Prevalence of Common Viral Skin Infections in Beach Volleyball Athletes.

Viral skin infections often affect the sports community. The aim of this study was to assess the rates, location sites, and seasons of appearance of common viral cutaneous diseases in beach volleyball athletes in Greece. Five hundred and forty-nine beach volleyball athletes participated in this study. The average age was 28.4 years. The viral infections were herpes simplex (type 1), molluscum contagiosum and warts. The measured parameters included: gender, age, the season when athletes may be more susceptible to infections and the location of infection in the body. Practicing information such as the number of training years, number of weekly trainings, and average hours of daily training was also recorded. Incidence rates correlated in relation to age: (a) warts (p < 0.001), molluscum contagiosum (p < 0.001), and herpes simplex (p = 0.001); (b) years of training: warts (p < 0.001), molluscum contagiosum (p < 0.001), and herpes simplex (p = 0.004); (c) average hours of daily training: molluscum contagiosum (p = 0.006) and herpes simplex (p < 0.010). The skin is the largest organ, and the risk of infection should not be underestimated. Prevention, early detection, recognition, and treatment are related to health and athletic performance, but also to the risk of transmission.

Recombination events and variability among full-length genomes of co-circulating molluscum contagiosum virus subtypes 1 and 2.

Molluscum contagiosum virus (MCV) is the sole member of the Molluscipoxvirus genus and causes a highly prevalent human disease of the skin characterized by the formation of a variable number of lesions that can persist for prolonged periods of time. Two major genotypes, subtype 1 and subtype 2, are recognized, although currently only a single complete genomic sequence corresponding to MCV subtype 1 is available. Using next-generation sequencing techniques, we report the complete genomic sequence of four new MCV isolates, including the first one derived from a subtype 2. Comparisons suggest a relatively distant evolutionary split between both MCV subtypes. Further, our data illustrate concurrent circulation of distinct viruses within a population and reveal the existence of recombination events among them. These results help identify a set of MCV genes with potentially relevant roles in molluscum contagiosum epidemiology and pathogenesis.

Rapid detection and typing of Molluscum contagiosum virus by FRET-based real-time PCR.

A fluorescence resonance energy transfer (FRET)-based real-time PCR (RT-PCR) was developed for very sensitive and specific detection of Molluscum contagiosum virus (MCV), as well as reliable differentiation of the two MCV subtype genetic lineages, MCV1 and MCV2, in a single reaction. The assay employs modified primers specific for the viral MC021L gene and uses two novel FRET hybridization probes to detect polymorphisms specific for each of the two subtypes. The sensitivity of the assay at a 95% detection level for both MCV subtypes was 3.3 DNA copies/reaction and the dynamic range was nine orders of magnitude, discriminating 10-10(9) viral genome equivalents/reaction. Post-amplification probe-specific dissociation analysis differentiated the two viral subtypes reliably in all tested concentrations. Testing of 43 tissue specimens clinically diagnosed as MCV lesions showed complete agreement with the results obtained with previously described MCV specific MC080R Taqman RT-PCR and MC021L whole gene sequencing. The novel assay is simple, robust and easy to perform, and may be of great value for clinical and epidemiological studies of MCV infections and related conditions.

Identification and genotyping of molluscum contagiosum virus from genital swab samples by real-time PCR and Pyrosequencing.

