Nucleic acid degradation after long-term dried blood spot storage.

Collecting and preserving biological samples in the field, particularly in remote areas in tropical forests, prior to laboratory analysis is challenging. Blood samples in many cases are used for nucleic acid-based species determination, genomics or pathogen research. In most cases, maintaining a cold chain is impossible and samples remain at ambient temperature for extended periods of time before controlled storage conditions become available. Dried blood spot (DBS) storage, blood stored on cellulose-based paper, has been widely applied to facilitate sample collection and preservation in the field for decades. However, it is unclear how long-term storage on this substrate affects nucleic acid concentration and integrity. We analysed nucleic acid quality from DBS stored on Whatman filter paper no. 3 and FTA cards for up to 15 years in comparison to cold-chain stored samples using four nucleic acid extraction methods. We examined the ability to identify viral sequences from samples of 12 free-ranging primates in the Amazon forest, using targeted hybridization capture, and determined if mitochondrial genomes could be retrieved. The results suggest that even after extended periods of storage, DBS will be suitable for some genomic applications but may be of limited use for viral pathogen research, particularly RNA viruses.

Toxoplasma gondii in a Remote Subsistence Hunting-Based Indigenous Community of the Peruvian Amazon.

Toxoplasma gondii is a ubiquitous zoonotic protozoan parasite that infects a wide variety range of warm-blooded animals. This study describes the epidemiological scenario of T. gondii in an indigenous community that relies on subsistence hunting in a well-conserved and isolated area of the Peruvian Amazon. The high seropositivity against T. gondii in humans (83.3% IgG and 6.1% IgM), wild mammals (30.45%, 17 species), peri-domestic rodents (10.0% Rattus sp.), and domestic animals (94.1% dogs and 100% cats) indicates the existence of a sylvatic cycle in the community under study. Individual age was found to be positively associated with IgG detection against T. gondii but not with IgM. It is estimated that each family consumed 5.67 infected animals per year with terrestrial species having higher infective rates than arboreal species. The main risk factors included improper handling and cooking of wild meat, poor hygiene practices, and feeding uncooked offal to domestic animals. This scenario results in a continuous process of infection and reinfection within the indigenous community with cats, dogs, and peri-domestic animals becoming infected through the ingestion of infected raw viscera. Our results emphasize the need to promote safe food handling practices and disposal of waste materials from hunted animals in such communities.

Phylogenetic congruence of Plasmodium spp. and wild ungulate hosts in the Peruvian Amazon.

Malaria parasites are known to infect a variety of vertebrate hosts, including ungulates. However, ungulates of Amazonia have not been investigated. We report for the first time, the presence of parasite lineages closely related to Plasmodium odocoilei clade 1 and clade 2 in free-ranging South American red-brocket deer (Mazama americana; 44.4%, 4/9) and gray-brocket deer (Mazama nemorivaga; 50.0%, 1/2). We performed PCR-based analysis of blood samples from 47 ungulates of five different species collected during subsistence hunting by an indigenous community in the Peruvian Amazon. We detected Plasmodium malariae/brasilianum lineage in a sample from red-brocket deer. However, no parasite DNA was detected in collared peccary (Pecari tajacu; 0.0%, 0/10), white-lipped peccary (Tayassu pecari; 0.0%, 0/15), and tapir (Tapirus terrestris; 0.0%, 0/11). Concordant phylogenetic analyses suggested a possible co-evolutionary relationship between the Plasmodium lineages found in American deer and their hosts.

Dengue and COVID-19 Co-Circulation in the Peruvian Amazon: A Population-Based Study.

The COVID-19 pandemic affected the main Amazon cities dramatically, with Iquitos City reporting the highest seroprevalence of anti-SARS-CoV-2 antibodies during the first COVID-19 wave worldwide. This phenomenon raised many questions about the possibility of a co-circulation of dengue and COVID-19 and its consequences. We carried out a population-based cohort study in Iquitos, Peru. We obtained a venous blood sample from a subset of 326 adults from the Iquitos COVID-19 cohort (August 13-18, 2020) to estimate the seroprevalence of anti-dengue virus (DENV) and anti-SARS-CoV-2 antibodies. We tested each serum sample for anti-DENV IgG (serotypes 1, 2, 3, and 4) and SARS-CoV-2 antibodies anti-spike IgG and IgM by ELISA. We estimated an anti-SARS-CoV-2 seroprevalence of 78.0% (95% CI, 73.0-82.0) and an anti-DENV seroprevalence of 88.0% (95% CI, 84.0-91.6), signifying a high seroprevalence of both diseases during the first wave of COVID-19 transmission in the city. The San Juan District had a lower anti-DENV antibody seroprevalence than the Belen District (prevalence ratio, 0.90; 95% CI, 0.82-0.98). However, we did not observe these differences in anti-SARS-CoV-2 antibody seroprevalence. Iquitos City presented one of the highest seroprevalence rates of anti-DENV and anti-SARS-CoV-2 antibodies worldwide, but with no correlation between their antibody levels.

