Exploring adenovirus in water environments: a systematic review and meta-analysis.

Adenoviruses (AdVs) have a significant impact in both medical and environmental contexts. The objective of this study was to investigate the prevalence of AdV in different water types, such as untreated and treated wastewater, surface water, groundwater, drinking water, and other water matrices. A total of 239 articles were included in this meta-analysis. Adenoviruses were detected in various waters worldwide. The overall prevalence in water was found to be 59.2%, with the highest prevalence in untreated wastewater (83.1%) and treated wastewater (75.3%), followed by "other water matrices" (53.4%), surface water (49.5%) drinking water (22.7%), and groundwater (18.5%). Most of the studies did not assess the viability of the viruses, leading to weak links between water contamination and risk. Both human and animal AdV were found in water environments. The findings suggest that water, including drinking water, could be a significant route of AdV transmission in both developed and developing economies.

Cutting efficiency of conventional and heat-treated nickel-titanium rotary or reciprocating glide path instruments.

AIM: To compare the cutting efficiency of several glide path files: HyFlex EDM Glidepath File (HEGF), One G (OG), R-Pilot (RP) and WaveOne Gold Glider (WOGG) at different cutting inclinations. METHODOLOGY: Cutting efficiency of 120 new HEGF, OG, RP and WOGG was tested at 90°, 70° and 45° inclination in relation to the sample. Depending on manufacturer instructions, instruments were tested in continuous rotation or reciprocating motion against standardized gypsum samples for 120 s using a customized apparatus. Cutting efficiency was determined by measuring the weight loss and the length of the sample cut. Data were analysed using two-way analysis of variance and Tukey t-test (P < 0.05). RESULTS: RP and WOGG had greater cutting ability than HEGF and OG in all tested angles. RP tested at 45° had significantly greater cutting efficiency than when tested at 90° and 70° (P < 0.05). OG was not significantly different in terms of cutting ability at 90°, 70° and 45° (P > 0.05). HEGF and WOGG had significantly greater cutting efficiency at 45° and 70° compared to 90° (P < 0.05), with no significant difference between 45° and 70° (P > 0.05). RP had the greatest cutting ability at all tested angles. CONCLUSIONS: Reciprocating glide path instruments had greater cutting efficiency compared to those in continuous rotation. An inclined insertion (45° or 70°) increased the cutting ability of glide path files except for One G conventional NiTi files.

Enteric viruses, somatic coliphages and Vibrio species in marine bathing and non-bathing waters in Italy.

Microbial safety of recreational waters is a significant public health issue. In this study we assessed the occurrence and quantity of enteric viruses in bathing and non-bathing waters in Italy, in parallel with microbial faecal indicators, somatic coliphages and Vibrio spp. Enteric viruses (aichivirus, norovirus and enterovirus) were detected in 55% of bathing water samples, including samples with bacterial indicator concentrations compliant with the European bathing water Directive. Aichivirus was the most frequent and abundant virus. Adenovirus was detected only in non-bathing waters. Somatic coliphages were identified in 50% bathing water samples, 80% of which showed simultaneous presence of viruses. Vibrio species were ubiquitous, with 9 species identified, including potential pathogens (V. cholerae, V. parahaemoylticus and V. vulnificus). This is the first study showing the occurrence and high concentration of Aichivirus in bathing waters and provides original information, useful in view of a future revision of the European Directive.

Evidence for swine and human papillomavirus in pig slurry in Italy.

AIMS: The diversity and the geographical distribution of swine papillomaviruses (PVs) are virtually unknown. The occurrence and the diversity of swine PV were therefore investigated in pig slurry collected in Italy, to contribute towards filling this gap in knowledge. METHODS AND RESULTS: Twenty-two slurry samples underwent analysis by nested PCR and DNA sequencing using published and newly designed specific primer pairs for Sus scrofa papillomavirus (SsPV) type 1 and 2 (SsPV1 and 2), along with degenerate PV-specific primers targeting the major coat protein L1 and the helicase protein E1. Overall, three samples (13·6%) were positive for SsPV1 by specific primers, and nucleotide (nt) sequences showed 99-100% nt identity with SsPV1 variant a (EF395818), while SsPV2 was not found in any sample. Using generic primers, eight samples (36·4%) were tested positive for human papillomavirus (HPV), and were characterized as follows: ß1-HPV8, ß1-HPV14, ß1-HPV206, ß2-HPV113, ß2-HPV120 and γ1-HPV173. Moreover, one unclassified γ-type was detected. CONCLUSIONS: Both swine and human PVs were detected in pig slurry in this study. The unexpected presence of HPV in pig waste could be explained as the result of an improper use of the sewage collection pits and/or with improper procedures of the operators. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the first detection of SsPV1 in Italy, along with the first detection of HPVs in pig slurry samples in Italy, and expands our knowledge about PV diversity and geographic distribution.

