Leishmania infantum infection serosurveillance in stray dogs inhabiting the Madrid community: 2007-2018.

BACKGROUND: Leishmaniosis is an endemic zoonotic disease in the Mediterranean basin caused by Leishmania infantum and transmitted by phlebotomine sandflies. While in dogs disease may be severe, leishmaniosis is also a public health concern as was shown in the largest outbreak of human leishmaniosis (HL) in Europe in 2009 occurring in the Madrid region. The aim of the present study was to assess the applicability of the Leishmaniosis Surveillance Program (LeishSP) established in Madrid in 1996 by examining trends in L. infantum seroprevalence and associated epidemiological risk factors based on data for the 2007-2018 period. METHODS: The study population consisted of 3225 stray dogs from 17 animal shelters collaborating with the LeishSP. Seroprevalences were recorded twice annually (April and November) from 2007 to 2018. In each yearly period, a minimum of 100 dogs were tested to detect dogs infected before and after the sandfly risk season in Madrid area. Each dog was subjected to the same protocol of blood sample collection and clinical examination to collect epidemiological data and clinical signs. Anti-Leishmania-specific IgG was determined by IFAT cut-off ≥ 1:100. RESULTS: Overall seroprevalence was 6.1% (198 positive dogs). Epidemiological data indicate a significantly higher seroprevalence in dogs > 4 years old, purebred dogs (Pit Bull and related breeds), and medium to large size dogs. There were no seroprevalence differences according to sex and/or season (April and November). In addition, no significant differences were observed according to whether dogs lived inside or outside the HL outbreak area. Remarkably, of 198 dogs testing positive for L. infantum, 64.6% had no clinical signs, indicating a high proportion of clinically healthy infected dogs that could be a potential source of infection. CONCLUSIONS: Results indicate a stable seroprevalence of L. infantum infection after 2006 in stray dogs in Madrid but with a recent slightly increasing trend. These observations support the need to continue with the LeishSP implemented by sanitary authorities of the Madrid Community as an early warning strategy for human and animal leishmaniosis and to enable continued assessment of the epidemiological role of dogs with subclinical infection in this important zoonotic disease.

Opportunistic feeding behaviour and Leishmania infantum detection in Phlebotomus perniciosus females collected in the human leishmaniasis focus of Madrid, Spain (2012-2018).

BACKGROUND: An outbreak of human leishmaniasis due to Leishmania infantum has been registered in an urban area of southwestern Madrid, Spain, since 2010. Entomological surveys carried out in the municipalities of Fuenlabrada, Leganés, Getafe and Humanes de Madrid showed that Phlebotomus perniciosus is the only potential vector. In this work, an intensive molecular surveillance was performed in P. perniciosus females captured in the region between 2012 and 2018. METHODOLOGY/PRINCIPAL FINDINGS: A total of 1805 P. perniciosus females were analyzed for Leishmania infection, and 1189 of them also for bloodmeal identification. Eleven different species of vertebrate were detected by amplification and subsequent sequencing of the 359 bp cytb fragment. The most prevalent blood source identified was hare (n = 553, 46.51%), followed by rabbit (n = 262, 21.95%). Less frequent were cat (n = 45, 3.80%), human (n = 34, 2.90%), pig (n = 14, 1.20%), horse (n = 11, 0.93%), sheep (n = 3, 0.25%), rhea (n = 3, 0.25%), partridge (n = 1, 0.09%) and chicken (n = 1, 0.09%). The distribution of the blood meal sources varied between the different locations. Regarding L. infantum detection, PCR amplification of a fragment of kDNA, cpb gene and ITS1 region showed 162 positive specimens (8.97%). The highest infection rate was found in the municipality of Leganés (15.17%). CONCLUSIONS: The results of this molecular survey in P. perniciosus, the only leishmaniasis vector in the outbreak occurred in southwestern Madrid region, showed its opportunistic blood-feeding behaviour, high infection rates and the differences between the different points. This study was an essential part of the intensive surveillance plan in the area and the results obtained have supported the implementation of control measures in the outbreak.

Leishmania sp. detection and blood-feeding behaviour of Sergentomyia minuta collected in the human leishmaniasis focus of southwestern Madrid, Spain (2012-2017).