Haemosporidians from a Neglected Group of Terrestrial Wild Birds in the Peruvian Amazonia.

Haemosporidians are a widespread group of blood parasites transmitted by vectors. Despite their relevance for bird conservation, few studies have been conducted in the Amazonia and even less in terrestrial wild birds. We analysed blood samples from 168 game birds, collected from 2008 to 2015 by subsistence hunters of an indigenous rural community in the Peruvian Amazonia. DNA was tested for Haemoproteus spp., Plasmodium spp. and Leucocytozoon spp. and positive amplicons were sequenced and curated for phylogenetic analysis. Haemosporidian prevalence was 72% overall, 66.7% for Haemoproteus spp. and 5.4% for Plasmodium spp. and respectively by bird species: Spix's Guan (Penelope jacquacu, n = 72) 87.5% and 0%, Razor-billed Curassow (Mitu tuberosum, n = 45) 77.8% and 6.7%, White-winged Trumpeter (Psophia leucoptera, n = 20) 6.3% and 12.5%, Blue-throated Piping-guan (Pipile cumanensis, n = 16) 73.3% and 6.7%, and Great Tinamou (Tinamus major, n = 15) 10% and 15%. Leucocytozoon spp. was not found. P. leucoptera and T. major were less likely to be infected with Haemoproteus spp. Fruit abundance had a negative association with Haemoproteus spp. prevalence and precipitation was negatively associated with Plasmodium spp. prevalence. The 106 sequences examined represented 29 lineages, 82.8% of them were new lineages (Plasmodium n = 3, Haemoproteus n = 21). Novel host-parasite associations and lineages were unveiled, including probably new species of Plasmodium spp. Our results highlight the scientific value of alternative sampling methods and the collaboration with local communities.

Lipid species affect morphology of endoplasmic reticulum: a sea urchin oocyte model of reversible manipulation.

The ER is a large multifunctional organelle of eukaryotic cells. Malfunction of the ER in various disease states, such as atherosclerosis, diabetes, cancer, Alzheimer's and Parkinson's and amyotrophic lateral sclerosis, often correlates with alterations in its morphology. The ER exhibits regionally variable membrane morphology that includes, at the extremes, large relatively flat surfaces and interconnected tubular structures highly curved in cross-section. ER morphology is controlled by shaping proteins that associate with membrane lipids. To investigate the role of these lipids, we developed a sea urchin oocyte model, a relatively quiescent cell in which the ER consists mostly of tubules. We altered levels of endogenous diacylglycerol (DAG), phosphatidylethanolamine (PtdEth), and phosphatidylcholine by microinjection of enzymes or lipid delivery by liposomes and evaluated shape changes with 2D and 3D confocal imaging and 3D electron microscopy. Decreases and increases in the levels of lipids such as DAG or PtdEth characterized by negative spontaneous curvature correlated with conversion to sheet structures or tubules, respectively. The effects of endogenous alterations of DAG were reversible upon exogenous delivery of lipids of negative spontaneous curvature. These data suggest that proteins require threshold amounts of such lipids and that localized deficiencies of the lipids could contribute to alterations of ER morphology. The oocyte modeling system should be beneficial to studies directed at understanding requirements of lipid species in interactions leading to alterations of organelle shaping.

Molecular Detection of Bartonella bacilliformis in Lutzomyia maranonensis in Cajamarca, Peru: A New Potential Vector of Carrion's Disease in Peru?