Influence of continuous rotation or reciprocation of Optimum Torque Reverse motion on cyclic fatigue resistance of nickel-titanium rotary instruments.

AIM: To evaluate the resistance to cyclic fatigue of ProTaper Next (PTN; Dentsply Sirona, Ballaigues, Switzerland), Revo-S (Micro-Mega, Besançon, France), Mtwo (Sweden & Martina, Padova, Italy), Twisted Files (TF, SybronEndo, Orange, CA, USA) and EndoWave (J Morita Corporation, Osaka, Japan) used in continuous rotation or in reciprocation of Optimum Torque Reverse motion (OTR). METHODOLOGY: A total of 120 nickel-titanium files were tested. Twenty-four instruments for each brand were divided into two groups (n = 12) on the basis of the motion tested: continuous rotation (Group 1) or reciprocation of OTR motion (Group 2). Resistance to cyclic fatigue was determined by recording time to fracture (TtF) in a stainless steel artificial canal with a 60° angle of curvature and 5 mm radius of curvature. The TtF data were analysed by using two-way analysis of variance (anova) and Bonferroni's post hoc tests at 0.05. RESULTS: Mtwo and TF had significantly higher TtF when compared with all other instruments, both in continuous rotation and in reciprocation of OTR motion (P < 0.0001 and P < 0.05, respectively). No difference was observed between Mtwo and TF (P > 0.05), in both motions. PTN was associated with higher cyclic fatigue resistance than Revo-S and EndoWave, both in continuous rotation and in reciprocation of OTR motions (P < 0.0001). No difference was observed between Revo-S and EndoWave, in both motions (P > 0.05). Reciprocating OTR motion improved TtF of all instruments (P < 0.0001). CONCLUSIONS: Reciprocation of OTR motion improved significantly cyclic fatigue resistance of all instruments tested compared with continuous rotation. Mtwo and TF had significantly higher cyclic fatigue than the other instruments, in both continuous rotation and reciprocation of OTR motion.

The impact of anthropogenic pressure on the virological quality of water from the Tiber River, Italy.

The objective of the present study was to assess the occurrence of major waterborne enteric viruses (enterovirus, norovirus, adenovirus, rotavirus, hepatitis A and E virus) along the Tiber River in Italy, in areas affected by different kinds of anthropogenic pressure (agricultural, urban, industrial and pristine). Moreover, in light of the recent abundant detection of human bocavirus in urban wastewater samples in Italy, the occurrence of this virus was also assessed. Virus detection was based on nested PCR followed by sequencing, and on real-time PCR. A correlation with anthropogenic pressure was observed. The urban and industrial areas were the most contaminated (100 and 75% of samples were positive for at least one virus respectively). The agricultural area was less contaminated, with 50% of samples positive. None of the samples collected in a pristine area were positive for viruses. The most frequently detected virus was human bocavirus, identified in 37·5% of samples, followed by norovirus and enterovirus (28% each) and adenovirus (21·6%). Rotavirus, and hepatitis A and E viruses were less common (<9%). Although Human Bocavirus is not considered a waterborne pathogen, the widespread contamination of river waters suggests that virus transmission via the water route should not be neglected. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this study constitutes the first attempt to assess the occurrence of enteric viruses in river waters, in areas differentially influenced by anthropogenic pressure. Enteric viruses (enterovirus, norovirus, adenovirus, rotavirus, hepatitis A and E viruses, and bocavirus) were widespread in the industrial and urban areas, and were less frequently detected in the agricultural area. Interestingly, human bocavirus was the most frequently detected virus, outnumbering even adenoviruses, known to be widespread in water environments. The widespread presence of bocavirus in surface waters suggests that a potential role of water in its transmission should not be excluded.

Detection of oncogenic viruses in water environments by a Luminex-based multiplex platform for high throughput screening of infectious agents.