Phlebotomine sand flies are the only known vectors of Leishmania spp. protozoan which causes leishmaniasis in 98 countries. In Spain, 11 sand fly species are described, but only Phlebotomus perniciosus and Phlebotomus ariasi are proven vectors of the disease. On the other hand, Sergentomyia minuta is one of the most abundant and ubiquitous sand flies in this territory, although scarce information is available about this species. Sand flies from this genus are known for their preference to feed on cold-blooded animals and are traditionally involved in the transmission of reptile Leishmania. However, studies have suggested that Sergentomyia spp. could be implicated in the transmission of human pathogenic Leishmania. This study analyses blood meal preferences and Leishmania sp. infection of S. minuta sand flies from the largest human leishmaniasis outbreak in Europe. Sand flies were collected during entomological surveillance carries out from 2012 to 2017 in the active season of these dipterans, from May to October. Molecular detection of Leishmania spp. showed 68 positive specimens of S. minuta out of 377 (18%). The analysis of blood meal preferences by amplification of 359 bp fragment of cytochrome b gene revealed that blood preference of S. minuta is not only limited to reptiles, but they also feed on mammals, including humans. Results suggest the presence of a Leishmania sp., related to Leishmania tarentolae, cycle in S. minuta from the studied area. Although there is no evidence about its incrimination in the L. infantum transmission more investigation is needed to elucidate the intravectorial cycle of Leishmania spp. in S. minuta sand flies, their feeding behaviour and their potential contribution in Leishmania spp. epidemiology in the country.

Newborns and low to moderate prenatal environmental lead exposure: might fathers be the key?

This study is part of the BioMadrid Project, a bio-monitoring study designed to assess pollutants in the environment surrounding children born in the Madrid region. Our aim in this report is to evaluate the association between prenatal lead exposure and fetal development using three biological samples (maternal and paternal blood lead at around 34 weeks of gestation as well as cord blood lead levels), three biomarkers of effect in cord blood peripheral lymphocytes (micronucleus in binucleated cells, nucleoplasmic bridges, and nuclear buds), and different anthropometrical characteristics at birth. Maternal and cord blood lead were not associated with newborn measurements or genotoxicity biomarkers. In contrast, increases in father blood lead were coupled with lower weight (mean difference (MD), -110.8 g; 95% confidence intervals (95%CI), -235.6 to 6.00; p < 0.10) and shorter abdominal (MD, -0.81 cm; 95%CI, -1.64 to 0.00; p < 0.05) and cephalic (MD, -0.32 cm; 95%CI, -0.65 to 0.00; p < 0.05) circumferences at birth as well as with the presence of nucleoplasmic bridges (odds ratio, 1.03; 95%CI, 1.00 to 1.06; p < 0.05) and nuclear buds (odds ratio, 1.02; 95%CI, 0.99 to 1.04; p < 0.10). These associations were mainly confined to female babies, in whom paternal lead was also inversely associated with length. Our results support the hypothesis that paternal lead exposure may be affecting the development of newborns.

Lead, mercury and cadmium in umbilical cord blood and its association with parental epidemiological variables and birth factors.

BACKGROUND: In Spain, few studies have evaluated prenatal exposure to heavy metals. The objective of this study was to describe lead, mercury and cadmium concentrations in blood from a sample of newborn-mother-father trios, as well as to investigate the association between metals in cord blood and parental variables. We also explored the relationship between cord blood metal concentrations and child characteristics at birth. METHODS: Metal correlations among family members were assessed using Spearman Rank Correlation Coefficient. Linear regression was used to explore the association between parental variables and log-transformed cord blood lead and cord blood mercury concentrations. In the case of cadmium, tobit regression was used due to the existence of samples below the detection limit. The association between cord blood metal concentrations and child characteristics at birth was evaluated using linear regression. RESULTS: Geometric means for lead, mercury and cadmium were 14.09 µg/L, 6.72 µg/L and 0.27 µg/L in newborns; 19.80 µg/L, 3.90 µg/L and 0.53 µg/L in pregnant women; and 33.00 µg/L, 5.38 µg/L and 0.49 µg/L in men. Positive correlations were found between metal concentrations among members of the trio. Lead and cadmium concentrations were 15% and 22% higher in newborns from mothers who smoked during pregnancy, while mercury concentrations were 25% higher in newborns from mothers with greater fish intake. Cord-blood lead levels showed seasonal periodicity, with lower concentrations observed in winter. Cord blood cadmium concentrations over 0.29 µg/L were associated with lower 1-minute and 5-minute Apgar scores. CONCLUSIONS: These results reinforce the need to establish biomonitoring programs in Spain, and provide support for tobacco smoke and fish consumption as important preventable sources of heavy metal exposure in newborns. Additionally, our findings support the hypothesis that cadmium exposure might be deleterious to fetal development.

