Hepatitis E Virus in Individuals Undergoing Heparin Therapy: An Observational Serological and Molecular Study

INTRODUCTION: Heparin is derived from swine and has been suggested as a possible source of HEV. To study the potential risk of HEV infection associated with heparin treatment, two groups of individuals were compared. Sera from heparinized (N=93) and non-heparinized individuals (N=111) were tested for markers of acute HEV infection and anti-HEV IgG seroprevalence. METHOD: An acute HEV case was defined by the presence of anti-HEV IgM and/or HEV RNA. From the 93 heparinized individuals, one was positive for IgM and IgG anti-HEV and two were positive for HEV RNA (for both ORF3 and ORF2), and there were a total of two (2.2%) cases of current or recent HEV infection. From the 111 non-heparinized individuals, three were positive for IgM anti-HEV, one was positive for both IgM and IgG anti-HEV, and none was positive for HEV RNA, and there were a total of three (2.7%) cases of current or recent HEV infection. The difference between HEV cases in the heparinized individuals and the non-heparinized individuals was not statistically significant (2.2% vs. 2.7%; p = 0.799). RESULTS: Concerning IgG anti-HEV, it was detected in 32 individuals from the heparinized group and in 18 from the non-heparinized control group. A statistically significant difference was observed in the presence of anti-HEV IgG in heparinized individuals and controls (p = 0.003). CONCLUSION: This study has not found any association between heparin treatment and acute HEV infection, but has shown the use of therapeutic heparin as a risk factor for IgG anti-HEV seropositivity.

Airborne SARS-CoV-2 is more frequently detected in environments related to children and elderly but likely non-infectious, Norway, 2022.

This study investigates the presence of SARS-CoV-2 in indoor and outdoor environments in two cities in Norway between April and May 2022. With the lifting of COVID-19 restrictions in the country and a focus on vaccination, this research aims to shed light on the potential for virus transmission in various settings. Air sampling was conducted in healthcare and non-healthcare facilities, covering locations frequented by individuals across different age groups. The study found that out of 31 air samples, only four showed the presence of SARS-CoV-2 RNA by RT-qPCR, with no viable virus detected after RNAse pre-treatment. These positive samples were primarily associated with environments involving children and the elderly. Notably, sequencing revealed mutations associated with increased infectivity in one of the samples. The results highlight the importance of considering children as potential sources of virus transmission, especially in settings with prolonged indoor exposure. As vaccination coverage increases globally, and with children still representing a substantial unvaccinated population, the study emphasizes the need to re-implement mask-wearing mandates indoors and in public transport to reduce virus transmission. The findings have implications for public health strategies to control COVID-19, particularly in the face of new variants and the potential for increased transmission during the autumn and winter seasons.

Hepatitis E Virus in the Iberian Peninsula: A Systematic Review.

One of the most frequent causes of acute viral hepatitis is hepatitis E virus (HEV) causing 20 million infections worldwide each year and 44,000 deaths. Studies on HEV in the Iberian Peninsula have been increasing through time with HEV infection being identified in humans and animals. The aim of the present systematic review was to compile and evaluate all the published data on HEV from studies performed in humans, animals and environmental samples in the Iberian Peninsula. The electronic databases Mendeley, PubMed, Scopus, and Web of Science were thoroughly searched, and research published up until February 01, 2023 were included. Resulting in a total of 151 eligible papers by full reading and application of PRISMA exclusion/inclusion criteria. Overall, the present review shows that several HEV genotypes, namely HEV-1, 3, 4, and 6 as well as Rocahepevirus, are circulating in humans, animals, and in the environment in the Iberian Peninsula. HEV-3 was the most common genotype circulating in humans in Portugal and Spain, as expected for developed countries, with HEV-1 only being detected in travelers and emigrants from HEV endemic regions. Spain is the biggest pork producer in Europe and given the high circulation of HEV in pigs, with HEV-3 being primarily associated to zoonotic transmission through consumption of swine meat and meat products, in our opinion, the introduction of an HEV surveillance system in swine and inclusion of HEV in diagnostic routines for acute and chronic human hepatitis would be important. Additionally, we propose that establishing a monitoring mechanism for HEV is crucial in order to gain a comprehensive understanding of the prevalence of this illness and the various strains present in the Iberian Peninsula, as well as their potential impact on public health.

