 22% for P. falciparum, and 4% were mixed infections, which correlates to the overall parasite prevalence in Colombia. This study showed that in the southern Pacific coast of Colombia (Bv and Tu), around 56% of the population have a Duffy-negative genotype, compared to the northern region (Ta) where the percentage of Duffy-negative genotype is around 3%.

Conclusions: Sub-microscopic infections are prevalent across different regions in Colombia, particularly in areas with relatively low transmission intensity. The poor microscopy results suggest the need for more sensitive diagnostic tools for detection of sub-microscopic infections. This study underscores the importance of conducting active case surveillance to more accurately determine malaria incidence, and highlights the need for updating the malaria guidelines to track and treat sub-microscopic malaria infections.
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http://dx.doi.org/10.1186/s12936-015-0711-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4438632PMC
May 2015

Recombinant Pvs48/45 antigen expressed in E. coli generates antibodies that block malaria transmission in Anopheles albimanus mosquitoes.

PLoS One 2015 16;10(3):e0119335. Epub 2015 Mar 16.

Malaria Vaccine and Drug Development Center, Cali, Colombia; Caucaseco Scientific Research Center, Cali, Colombia; Primates Center Foundation, Cali, Colombia.

Transmission of malaria parasites from humans to Anopheles mosquitoes can be inhibited by specific antibodies elicited during malaria infection, which target surface Plasmodium gametocyte/gamete proteins. Some of these proteins may have potential for vaccine development. Pvs48/45 is a P. vivax gametocyte surface antigen orthologous to Pfs48/45, which may play a role during parasite fertilization and thus has potential for transmission blocking (TB) activity. Here we describe the expression of a recombinant Pvs48/45 protein expressed in Escherichia coli as a ∼60kDa construct which we tested for antigenicity using human sera and for its immunogenicity and transmission blocking activity of specific anti-mouse and anti-monkey Pvs48/45 antibodies. The protein reacted with sera of individuals from malaria-endemic areas and in addition induced specific IgG antibody responses in BALB/c mice and Aotus l. griseimembra monkeys. Sera from both immunized animal species recognized native P. vivax protein in Western blot (WB) and immunofluorescence assays. Moreover, sera from immunized mice and monkeys produced significant inhibition of parasite transmission to An. Albimanus mosquitoes as shown by membrane feeding assays. Results indicate the presence of reactive epitopes in the Pvs48/45 recombinant product that induce antibodies with TB activity. Further testing of this protein is ongoing to determine its vaccine potential.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0119335PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361554PMC
February 2016

Evaluation of the loop mediated isothermal DNA amplification (LAMP) kit for malaria diagnosis in P. vivax endemic settings of Colombia.

PLoS Negl Trop Dis 2015 Jan 8;9(1):e3453. Epub 2015 Jan 8.

Caucaseco Scientific Research Center, Cali, Colombia.

Background: Most commonly used malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections which are frequent in low transmission settings. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too laborious for field deployment. In this study, the applicability of a malaria diagnosis kit based on loop-mediated isothermal amplification (mLAMP) was assessed in malaria endemic areas of Colombia with Plasmodium vivax predominance.

Methodology/principal Findings: First, a passive case detection (PCD) study on 278 febrile patients recruited in Tierralta (department of Cordoba) was conducted to assess the diagnostic performance of the mLAMP method. Second, an active case detection (ACD) study on 980 volunteers was conducted in 10 sentinel sites with different epidemiological profiles. Whole blood samples were processed for microscopic and mLAMP diagnosis. Additionally RT-PCR and nested RT-PCR were used as reference tests. In the PCD study, P. falciparum accounted for 23.9% and P. vivax for 76.1% of the infections and no cases of mixed-infections were identified. Microscopy sensitivity for P. falciparum and P. vivax were 100% and 86.1%, respectively. mLAMP sensitivity for P. falciparum and P. vivax was 100% and 91.4%, respectively. In the ACD study, mLAMP detected 65 times more cases than microscopy. A high proportion (98.0%) of the infections detected by mLAMP was from volunteers without symptoms.

Conclusions/significance: mLAMP sensitivity and specificity were comparable to RT-PCR. LAMP was significantly superior to microscopy and in P. vivax low-endemicity settings and under minimum infrastructure conditions, it displayed sensitivity and specificity similar to that of single-well RT-PCR for detection of both P. falciparum and P. vivax infections. Here, the dramatically increased detection of asymptomatic malaria infections by mLAMP demonstrates the usefulness of this new tool for diagnosis, surveillance, and screening in elimination strategies.
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http://dx.doi.org/10.1371/journal.pntd.0003453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287555PMC
January 2015

Knowledge, attitudes and practices of malaria in Colombia.

Malar J 2014 May 1;13:165. Epub 2014 May 1.

Caucaseco Scientific Research Center, Cali, Colombia.

Background: Although Colombia has witnessed an important decrease in malaria transmission, the disease remains a public health problem with an estimated ~10 million people currently living in areas with malaria risk and ~61,000 cases reported in 2012. This study aimed to determine and compare the level of knowledge, attitudes and practices (KAP) about malaria in three endemic communities of Colombia to provide the knowledge framework for development of new intervention strategies for malaria elimination.

Methods: A cross-sectional KAP survey was conducted in the municipalities of Tierralta, Buenaventura and Tumaco, categorized according to high risk (HR) and moderate risk (MR) based on the annual parasite index (API). Surveys were managed using REDCap and analysed using MATLAB and GraphPad Prism.