Detection of Leishmania infantum and identification of blood meals in Phlebotomus perniciosus from a focus of human leishmaniasis in Madrid, Spain.

Since 2010, the number of cases of both human visceral leishmaniasis and cutaneous leishmaniasis in southwestern Madrid region (Spain) and more specifically in the town of Fuenlabrada has increased. Direct xenodiagnosis of leishmaniasis proved that hares (Lepus granatensis) from this focus are able to infect with Leishmania infantum colonized Phlebotomus perniciosus. To a better understanding of this focus of leishmaniasis, we conducted an entomological survey using CDC light traps, at the end of the seasonal transmission period of 2011 before the beginning of control measures of the disease, to study the phlebotomine sand flies species involved. Detection of Leishmania DNA in the sand flies captured was studied by kDNA-PCR and cpb-PCR. In addition, blood fed and gravid female P. perniciosus were analysed by a PCR based in vertebrate cytochrome b (cyt b) gene. Taxonomic identification of captured sand flies (n = 174) as P. perniciosus (n = 171) and Sergentomyia minuta (n = 3) together with the analysis of blood feeding in ten sand flies that shows a high preference for hares (n = 6), followed by humans (n = 3), and cats (n = 1) confirm a strong association between P. perniciosus hares and humans in the focus. Moreover, 79 out of 135 (58.5 %) P. perniciosus were positive to L. infantum by PCR approaches. These data support the increase of human leishmaniasis cases in the area and the existence of an unusual sylvatic cycle alternative to the classical domestic one, where the dog is the main reservoir of L. infantum.

Mercury, lead and cadmium in human milk in relation to diet, lifestyle habits and sociodemographic variables in Madrid (Spain).

BACKGROUND: Although breastfeeding is the ideal way of nurturing infants, it can be a source of exposure to toxicants. This study reports the concentration of Hg, Pb and Cd in breast milk from a sample of women drawn from the general population of the Madrid Region, and explores the association between metal levels and socio-demographic factors, lifestyle habits, diet and environmental exposures, including tobacco smoke, exposure at home and occupational exposures. METHODS: Breast milk was obtained from 100 women (20 mL) at around the third week postpartum. Pb, Cd and Hg levels were determined using Atomic Absorption Spectrometry. Metal levels were log-transformed due to non-normal distribution. Their association with the variables collected by questionnaire was assessed using linear regression models. Separate models were fitted for Hg, Pb and Cd, using univariate linear regression in a first step. Secondly, multivariate linear regression models were adjusted introducing potential confounders specific for each metal. Finally, a test for trend was performed in order to evaluate possible dose-response relationships between metal levels and changes in variables categories. RESULTS: Geometric mean Hg, Pb and Cd content in milk were 0.53 µg L(-1), 15.56 µg L(-1), and 1.31 µg L(-1), respectively. Decreases in Hg levels in older women and in those with a previous history of pregnancies and lactations suggested clearance of this metal over lifetime, though differences were not statistically significant, probably due to limited sample size. Lead concentrations increased with greater exposure to motor vehicle traffic and higher potato consumption. Increased Cd levels were associated with type of lactation and tended to increase with tobacco smoking. CONCLUSIONS: Surveillance for the presence of heavy metals in human milk is needed. Smoking and dietary habits are the main factors linked to heavy metal levels in breast milk. Our results reinforce the need to strengthen national food safety programs and to further promote avoidance of unhealthy behaviors such as smoking during pregnancy.

Cytogenetic status in newborns and their parents in Madrid: the BioMadrid study.

Monitoring cytogenetic damage is frequently used to assess population exposure to environmental mutagens. The cytokinesis-block micronucleus assay is one of the most widely used methods employed in these studies. In the present study we used this assay to assess the baseline frequency of micronuclei in a healthy population of father-pregnant woman-newborn trios drawn from two Madrid areas. We also investigated the association between micronucleus frequency and specific socioeconomic, environmental, and demographic factors collected by questionnaire. Mercury, arsenic, lead, and cadmium blood levels were measured by atomic absorption spectrometry. The association between micronucleated cell frequency and the variables collected by questionnaire, as well as, the risk associated with the presence of elevated levels of metals in blood, was estimated using Poisson models, taking the number of micronucleated cells in 1,000 binucleated cells (MNBCs) as the dependent variable. Separate analyses were conducted for the 110 newborns, 136 pregnant women, and 134 fathers in whom micronuclei could be assessed. The mean number of micronucleated cells per 1,000 binucleated cells was 3.9, 6.5, and 6.1 respectively. Our results show a statistically significant correlation in MNBC frequency between fathers and mothers, and between parents and newborns. Elevated blood mercury levels in fathers were associated with significantly higher MNBC frequency, compared with fathers who had normal mercury levels (RR:1.21; 95%CI:1.02-1.43). This last result suggests the need to implement greater control over populations which, by reason of their occupation or life style, are among those most exposed to this metal.