Diagnosing congenital cytomegalovirus infections using archived dried blood spots: A 15-year observational study, Portugal.

BACKGROUND: Cytomegalovirus (CMV) is a leading cause of congenital infections. Dried blood spots (DBS) collected in the first week of life (Guthrie cards) have been used in the diagnosis of CMV infection outside the three-week window period following birth. The present work summarizes the results of a 15-year observational study in which DBS from 1388 children were used for a late diagnosis of congenital CMV infection. METHODS: Three groups of children were studied: (i) symptomatic (with symptoms at birth or late sequelae) (N = 779); (ii) born to mothers with serological profile of primary CMV infection (N = 75); (iii) without any information (N = 534). A highly sensitive method of DNA extraction (heat-induced) from the DBS was used. CMV DNA was detected by a nested PCR. RESULTS: In total CMV DNA was detected in 7.5% (104/1388) of children. Symptomatic children showed a low rate of CMV DNA detection (6.7%) than children born to mothers with serological profile of primary CMV infection (13.3%) (p = 0.034). Sensorial hearing loss and encephalopathy were the two clinical manifestations with the highest CMV detection rate (18.3% and 11.1%, respectively). Children whose mothers had a confirmed primary infection showed a higher rate of CMV detection (35.3%) when compared with children whose mothers had a not confirmed primary infection (6.9%) (p = 0.007). CONCLUSION: The present work emphasises the importance of testing DBS in symptomatic children even a long time after symptoms onset and in children born to mothers with serologic diagnosis of maternal primary CMV infection when they miss the diagnosis during the three-week window following birth.

A Systematic Review of Hepatitis E Virus Detection in Camels.

Hepatitis E virus (HEV) represents a major cause of acute hepatitis and is considered an emerging public health problem around the world. In the Middle East's and Africa's arid regions, where camels frequently interact with human populations and camel-derived food products are a component of the food chain, camel-borne zoonotic HEV infection is a potential threat. To date, no review paper has been published on HEV in camels. As such, the purpose of the current work is to provide a scientific review of the identification of HEV genotypes seven and eight in camels worldwide to have a better understanding of the current status of this topic and to identify gaps in the current knowledge. Searches were carried out in the electronic databases PubMed, Mendeley, Web of Science, and Scopus, including studies published until 31 December 2022 (n = 435). Once the databases were checked for duplicate papers (n = 307), the exclusion criteria were applied to remove any research that was not relevant (n = 118). As a result, only 10 papers were found to be eligible for the study. Additionally, in eight of the ten studies, the rates of HEV infection were found to be between 0.6% and 2.2% in both stool and serum samples. Furthermore, four studies detected HEV genotype seven in dromedary camels, and two studies have shown HEV genotype eight in Bactrian camels. Interestingly, these genotypes were recently reported in camels from the Middle East and China, where one human infection with HEV genotype seven has been associated with the consumption of contaminated camel meat and milk. In conclusion, more research will be needed to determine the prevalence of HEV infection in camels around the world as well as the risk of foodborne transmission of contaminated camel products. As camels are utility animals in several countries, HEV in these animals may pose a potential risk to public health.

A Systematic Review and Meta-Analysis on Hepatitis E Virus Detection in Farmed Ruminants.