Recent studies documented the detection of viruses strongly associated with human cancer in urban sewages and other water environments worldwide. The aim of this study was to estimate the occurrence of human oncogenic viruses in environmental samples (sewage, river, marine, and pool/spa water) using highly sensitive and specific multiplex bead-based assays (Luminex technology). A total of 33 samples were analysed for 140 oncogenic viral agents, including mucosal and cutaneous human papillomaviruses (HPVs), human polyomaviruses (HPyV), human herpesviruses (HHV) and mouse mammary tumour virus (MMTV). Eighty-eight percent of the samples tested positive for at least one viral pathogen and the simultaneous presence of more than one virus was frequent (mean number of positivities/sample = 3.03). A total of 30 different Alpha, Beta and Gamma HPVs were detected, including mucosal and cutaneous types. The high-risk type HPV16 was the most frequently detected virus, identified in 73% of the samples. Of the 12 HPyVs tested, only two (BKPyV and MCPyV) were detected. At least one of these two was present in 48% of the samples. MMTV was detected in 21% of the samples, while herpesviruses - HHV-6 and HHV-1 - were detected in two samples (6%). The present study is the first to provide a comprehensive picture of the occurrence of oncogenic viruses belonging to different families and species in diverse water environments, and the first to successfully use, in environmental samples, a Luminex-based multiplex platform for high throughput screening of infectious agents. Our findings, showing that oncogenic viruses are ubiquitous in water environments, pave the way for future studies on the fate of these pathogens in water environments as well as on their potential for transmission via the waterborne route.

Molecular characterization of human adenoviruses in urban wastewaters using next generation and Sanger sequencing.

Human adenoviruses (HAdVs) are of major public health importance and are associated with a variety of clinical manifestations, including gastroenteritis, respiratory, ocular and urinary tract infections. To study the occurrence, prevalence and diversity of HAdV species and types circulating in Italy, we conducted a large-scale molecular-epidemiological investigation, a yearlong monitoring of 22 wastewater treatment plants, covering 10 Italian regions, representative of northern, central, and southern Italy. A total of 141 raw sewage samples were collected from January to December 2013, and processed to detect and characterize by phylogenetic analysis a fragment of the hexon coding region of HAdVs. Nested PCR results showed the presence of HAdVs in 85 out of 141 samples (60% of samples). Fifty-nine samples were characterized by conventional Sanger sequencing as belonging to four HAdV species and four types: A (type 12, 5 samples), B (type 3, 8 samples), C (type 5, 1 sample) and F (type 41, 45 samples). The remaining 26 samples could not be characterized because of uninterpretable (mixed) electropherograms suggesting the presence of multiple species and/or types. Pools of characterized and uncharacterized PCR amplicons were further analyzed by next-generation sequencing (NGS). NGS results revealed a marked HAdV diversity with 16 additional types detected beyond the four types found by Sanger sequencing. Overall, 19 types were identified, belonging to HAdV species A-F: types 12 and 31 (species A), type 3 (species B), types 1, 2, and 5 (species C), types 9, 17, 24, 26, 37, 38, 42, 44, 48, and 70 (species D), type 4 (species E), and types 40 and 41(species F). An untypeable HAdV was also detected, showing similar percentages of identity with more than one prototype (types 15, 30, 56, and 59). Our findings documented the circulation of a wide variety of species and types in raw sewage, potentially able to affect other surface water environments and hence human health. Next-generation sequencing proved to be an effective strategy for HAdV genotyping in wastewater samples. It was able to detect a wide range of "less prevalent" types unidentified by conventional Sanger sequencing, confirming that studies based on conventional technologies may grossly underestimate the existence of some, possibly less common, types. Knowledge of the distribution of HAdV species and types would improve our understanding of waterborne HAdV-related health risks.

One-year Surveillance of Human Enteric Viruses in Raw and Treated Wastewaters, Downstream River Waters, and Drinking Waters.