Results: A total of 267 residents, mostly women (74%) were surveyed. Although no differences were observed on the knowledge of classical malaria symptoms between HR and MR regions, significant differences were found in knowledge and attitudes about transmission mechanisms, anti-malarial use and malaria diagnosis. Most responders in both regions (93.5% in MR, and 94.3% in HR areas) indicated use of insecticide-treated nets (ITNs) to protect themselves from malaria, and 75.5% of responders in HR indicated they did nothing to prevent malaria transmission outdoors. Despite a high level of knowledge in the study regions, significant gaps persisted relating to practices. Self-medication and poor adherence to treatment, as well as lack of both indoor and outdoor vector control measures, were significantly associated with higher malaria risk.

Conclusions: Although significant efforts are currently being made by the Ministry of Health to use community education as one of the main components of the control strategy, these generic education programmes may not be applicable to all endemic regions of Colombia given the substantial geographic, ethnic and cultural diversity.
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http://dx.doi.org/10.1186/1475-2875-13-165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113137PMC
May 2014

Field evaluation of an automated RDT reader and data management device for Plasmodium falciparum/Plasmodium vivax malaria in endemic areas of Colombia.

Malar J 2014 Mar 10;13:87. Epub 2014 Mar 10.

Caucaseco Scientific Research Center, Cali, Colombia.

Background: Massive implementation of malaria diagnostics in low-resource countries is regarded as a pivotal strategy in control and elimination efforts. Although malaria rapid diagnostic tests (RDTs) are considered a viable alternative, there are still obstacles to the widespread implementation of this strategy, such as reporting constraints and lack of proper quality assurance of RDT-based programmes at point-of-care (POC).

Methods: A prospective cohort of patients, seeking routine care for febrile episodes at health centres in malaria-endemic areas of Colombia, was used to assess the diagnostic performance of a device based on smartphone technology (Deki ReaderTM, former codename "GenZero"), that assists users at POC to process RDTs. After informed consent, patients were enrolled into the study and blood samples were collected for thick blood smear (TBS) and RDT. The RDT results were interpreted by both visual inspection and Deki Reader device and concordance between visual and device interpretation was measured. Microscopy corrected by real-time polymerase chain reaction (PCR) and microscopy were "gold standard" tests to assess the diagnostic performance.

Results: In total, 1,807 patients were enrolled at seven health centres in malaria-endemic areas of Colombia. Thirty-three Plasmodium falciparum and 100 Plasmodium vivax cases were positive by corrected microscopy. Both sensitivity and specificity were 93.9% (95% CI 69.7-95.2) and 98.7% (95% CI 98.5-99.4) for P. falciparum, and 98.0% (95% CI 90.3-98.9) and 97.9% (95% CI 97.1-98.5) for P. vivax. Percentage concordance between visual and device interpretation of RDT was 98.5% and 99.0% for P. vivax and P. falciparum, respectively.The RDT, when compared to TBS, showed high sensitivity and specificity for P. falciparum in both visual and device interpretation, and good overall diagnostic performance for P. vivax. Comparison between PCR as gold standard and visual and device interpretation showed acceptable overall performance for both species.

Conclusions: The diagnostic performance of the Deki Reader was comparable to visual interpretation of RDTs (without significant differences) for both malaria species. Providing standardized automated interpretation of RDTs at POC in remote areas, in addition to almost real-time reporting of cases and enabling quality control would greatly benefit large-scale implementation of RDT-based malaria diagnostic programmes.
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http://dx.doi.org/10.1186/1475-2875-13-87DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3995821PMC
March 2014

Characterization of a malaria outbreak in Colombia in 2010.

Malar J 2013 Sep 17;12:330. Epub 2013 Sep 17.

National Institute of Health of Colombia, Bogotá, Colombia.

Background: Although malaria has presented a significant reduction in morbidity and mortality worldwide during the last decade, it remains a serious global public health problem. In Colombia, during this period, many factors have contributed to sustained disease transmission, with significant fluctuations in an overall downward trend in the number of reported malaria cases. Despite its epidemiological importance, few studies have used surveillance data to describe the malaria situation in Colombia. This study aims to describe the characteristics of malaria cases reported during 2010 to the Public Health Surveillance System (SIVIGILA) of the National Institute of Health (INS) of Colombia.

Methods: A descriptive study was conducted using malaria information from SIVIGILA 2010. Cases, frequencies, proportions, ratio and measures of central tendency and data dispersion were calculated. In addition, the annual parasite index (API) and the differences between the variables reported in 2009 and 2010 were estimated.

Results: A total of 117,108 cases were recorded by SIVIGILA in 2010 for a national API of 10.5/1,000 habitants, with a greater number of cases occurring during the first half of the year. More than 90% of cases were reported in seven departments (=states): Antioquia: 46,476 (39.7%); Chocó: 22,493 (19.2%); Cordoba: 20,182 (17.2%); Valle: 6,360 (5.4%); Guaviare: 5,876 (5.0%); Nariño: 4,085 (3.5%); and Bolivar: 3,590 (3.1%). Plasmodium vivax represented ~71% of the cases; Plasmodium falciparum ~28%; and few infrequent cases caused by Plasmodium malariae.

Conclusions: Overall, a greater incidence was found in men (65%) than in women (35%). Although about a third of cases occurred in children <15 years, most of these cases occurred in children >5 years of age. The ethnic distribution indicated that about 68% of the cases occurred in mestizos and whites, followed by 23% in Afro-descendants, and the remainder (9%) in indigenous communities. In over half of the cases, consultation occurred early, with 623 complicated and 23 fatal cases. However, the overall incidence increased, corresponding to an epidemic burst and indicating the need to strengthen prevention and control activities as well as surveillance to reduce the risk of outbreaks and the consequent economic and social impact.
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http://dx.doi.org/10.1186/1475-2875-12-330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3848715PMC
September 2013
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