Swine are widely recognized as the main reservoir of zoonotic HEV; however, a growing body of data on the HEV prevalence in farmed ruminants of different species also points to a potential route for HEV transmission through ruminants and ruminant products and by-products. Definite information on the zoonotic potential of ruminants is still absent or unclear, compelling the necessity for increasing knowledge on this. The aim of the current study was to analyze the state-of-the-art in this research topic and provide a summary of HEV detection and characterization in farmed ruminants. A total of 1567 papers were retrieved from four search databases that resulted in 35 eligible papers after application of exclusion/inclusion criteria. Studies on HEV in farmed ruminants were mainly based on the detection of HEV RNA and were reported in Africa (n = 1), America (n = 3), Asia (n = 18) and Europe (n = 13), and focused on a variety of ruminants species, namely cow, goat, sheep, deer, buffalo and yak. The overall pooled prevalence of HEV was 0.02% (0.01-0.03, 95% CI). The subgroup pooled prevalence of HEV RNA was 0.01% (0.00-0.02, 95% CI) in cow milk, stool, serum, liver, intestinal, bile, blood, spleen and rectal swab samples; 0.09% (0.02-0.18, 95% CI) in goat serum, bile, stool, milk, liver, rectal swab and blood samples; 0.01% (0.00-0.04, 95% CI) in sheep stool, serum, milk, blood and liver samples. Most of the HEV genotypes found in farmed ruminants belonged to the zoonotic HEV-3 (subtypes 3a, 3c) and HEV-4 (subtype 4d, 4h), with Rocahepevirus also found. The wide HEV circulation observed in different farmed ruminants raises concerns for the possibility of HEV transmission through products from infected ruminants and alerts for the potential zoonotic route for HEV in ruminant products, such as meat and dairy products. Also, contact exposure to infected farmed animals could be a risk factor. Further research should be conducted in order to understand the circulation of HEV in these animals and its zoonotic potential, as there is currently a lack of data on this topic.

A One-Year Systematic Study to Assess the Microbiological Profile in Oysters from a Commercial Harvesting Area in Portugal.

As filter-feeding animals farmed in water bodies exposed to anthropogenic influences, oysters can be both useful bioremediators and high-risk foodstuffs, considering that they are typically consumed raw. Understanding the dynamic of bacterial and viral load in Pacific oyster (Crassostrea gigas) tissues, hemolymph, outer shell surface biofilm, and farming water is therefore of great importance for microbiological risk assessment. A one-year survey of oysters collected from a class B production area (Canal de Mira, on the Portuguese western coast) revealed that these bivalve mollusks have a good depurating capacity with regard to bacteria, as Salmonella spp. and viable enterococci were not detected in any oyster flesh (edible portion) samples, despite the fact that these bacteria have regularly been found in the farming waters. Furthermore, the level of Escherichia coli contamination was clearly below the legal limit in oysters reared in a class B area (>230-≤4600 MPN E. coli/100 g). On the contrary, norovirus was repeatedly detected in the digestive glands of oysters sampled in autumn, winter, and spring. However, their presence in farming waters was only detected during winter.

SARS-CoV-2 in outdoor air following the third wave lockdown release, Portugal, 2021.

Aiming to contribute with more data on the presence of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in outdoor environments, we performed air sampling in outdoor terraces from restaurants in three major cities of Portugal in April 2021, following the third wave lockdown release in the country. Air samples (n=19) were collected in 19 restaurant terraces during lunch time. Each air sample was collected using a Coriolis Compact air sampler, followed by RNA extraction and real-time quantitative PCR for the detection of viral RNA. Viral viability was also assessed through RNAse pre-treatment of samples. Only one of the 19 air samples was positive for SARS-CoV-2 RNA, with 7337 gene copies m-3 for the genomic region N2, with no viable virus in this sample. The low number of positive samples found in this study is not surprising, as sampling took place in outdoor settings where air circulation is optimal, and aerosols are rapidly dispersed by the air currents. These results are consistent with previous reports stating that transmission of SARS-CoV-2 in outdoor spaces is low, although current evidence shows an association of exposures in settings where drinking and eating is possible on-site with an increased risk in acquiring SARS-CoV-2 infection. Moreover, the minimal infectious dose for SARS-CoV-2 still needs to be determined so that the real risk of infection in different environments can be accurately established.