Human enteric viruses are a major cause of waterborne diseases, and can be transmitted by contaminated water of all kinds, including drinking and recreational water. The objectives of the present study were to assess the occurrence of enteric viruses (enterovirus, norovirus, adenovirus, hepatitis A and E virus) in raw and treated wastewaters, in rivers receiving wastewater discharges, and in drinking waters. Wastewater treatment plants' (WWTP) pathogen removal efficiencies by adenovirus quantitative real-time PCR and the presence of infectious enterovirus, by cell culture assays, in treated wastewaters and in surface waters were also evaluated. A total of 90 water samples were collected: raw and treated wastewaters (treated effluents and ultrafiltered water reused for industrial purposes), water from two rivers receiving treated discharges, and drinking water. Nested PCR assays were used for the identification of viral DNA/RNA, followed by direct amplicon sequencing. All raw sewage samples (21/21), 61.9 % of treated wastewater samples (13/21), and 25 % of ultrafiltered water samples (3/12) were contaminated with at least one viral family. Multiple virus families and genera were frequently detected. Mean positive PCRs per sample decreased significantly from raw to treated sewage and to ultrafiltered waters. Moreover, quantitative adenovirus data showed a reduction in excess of 99 % in viral genome copies following wastewater treatment. In surface waters, 78.6 % (22/28) of samples tested positive for one or more viruses by molecular methods, but enterovirus-specific infectivity assays did not reveal infectious particles in these samples. All drinking water samples tested negative for all viruses, demonstrating the effectiveness of treatment in removing viral pathogens from drinking water. Integrated strategies to manage water from all sources are crucial to ensure water quality.

Human bocavirus in children with acute gastroenteritis in Albania.

Human Bocavirus (HBoV) has been recently identified in association with acute viral gastroenteritis (AGE). The objective of this work was to investigate the prevalence of HBoV in children with AGE in Albania. Stool specimens collected from 142 children were analyzed by amplification of partial NP1 and Vp1/Vp2 genes. HBoV was detected in 13 samples (9.1%), 12 HBoV-1 and one HBoV-2. All HBoV-positive patients were co-infected with rotavirus and/or adenovirus, a finding which might indicate that there is no clear causal association of this agent with diarrhea. Further investigation is needed to assess the pathogenic role of HBoV in childhood diarrhea.

Genetic Diversity of Human Adenovirus in Children with Acute Gastroenteritis, Albania, 2013-2015.

The objectives of the present study were to assess the occurrence of human adenoviruses (HAdVs) in paediatric patients with gastroenteritis in Albania and to characterize HAdV strains. Faecal specimens from children admitted with acute gastroenteritis to the Paediatric Hospital in Tirana were screened for HAdV, using broad-range primers targeting the hexon gene, in combination with species-specific primers targeting the fiber gene. Phylogenetic analysis was then performed to assess the genetic relationships among the different sequences and between the sequences of the samples and those of the prototype strains. Adenovirus DNA was detected in 33/142 samples (23.2%); 14 belonged to species F (13 HAdV-41 and 1 HAdV-40), 13 to species C (1 HAdV-1, 8 HAdV-2, and 4 HAdV-5), 5 to species B (HAdV-3), and 1 to species A (HAdV-12). Rotavirus coinfection was present in 9/33 (27.2%) positive samples. In the remaining 24 positive samples (12 enteric--F species; 12 nonenteric--A, B, or C species), HAdVs were detected as unique viral pathogens, suggesting that HAdV may be an important cause of diarrhoea in children requiring hospitalization. This is the first study investigating the presence of human adenoviruses (species A-G) as etiologic agents of viral gastroenteritis in children in Albania.

First detection of papillomaviruses and polyomaviruses in swimming pool waters: unrecognized recreational water-related pathogens?

AIMS: Viral outbreaks associated with swimming pools have been described worldwide. The objective of this study was to examine the extent of viral contamination in indoor and outdoor swimming pools. METHODS AND RESULTS: Pools were examined for the presence of human enteric viruses (adenovirus, norovirus and enterovirus) and nonenteric viruses (papillomavirus and polyomavirus-BK, JC, KI, WU and Merkel cell). Bacteriological parameters were also evaluated. The analysed pool waters met microbiological quality standards. Enteric viruses were not detected. On the other hand, papillomaviruses (HPV8, 12, 23, 25, 120 and unclassified HPVs) and polyomaviruses (JC and Merkel cell polyomaviruses) were detected in 9/14 samples (64%). The number of HPV DNA copies in pool waters, measured by quantitative Real-time PCR, ranged from 1.27E+04 to 1.13E+05/10L. CONCLUSION: Results show that a variety of nonenteric viruses may be discharged in pool waters by various secretions and excretions from infected individuals or asymptomatic carriers. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report on human papillomaviruses and polyomaviruses in swimming pools. The likelihood that these viruses can be transmitted by recreational activities deserves to be explored in future studies.

First Detection of Human Papillomaviruses and Human Polyomaviruses in River Waters in Italy.