Biomonitoring of exposure to environmental pollutants in newborns and their parents in Madrid, Spain (BioMadrid): study design and field work results.

In Spain environmental surveillance has mainly relied on measures of selected pollutants in air, water, food and soil. A study was conducted in Madrid to assess the feasibility of implementing a surveillance system of exposure among the general population to specific environmental pollutants, using bio-markers. The project was basically focused on the environment surrounding newborns. Hence, the study population was made up of 145 triplets of pregnant women at around 8 months' gestation, their partners, and newborns from two areas, representing the two main types of urban environments in the region, i.e., the City of Madrid and its outlying metropolitan belt. Multiple biologic substrates were collected from each participant in order to assess the most suitable samples for an environmental surveillance system. The selected contaminants represent the main agents to which a population like that of Madrid is exposed every day, including certain heavy metals, persistent organic pollutants and polycyclic aromatic hydrocarbons, as well as micronuclei in peripheral blood, a commonly used unspecific index of cytogenetic damage. In addition, passive air samplers were placed around subjects' place of residence. This paper reports in detail on the design and response rates, summarizes field work results, and discusses some lessons learned.

Biomonitorización de la exposición a contaminantes ambientales en recién nacidos y sus progenitores en Madrid [BioMadrid]: diseño del estudio y resultados del trabajo de campo / Biomonitoring of exposure to environmental pollutants in newborns and their parents in Madrid, Spain (BioMadrid): study design and field work results

In Spain environmental surveillance has mainly relied on measuresof selected pollutants in air, water, food and soil. A studywas conducted in Madrid to assess the feasibility of implementinga surveillance system of exposure among the generalpopulation to specific environmental pollutants, using biomarkers.The project was basically focused on the environmentsurrounding newborns. Hence, the study population was madeup of 145 triplets of pregnant women at around 8 months’ gestation,their partners, and newborns from two areas, representingthe two main types of urban environments in the region,i.e., the City of Madrid and its outlying metropolitan belt.Multiple biologic substrates were collected from each participantin order to assess the most suitable samples for an environmentalsurveillance system. The selected contaminantsrepresent the main agents to which a population like that ofMadrid is exposed every day, including certain heavy metals,persistent organic pollutants and polycyclic aromatic hydrocarbons,as well as micronuclei in peripheral blood, a commonlyused unspecific index of cytogenetic damage. In addition,passive air samplers were placed around subjects’ placeof residence. This paper reports in detail on the design andresponse rates, summarizes field work results, and discussessome lessons learned(AU)

En España, la vigilancia medioambiental se basa principalmenteen medidas de ciertos contaminantes en muestrasde aire, agua, alimentos y suelos. En Madrid se ha realizadoun estudio para valorar la posibilidad de poner en marchaun sistema de vigilancia de exposiciones a contaminantesambientales en la población general utilizando biomarcadores.El proyecto ha tenido como eje el estudio del entornode los recién nacidos. Por tanto, la población de estudio laconstituyen 145 ®tríos» formados por mujeres en su octavomes de embarazo, sus parejas y los recién nacidos de dosáreas geográficas, que representan los dos principales entornosurbanos de la región, es decir, Madrid capital y su áreametropolitana. Se recogieron múltiples sustratos biológicosde cada participante con el objeto de valorar las muestrasmás adecuadas para un sistema de vigilancia de exposicionesambientales. Los contaminantes elegidos representan losprincipales agentes tóxicos a los que una población como lade Madrid está expuesta diariamente, e incluyen metales pesados,contaminantes orgánicos persistentes e hidrocarburosaromáticos policíclicos; se ha añadido también una medidainespecífica de daño citogenético, los micronúcleos ensangre periférica. Además, se han colocado muestreadorespasivos de aire en los alrededores del domicilio de los participantes.Este artículo describe en detalle el diseño del estudioy la tasa de respuesta, resume los resultados del trabajode campo y comenta algunas enseñanzas prácticas deéste(AU)