Detection of hepatitis E virus in milk: Current evidence for viral excretion in a wide range of mammalian hosts.

Infection with hepatitis E virus (HEV) is common in both developing and industrialized nations. Genotypes 3 and 4 are increasingly being reported, particularly in high-income countries where the precise extent of HEV transmission via food is currently unclear. Recently, HEV has been found to be excreted in milk; however, data on the potential milk-borne transmission is still lacking or conflicting and warrants further research on the topic. As such, the aim of the present study was to review the current scientific knowledge and to summarize the existing studies in which HEV has been detected in milk. Exhaustive searches were carried out in Mendeley, PubMed, Scopus and Web of Science. A total of 157 papers were retrieved from the four electronic databases. After removing duplicate articles from the databases (n = 30), exclusion criteria identified unrelated research (n = 115). This allowed the identification of 12 eligible papers. To date, studies on HEV detection in milk were mostly from China (n = 5), followed by Egypt (n = 2), Germany (n = 1), Belgium and Holland (n = 1), Turkey (n = 1), Czech Republic (n = 1) and Spain (n = 1) and were focused on a variety of animals (cow, goat, donkey, buffalo, sheep and camel) and humans. Four out of the 12 eligible studies did not find any evidence of HEV in milk. Moreover, 3 out of the 12 studies detected low rates of HEV (0.2-1.8%) and two were based on a low sample size (n = 1 and n = 4). Interestingly, one study showed very high detection rates and also detected HEV genotype 1 in an animal milk sample, an unusual finding since it only occurs in humans, deserving further studies for confirmation and characterization. Two studies detected high prevalence of HEV genotype 4 in bovine samples from China, with one showing indication of the presence of infectious HEV in milk. To date, there is still a small amount of available data on the HEV presence in milk, posing important questions regarding both animal and human health. Thus, further efforts on this potentially underestimated zoonotic route for HEV should be given, warranting further studies on the topic.

Crimean-Congo hemorrhagic fever virus circulating among sheep of Portugal: a nationwide serosurvey assessment.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a widespread zoonotic pathogen that can cause mild to severe hemorrhagic disease in humans. CCHFV may be transmitted through direct contact with tissue or blood of viremic animals; however, the primary transmission route is through infected tick bites. CCHFV RNA has been detected in ticks feeding on domestic and wild animals in western Spain, suggesting an established circulation of CCHFV in Western Europe. Ruminants have been recognized as important CCHFV reservoirs and have been linked to human cases in endemic regions. Given the emergence of CCHF in neighboring Spain, and a report of two CCHFV seropositive humans in southern Portugal in 1985, we investigated the potential circulation of this virus in the country by performing a nationwide anti-CCHFV IgG serosurvey in sentinel sheep of Portugal. Sera (n = 459) randomly selected from widely distributed farms (n = 20) of Portugal were tested using a commercial double-antigen enzyme-linked immunosorbent assay, yielding an overall seroprevalence of 0.4% (95% confidence interval [CI] 0.04-1.56%). Positive sheep were from the southern region of Portugal (Alentejo region), which raise the seroprevalence of this region to 0.74% (95% CI 0.09-2.66%). This is the first study reporting the presence of CCHFV antibodies in sheep of Portugal, thus suggesting a geographical expansion of CCHFV to this country. It seems likely that CCHFV may exist focally in southern Portugal.

Detection of SARS-CoV-2 in the Indoor and Outdoor Areas of Urban Public Transport Systems of Three Major Cities of Portugal in 2021.