Waterborne exposure to human viruses is possible through contact with contaminated water environments and can result in infections associated with a wide range of illnesses, including gastrointestinal, respiratory, ear, ocular, and skin infections. Recently, the occurrence in water environments of two groups of human viruses-both known with oncogenic potential, human polyomaviruses (HPyVs) and papillomaviruses (HPVs)-has been reported worldwide. These viruses, responsible for highly prevalent infections worldwide, have recently been proposed as potentially emerging waterborne pathogens. The objective of the present study was to examine the occurrence of HPyVs and HPVs in surface waters, by monitoring two rivers in Northwestern Italy, by nested PCR assays and sequencing. HPyVs (JC, BK, and Merkel cell polyomavirus) were detected in 10/25 (40%) samples. HPVs (HPV8, 17, 21, 25, 32, 80, 99, 105, and putative new HPVs) were identified in 14/25 (56%) river samples. The number of HPV DNA copies in waters was measured by quantitative real-time PCR. To our knowledge, this is the first detection and quantification of HPVs in surface waters. The possibility that HPyVs and HPVs can be transmitted by the waterborne route deserves to be explored in future studies.

Influence of cyclic torsional preloading on cyclic fatigue resistance of nickel - titanium instruments.

AIM: To evaluate the effect of different torsional preloads on cyclic fatigue resistance of endodontic rotary instruments constructed from conventional nickel-titanium (NiTi), M-Wire or CM-Wire. METHODOLOGY: Eighty new size 25, 0.06 taper Mtwo instruments (Sweden & Martina), size 25, 0.06 taper HyFlex CM (Coltene/Whaledent, Inc) and X2 ProTaper Next (Dentsply Maillefer) were used. The Torque and distortion angles at failure of new instruments (n = 10) were measured, and 0% (n = 10), 25%, 50% and 75% (n = 20) of the mean ultimate torsional strength as preloading condition were applied according to ISO 3630-1 for each brand. The twenty files tested for every extent of preload were subjected to 20 or 40 torsional cycles (n = 10). After torsional preloading, the number of cycles to failure was evaluated in a simulated canal with 60° angle of curvature and 5 mm of radius of curvature. Data were analysed using two-way analysis of variance. The fracture surface of each fragment was examined with a scanning electron microscope (SEM). Data were analysed by two-way analyses of variance. RESULTS: Preload repetitions did not influence the cyclic fatigue of the three brands; however, the 25%, 50% and 75% torsional preloading significantly reduced the fatigue resistance of all instruments tested (P < 0.01, P < 0.001 and P < 0.0001, respectively) except for the HyFlex CM preloaded with 25% of the maximum torsional strength (P > 0.05). CONCLUSIONS: Torsional preloads reduced the cyclic fatigue resistance of conventional and treated (M-wire and CM-wire) NiTi rotary instruments except for size 25, 0.06 taper HyFlex CM instruments with a 25% of torsional preloading.

Frequent and abundant Merkel cell polyomavirus detection in urban wastewaters in Italy.

Viruses strongly associated with human cancer have recently been detected in urban sewages and other water environments worldwide. The aim of the present study was to assess the presence of Merkel cell polyomavirus (MCPyV), a newly discovered, potentially oncogenic human virus, in urban sewage samples collected at wastewater treatment plants (WTPs) in Italy. A total of 131 raw sewage samples were collected from 21 WTPs in nine Italian regions and analyzed by both qualitative (PCR/nested) and quantitative (Real-Time qRT-PCR) methods. Of these, 66 samples (50.3 %) were positive for MCPyV by the qualitative assay. Quantitative data showed high viral loads in wastewaters (mean, 1.5E + 05 genome copies/liter). High concentrations of MCPyV were found in all WTPs under study, suggesting a wide circulation of the virus and thus the need for further studies to assess possible waterborne MCPyV transmission.

Oncogenic Papillomavirus and Polyomavirus in Water Environments: Is There a Potential for Waterborne Transmission?