Carrion's disease is a neglected, vector-borne illness that affects Colombia, Ecuador, and especially Peru. The phlebotomine sand flies Lutzomyia verrucarum and Lutzomyia peruensis are the main illness vectors described, although other species may be implicated in endemic areas such as some northern Peruvian regions, in which Carrion's disease vector has not been established. The aim of this study was to evaluate the presence of Bartonella bacilliformis DNA in Lutzomyia maranonensis from Cajamarca, northern Peru. This sand fly has not been defined as a vector yet. Centers for Disease Control and Prevention light traps were used to collect adult phlebotomine sand flies from 2007 to 2008 in the Cajamarca department. Female specimens were identified using morphological keys and were grouped into pools of five sand flies, taking into account district and sampling site (intradomicile or peridomicile). DNA was extracted, and then conventional and real-time polymerase chain reaction (RT-PCR) were performed to detect B. bacilliformis and subsequently confirmed by sequencing. A total of 383 specimens of L. maranonensis species were analyzed. Two of 76 pools were positive for B. bacilliformis by sequencing; all positives pools were from Querocotillo district. In addition, Mesorhizobium spp. were identified in two pools of sand flies, which is an α-proteobacteria phylogenetically very close to B. bacilliformis. This study presents molecular evidence that suggests L. maranonensis is naturally infected by B. bacilliformis in the Cajamarca department. Further research should determine if L. maranonensis is a vector and could transmit B. bacilliformis.

Obesity is an independent risk factor for heart failure: Zona Franca Cohort study.

BACKGROUND: Heart failure (HF) is a major problem in developed countries. However, its relationship with obesity remains unclear, especially in low-risk populations. The objective of the study was to analyze the relationship between obesity and HF in a low-risk Mediterranean population. HYPOTHESIS: Obesity is an independent predictor for HF. METHODS: A prospective community-based population cohort study with 10 years' follow-up was conducted at 2 healthcare centers in the city of Barcelona, Spain. From a registered population of 35 275, the study included 932 randomly selected patients without HF, age 35-84 years. Obesity was defined as body mass index (BMI) ≥30 and HF according to European Society of Cardiology guidelines, confirmed by echocardiography. Cox proportional hazards regression was used to examine the association between obesity and heart failure. RESULTS: The difference in HF incidence between obese subjects (4.7%) and nonobese subjects (1.6%) was 3.1% (95% confidence interval [CI]: 0.7-5.5). In the unadjusted model, incident HF was significantly associated with BMI: the hazard ratio [HR] was 1.09 for every 1 kg/m² increase (95% CI: 1.05-1.14) and 3.01 for BMI ≥30 (95% CI: 1.34-6.77). After adjusting for age, sex, hypertension, ischemic heart disease, and diabetes mellitus, the results were similar: HR 1.06 (95% CI: 1.01-1.10) and HR 2.45 for BMI ≥30 (95% CI: 1.02-5.61). Overweight was not associated with HF in any of the models. The population-attributable risk of HF due to obesity was 43.0% (95% CI: 13.9-74.9). CONCLUSIONS: High rate differences, HRs, and attributable risk indicate that obesity is an important risk factor for incident HF.

The epidemiology of cardiovascular disease in primary care. the Zona Franca Cohort study in Barcelona, Spain.

INTRODUCTION AND OBJECTIVES: Knowledge about the incidence of cardiovascular disease in Spain is limited, particularly about emergent conditions such as peripheral arterial disease and heart failure. The objective of this study was to determine the incidence of these conditions in a primary care setting. METHODS: A prospective population-based cohort study was carried out in two health centers in Barcelona, Spain. It included 931 randomly selected individuals, aged 35 to 84 years, without cardiovascular disease who were followed for 10 years. We assessed the incidence of ischemic heart disease (i.e. myocardial infarction and angina), cerebrovascular disease (i.e. stroke and transient ischemic attack), peripheral arterial disease of the lower extremities, and heart failure. RESULTS: The most frequently occurring condition was ischemic heart disease (cumulative incidence, 6.1%), followed by cerebrovascular disease (4.3%), heart failure (3.0%), and peripheral arterial disease (1.9%). These conditions were more frequent in patients aged >65 years (cumulative incidence, 9.6%, 8.9%, 4.6% and 3.1%, respectively). The most frequent comorbid conditions were ischemic heart disease and cerebrovascular disease. The incidence of acute myocardial infarction was greater in men (P< .001), but there was no significant difference for other conditions. The adjusted incidence (European population) per 100,000 inhabitants per year in men and women, respectively, was: 605 and 115 for acute myocardial infarction; 238 and 220 for angina; 300 and 327 for stroke; 125 and 115 for transient ischemic attack; 136 and 178 for peripheral arterial disease; and 219 and 267 for heart failure. CONCLUSIONS: The incidence of cardiovascular disease was lower than in other developed countries. Disease occurred more frequently in patients aged >65 years. Ischemic heart disease remained the most common, with heart failure being a notable emergent condition.