Airborne transmission is mainly associated with poorly ventilated and crowded indoor environments where people stay for long periods of time. As such, public transport is often perceived as having a high risk for the transmission of SARS-CoV-2. Considering that data on the detection of SARS-CoV-2 in public transport systems are scarce, we performed air sampling for SARS-CoV-2 in indoor and outdoor spaces of public transport systems in Portugal. Air (n = 31) and surface (n = 70) samples were collected using a Coriolis® Compact microbial air sampler and sterile flocked plastic swabs, respectively. Samples were extracted and analyzed through RT-qPCR. Only two air samples from an outdoor and a partially open space were found to be positive for SARS-CoV-2 RNA. No positive surface samples were detected. These results indicate that the viral concentration in ambient air in public transport systems is linked to the number of people present in that environment and whether they are wearing properly fitting masks. Considering the current lifting of COVID-19 restrictions around the world, it is essential that people continue to wear masks in both indoor and outdoor environments, especially in crowded spaces. More studies on this topic are needed to fully elucidate the real risk of infection in outdoor spaces.

Occurrence and molecular characterization of human pegivirus-1 (HPgV-1) viremia in healthy volunteer blood donors from Northern Portugal.

Human pegivirus-1 (HPgV-1) is a member of the Flaviviridae family and the Pegivirus genus. Despite having been discovered 25 years ago, there is still much to know regarding HPgV-1 clinical impact, as this virus is currently not associated with any pathology. Yet, HPgV-1 prevalence and molecular characterization are still unknown in many countries, including Portugal. To fill in this knowledge gap, this study aimed to determine the occurrence and molecular characterization of HPgV-1 in a group of healthy blood donors from the north of Portugal. Blood samples from 465 Portuguese blood donors were collected from a major Hospital Center in the north of Portugal. RNA was extracted and an initial nested RT-PCR was performed targeting the conserved 5'-untranslated region  region of the HPgV-1 genome. A second nested RT-PCR targeting the E2 region was performed for genotyping. Only one sample tested positive for HPgV-1 RNA, resulting in a prevalence of approximately 0.22%. Phylogenetic analyses confirmed the characterization as genotype 2, the most prevalent in Europe.

Evidence of Air and Surface Contamination with SARS-CoV-2 in a Major Hospital in Portugal.

As the third wave of the COVID-19 pandemic hit Portugal, it forced the country to reintroduce lockdown measures due to hospitals reaching their full capacities. Under these circumstances, environmental contamination by SARS-CoV-2 in different areas of one of Portugal's major Hospitals was assessed between 21 January and 11 February 2021. Air samples (n = 44) were collected from eleven different areas of the Hospital (four COVID-19 and seven non-COVID-19 areas) using Coriolis® µ and Coriolis® Compact cyclone air sampling devices. Surface sampling was also performed (n = 17) on four areas (one COVID-19 and three non-COVID-19 areas). RNA extraction followed by a one-step RT-qPCR adapted for quantitative purposes were performed. Of the 44 air samples, two were positive for SARS-CoV-2 RNA (6575 copies/m3 and 6662.5 copies/m3, respectively). Of the 17 surface samples, three were positive for SARS-CoV-2 RNA (200.6 copies/cm2, 179.2 copies/cm2, and 201.7 copies/cm2, respectively). SARS-CoV-2 environmental contamination was found both in air and on surfaces in both COVID-19 and non-COVID-19 areas. Moreover, our results suggest that longer collection sessions are needed to detect point contaminations. This reinforces the need to remain cautious at all times, not only when in close contact with infected individuals. Hand hygiene and other standard transmission-prevention guidelines should be continuously followed to avoid nosocomial COVID-19.

Systematic Review of Hepatitis E Virus in Brazil: A One-Health Approach of the Human-Animal-Environment Triad.