Waterborne exposure to human viruses through contact with sewage-contaminated water environments can result in infections associated with a wide range of illnesses. Gastrointestinal symptoms are the most commonly encountered manifestations of waterborne viral illness. Respiratory diseases, neurological diseases and paralysis can also occur. Whether viral infections resulting in health outcomes like cancer might also be transmitted by the waterborne route is unknown. Recently, viruses belonging to two oncogenic groups-Human Papillomaviruses (HPVs) and Human Polyomaviruses (HPyVs)-have been detected in urban sewages worldwide. The latter have also been identified in other water environments. HPVs are epitheliotropic viruses responsible for several diseases of skin and mucosae, from common warts to squamous intraepithelial lesions that can either heal or progress to invasive carcinoma of the cervix, vulva, vagina, penis, anus or oropharynx. Human PyVs infect different tissues and organs, causing infections that are usually subclinical in immunocompetent individuals but can be serious in immunocompromised hosts. These pathogens belong to a family of DNA tumour viruses. Merkel cell polyomavirus, a HPyV identified in recent years, has attracted much attention due to its link with a rare and aggressive form of human cancer. Merkel cell carcinoma, the incidence of which has tripled over the past two decades. JC polyomavirus and BK polyomavirus are also potentially oncogenic. The observed abundance and wide dissemination of HPVs and HPyVs in water environments strongly suggest the need to shed light on the fate of these viruses in water environments and to elucidate their potential for waterborne transmission. Such information is essential for the improvement of wastewater management programs in terms of both sewage treatment and water quality surveillance.

Improving the prediction of malignancy in cytologically suspicious thyroid nodules.

BACKGROUND: Fine needle cytology aspirates (FNA) classified as THY4 are a heterogeneous group suspicious for malignancy [papillary thyroid cancer (PTC)], which is confirmed in 50-80% of cases after surgery. AIM: To better stratify THY4 FNA specimens for the relative risk of malignancy. METHODS: We retrospectively analyzed 78 thyroid nodules classified as THY4 because of the presence of atypical cells, hypercellular trabeculae and/or intranuclear inclusions (ICI), in the absence of papillae. Two subgroups were identified: group 1 (38 nodules), showing ICI with (no.=17) or without (no.=21) trabeculae and cellular atypia, and group 2 (40 nodules), showing trabeculae and atypia but without ICI. RESULTS: PTC was detected at histology in 56/78 of the patients (71.8%). Malignancy occurred in 36/38 (94.7%) of the patients in group 1 and in 20/40 (50.0%) of the patients in group 2. Therefore, the positive predictive value (PPV) for PTC was 97.3% in the ICI+ specimens (group 1), with a sensitivity of 64.3% and specificity of 95.2%. When only ICI was present, without atypia and trabeculae, the PPV and specificity were similar (95.0 and 95.2%, respectively), but the sensitivity was decreased (48.7%). In specimens without ICI (group 2), the PPV was only 50.0%; however, combined with young age (<40 yr) and male gender, it reached a value similar to that of group1. CONCLUSIONS: In ICI+ specimens compared to ICI-, the risk of PTC is nearly doubled, since PPV increases from 50.0% to 97.3%. This observation suggests that surgery should be considered mandatory in all lesions classified THY4 at FNA, although the relevant difference in terms of cancer risk between ICI- vs ICI+ nodules might be an useful information for both the clinician and the patient.

Survival characteristics and prognostic variables of dogs with preclinical chronic degenerative mitral valve disease attributable to myxomatous degeneration.

BACKGROUND: Preclinical myxomatous mitral valve degeneration (MMVD) includes a heterogeneous group of dogs. Therefore, identifying risk factors for progression of the disease is of clinical importance. OBJECTIVES: To investigate survival time and risk factors for clinical and echocardiographic variables taken at initial examination for clinical progression in preclinical MMVD dogs. ANIMALS: A total of 256 dogs with stage B1 or B2 MMVD. MATERIALS AND METHODS: Medical records of 256 dogs with preclinical MMVD were reviewed retrospectively. Long-term outcome was assessed by telephone interview. Dogs alive at the time of phone interview were asked to return to the hospital for re-evaluation of their cardiac status. RESULTS: Seventy of 256 (27.3%) dogs died during the observation period. The median survival time, regardless of cause of death, was 588 (range 75-1,668) days. The presence of a murmur was associated with an increased risk of death (AHR 2.14; 95% CI 1.12, 4.11; P = 0.022). Thirty (12%) deaths were considered cardiac related. LA/Ao > 1.4 was the only negative predictor (AHR 2.64; 1.13, 6.13; P = 0.024) for cardiac-related deaths. Eighty-three dogs were re-examined, of which 34 progressed to a more advanced stage of MMVD. The presence of Emax > 1.2 (AHR 2.75; 95% CI 1.01, 7.48; P = 0.047) and cough (AHR 7.89; 95% CI 3.18, 20.07; P < 0.001) were significant in the multivariate analysis. CONCLUSIONS AND CLINICAL IMPORTANCE: Preclinical MMVD represents a relatively benign condition in dogs. Clinicians might find stratification of this dog population according to risk factors based on clinical and echocardiographic findings helpful in determining treatment.