Epidemiología de las enfermedades cardiovasculares en atención primaria. Estudio cohorte zona franca de barcelona / The epidemiology of cardiovascular disease in primary care. The zona franca cohort study in Barcelona, Spain

Introducción y objetivos. La incidencia de las enfermedades cardiovasculares no se conoce suficientemente en España, sobre todo en enfermedades emergentes como la arteriopatía periférica y la insuficiencia cardiaca. El objetivo es estudiar la incidencia de estas enfermedades en atención primaria. Métodos. Estudio prospectivo de cohortes de base poblacional, realizado en dos centros de salud de Barcelona (España). Se incluyó a 931 sujetos entre 35 y 84 años, sin enfermedades cardiovasculares, seleccionados aleatoriamente y seguidos 10 años. Se estudió la incidencia de cardiopatía isquémica (infarto agudo de miocardio y angina), enfermedad cerebrovascular (ictus y accidente isquémico transitorio), arteriopatía periférica de extremidades inferiores e insuficiencia cardiaca. Resultados. La enfermedad más frecuente fue la cardiopatía isquémica (incidencia acumulada, 6,1%), seguida de la enfermedad cerebrovascular (4,3%), la insuficiencia cardiaca (3%) y la arteriopatía periférica (1,9%). Fueron más frecuentes en mayores de 65 años (incidencias acumuladas, 9,6, 8,9, 4,6 y 3,1% respectivamente). La superposición más frecuente fue entre cardiopatía isquémica y enfermedad cerebrovascular. La incidencia de infarto agudo de miocardio fue superior en varones (p < 0,001), sin diferencias significativas en otras enfermedades. Las tasas ajustadas (población europea) cada 100.000 habitantes/año en varones y mujeres respectivamente fueron: infarto agudo de miocardio, 605 y 115; angina, 238 y 220; ictus, 200 y 327; accidente isquémico transitorio, 125 y 115; arteriopatía periférica, 136 y 178, e insuficiencia cardiaca, 219 y 267. Conclusiones. La incidencia de enfermedades cardiovasculares es inferior que en otros países desarrollados y son especialmente frecuentes en mayores de 65 años. La cardiopatía isquémica sigue siendo la más frecuente y destaca la insuficiencia cardiaca como enfermedad emergente (AU)

Introduction and objectives. Knowledge about the incidence of cardiovascular disease in Spain is limited, particularly about emergent conditions such as peripheral arterial disease and heart failure. The objective of this study was to determine the incidence of these conditions in a primary care setting. Methods. A prospective population-based cohort study was carried out in two health centers in Barcelona, Spain. It included 931 randomly selected individuals, aged 35 to 84 years, without cardiovascular disease who were followed for 10 years. We assessed the incidence of ischemic heart disease (i.e. myocardial infarction and angina), cerebrovascular disease (i.e. stroke and transient ischemic attack), peripheral arterial disease of the lower extremities, and heart failure. Results. The most frequently occurring condition was ischemic heart disease (cumulative incidence, 6.1%), followed by cerebrovascular disease (4.3%), heart failure (3.0%), and peripheral arterial disease (1.9%). These conditions were more frequent in patients aged >65 years (cumulative incidence, 9.6%, 8.9%, 4.6% and 3.1%, respectively). The most frequent comorbid conditions were ischemic heart disease and cerebrovascular disease. The incidence of acute myocardial infarction was greater in men (P<.001), but there was no significant difference for other conditions. The adjusted incidence (European population) per 100,000 inhabitants per year in men and women, respectively, was: 605 and 115 for acute myocardial infarction; 238 and 220 for angina; 300 and 327 for stroke; 125 and 115 for transient ischemic attack; 136 and 178 for peripheral arterial disease; and 219 and 267 for heart failure. Conclusions. The incidence of cardiovascular disease was lower than in other developed countries. Disease occurred more frequently in patients aged >65 years. Ischemic heart disease remained the most common, with heart failure being a notable emergent condition (AU)