Brazil is the fifth largest country in the world with diverse socioeconomic and sanitary conditions, also being the fourth largest pig producer in the world. The aim of the present systematic review was to collect and summarize all HEV published data from Brazil (from 1995 to October 2020) performed in humans, animals, and the environment, in a One Health perspective. A total of 2173 papers were retrieved from five search databases (LILACs, Mendeley, PubMed, Scopus, and Web of Science) resulting in 71 eligible papers after application of exclusion/inclusion criteria. Data shows that HEV genotype 3 (HEV-3) was the only retrieved genotype in humans, animals, and environment in Brazil. The South region showed the highest human seroprevalence and also the highest pig density and industry, suggesting a zoonotic link. HEV-1 and 2 were not detected in Brazil, despite the low sanitary conditions of some regions. From the present review we infer that HEV epidemiology in Brazil is similar to that of industrialized countries (only HEV-3, swine reservoirs, no waterborne transmission, no association with low sanitary conditions). Hence, we alert for the implementation of HEV surveillance systems in swine and for the consideration of HEV in the diagnostic routine of acute and chronic hepatitis in humans.

Surface contamination with SARS-CoV-2: A systematic review.

Little is known about contaminated surfaces as a route of transmission for SARS-CoV- 2 and a systematic review is missing and urgently needed to provide guidelines for future research studies. As such, the aim of the present study was to review the current scientific knowledge and to summarize the existing studies in which SARS-CoV-2 has been detected in inanimate surfaces. This systematic review includes studies since the emergence of SARS-CoV-2, available in PubMed/MEDLINE and Scopus. Duplicate publications were removed, and exclusion criteria was applied to eliminate unrelated studies, resulting in 37 eligible publications. The present study provides the first overview of SARS-CoV-2 detection in surfaces. The highest detection rates occurred in hospitals and healthcare facilities with COVID-19 patients. Contamination with SARS-CoV-2 on surfaces was detected in a wide range of facilities and surfaces. There is a lack of studies performing viability testing for SARS-CoV-2 recovered from surfaces, and consequently it is not yet possible to assess the potential for transmission via surfaces.

Airborne spread of infectious SARS-CoV-2: Moving forward using lessons from SARS-CoV and MERS-CoV.

BACKGROUND: Although an increasing body of data reports the detection of SARS-CoV-2 RNA in air, this does not correlate to the presence of infectious viruses, thus not evaluating the risk for airborne COVID-19. Hence there is a marked knowledge gap that requires urgent attention. Therefore, in this systematic review, viability/stability of airborne SARS-CoV-2, SARS-CoV and MERS-CoV viruses is discussed. METHODS: A systematic literature review was performed on PubMed/MEDLINE, Web of Science and Scopus to assess the stability and viability of SARS-CoV, MERS-CoV and SARS-CoV-2 on air samples. RESULTS AND DISCUSSION: The initial search identified 27 articles. Following screening of titles and abstracts and removing duplicates, 11 articles were considered relevant. Temperatures ranging from 20 °C to 25 °C and relative humidity ranging from 40% to 50% were reported to have a protective effect on viral viability for airborne SARS-CoV and MERS-CoV. As no data is yet available on the conditions influencing viability for airborne SARS-CoV-2, and given the genetic similarity to SARS-CoV and MERS-CoV, one could extrapolate that the same conditions would apply. Nonetheless, the effect of these conditions seems to be residual considering the increasing number of cases in the south of USA, Brazil and India, where high temperatures and humidities have been observed. CONCLUSION: Higher temperatures and high relative humidity can have a modest effect on SARS-CoV-2 viability in the environment, as reported in previous studies to this date. However, these studies are experimental, and do not support the fact that the virus has efficiently spread in the tropical regions of the globe, with other transmission routes such as the contact and droplet ones probably being responsible for the majority of cases reported in these regions, along with other factors such as human mobility patterns and contact rates. Further studies are needed to investigate the extent of aerosol transmission of SARS-CoV-2 as this would have important implications for public health and infection-control policies.

Hepatitis E virus genotype 3 in echinoderms: First report of sea urchin (Paracentrotus lividus) contamination.

Hepatitis E virus (HEV) deriving from manure application runoffs and faecal waste spill over of swine and human origin bypass wastewater treatment plants and contaminate coastal waters. Shellfish bioaccumulate enteric viruses such as HEV from fecally contaminated coastal waters and under current European Regulations, shellfish sanitary status surveillance is mandatory but only by means of bacterial faecal indicators. The sea urchins are under the same regulations and their vulnerability to fecal contamination has been pointed out. Since they are consumed raw and with no steps to control/reduce hazards, sea urchin contamination with enteric viruses can represent a food safety risk. Hence, the aim of the present study was to screen sea urchin gonads destined for human consumption for the presence of HEV. HEV was detected and quantified in gonads of sea urchins collected in north Portugal by a reverse transcription-quantitative PCR (RT-qPCR) assay targeting the ORF3 region, followed by genotyping by a nested RT-PCR targeting the ORF2 region. Sequencing and phylogenetic analysis clustered the HEV sequence within genotype 3, subgenotype e. This the first study reporting HEV contamination of sea urchins. We hypothesize that like shellfish, sea urchins can also be a food vehicle for HEV transmission to humans.

Mumps antibodies in the cord blood: Association with maternal recall of mumps. Possible consequences for vaccination strategies.

The present study aims to contribute to the evaluation of the serological impact of vaccination against mumps in Portugal, measuring anti-mumps IgG (MuIgG) levels in cord sera and the corresponding proportions of seropositive newborns, and their association with potential predictive variables. The data from this study came from 198 umbilical cord sera. Detailed vaccination records were available for all mothers. MuIgG were measured in the sera, using a commercial immunoassay. The geometric mean concentration (GMC) of MuIgG was 31.7 RU/ml. Seropositive/immune sera (concentration ≥16 RU/ml) were 75.3%. While 49 mothers were "unsure" about ever having had mumps, 46 said they had had the disease and 103 said they had not had it. Eighty eight women did not receive a single dose of MMR while the other received 1 or 2 doses, with different combinations of vaccine strains. This study found that recalling mumps was predictive of higher MuIgG GMC and seropositivity. Maternal age and vaccination status were not associated with GMC or seropositivity. Nevertheless, in the small subset of newborns from vaccinated mothers not recalling mumps, receiving two doses was predictive of higher GMC than just receiving one. Maternal recall of mumps is highly predictive of seropositivity while not recalling the disease results in numerous false-negatives. This is consistent with other studies and with the fact that infection with mumps virus can result in a wide range of clinical manifestations. We agree on the need for further research to support a recommendation of a three (or more)-dose MMR strategy but we also believe that evidence is fast accumulating in favour of a higher dose strategy. The issue of waning immunity due to vaccines when vaccination succeeds in controlling (and nationally eliminating) target diseases like measles and mumps must be urgently taken into account.

Development and characterization of PLGA nanoparticles containing 1,3-dihydroxy-2-methylxanthone with improved antitumor activity on a human breast cancer cell line.

Interest on xanthones has been growing considerably due to their broad spectrum of biological activities. 1,3-Dihydroxy-2-methylxanthone (DHMXAN) showed a significant inhibitory effect on the growth of MCF-7 cancer cells. DHMXAN-loaded nanosphere and nanocapsule formulations were prepared by the solvent displacement technique with the aim of improving the delivery of this poorly water-soluble compound. Moreover, we investigated the usefulness of this nanotechnology-based approach on the improvement of compound's antitumor activity. Nanosphere and nanocapsule mean diameters ranged from 117 ± 8 to 138 ± 5 nm and from 266 ± 7 to 286 ± 22 nm, respectively, and both systems exhibited negative surface charge. Incorporation efficiency values of DHMXAN in nanospheres and nanocapsules were higher than 30% (30.9 ± 3.3 to 38.8 ± 3.6%) and 80% (85 ± 7 to 88 ± 6%), respectively. The release study of DHMXAN from nanocapsules suggests that drug release is mainly governed by its partition between the oil core and the external aqueous medium. The effect of different nanoparticulate formulations on the growth of human breast cancer cell line MCF-7 was evaluated using the sulforhodamine B (SRB) assay. Incorporation of DHMXAN in nanospheres and nanocapsules afforded a marked potentiation of the compound inhibitory effect.