BACKGROUND: Laboratory diagnosis of molluscum contagiosum virus (MCV) is important as lesions can be confused with those caused by Cryptococcus neoformans, herpes simplex virus, human papillomavirus, and varicella-zoster virus. OBJECTIVES: To develop a rapid method for identifying patients infected with MCV via swab sampling. STUDY DESIGN: Two dual-labeled probe real-time PCR assays, one homologous to the p43K gene and one to the MC080R gene, were designed. The p43K PCR was designed to be used in conjunction with Pyrosequencing for confirmation of PCR products and discrimination between MCV1 and MCV2. RESULTS: Both PCR assays were optimized with respect to reaction components, thermocycling parameters, and primer and probe concentrations. The specificities of both PCR assays were confirmed by non-amplification of 38 known human pathogens. Sensitivity assays demonstrated detection of as few as 10 copies per reaction. Testing 703 swabs, concordance between the two real-time PCR assays was 99.9%. Under the developed conditions, Pyrosequencing of the p43K PCR product was capable of providing enough nucleotide sequence to definitively differentiate MCV1 and MCV2. CONCLUSIONS: These real-time PCR assays can be used for the rapid, sensitive, and specific detection of MCV and, when combined with Pyrosequencing, can further discriminate between MCV1 and MCV2.

Efficacy of pulsed dyed laser (585 nm) in the treatment of molluscum contagiosum subtype 1.

Molluscum contagiosum is a common cutaneous disease that may be difficult to treat when there are multiple lesions; especially in children. This study was conducted to determine the efficacy of pulsed dye laser (585 nm) in the treatment of molluscum contagiosum in 20 children. In the treated group, 70.5% of lesions healed after the first treatment; the remaining 10.6% after the second treatment (2 weeks later). The overall cure rate was significantly different from the control group (p< 0.01). The therapy was also well tolerated. Only mild transient hypopigmentation and erythema were observed. None encountered infectious events. In conclusion, pulsed dye laser is a good alternative treatment for molluscum contagiosum due to high efficacy and mild transient side effects.

Detection of Molluscum contagiosum virus (MCV) subtype I as a single dominant virus subtype in Molluscum lesions from a Turkish population.

BACKGROUND: Molluscum contagiosum has a worldwide occurrence and its primary mode of transmission is via direct human contact including sexual means. The aim of the study was to implement a polymerase chain reaction-based assay for detection and subtyping of Molluscum contagiosum virus (MCV) in skin lesions diagnosed with molluscum contagiosum in a large regional teaching hospital in Turkey. METHODS: For this purpose, a total of 61 patients were included in the study. Randomly selected single lesion from each patient was used to extract DNA material and a specific PCR reaction amplifying 393-bp- and 575-bp-long regions from MCV genome was used in the detection. Subtyping was carried out by digestion of the amplified 575-bp product with restriction endonuclease enzyme BamHI. Both amplified and restriction enzyme digested products visualized on agarose gel electrophoresis. RESULTS: All 61 molluscum cases (100%) included in the study contained MCV genetic material as demonstrated by the presence of 393- and 575-bp-long PCR amplified products. Restriction enzyme BamHI digestion of the 575-bp-long amplicon indicated that the infecting subtype in all the cases (100%) was MCV subtype I. CONCLUSIONS: Results of this study demonstrate that subtype I is the only infecting strain dominant in our region. Because the only consecutive molluscum patients admitted to our hospital were included in the study, our data do not rule out the possibility that other genotypes might be present in the Turkish population. However, it is not unreasonable to conclude that similar trends exist in the rest of the country. Results also show that a molecular-based diagnostic assay would be feasible in cases where diagnosis was deemed necessary.

Molecular epidemiology of molluscum contagiosum virus and analysis of the host-serum antibody response in Spanish HIV-negative patients.

Molluscum contagiosum virus (MCV) lesions from Spanish human immunodeficiency virus (HIV)-negative patients were clinically examined and analyzed for virus detection and typing. In a study of 147 patients, 97 (66%) were children under 10 years, of whom 49% had atopic dermatitis. MCV lesions were morphologically indistinguishable among the different age groups, but atopic patients presented larger lesions compared with patients without the disorder. In adults, lesions were observed mainly on the genitals. MCVI was the predominant subtype. The deduced MCVI/MCVII ratio (146:1) was much higher than that found in other geographical areas. Protein preparations of the virus-induced lesions were immunoblotted with sera from 25 MCVI patients. The host-serum antibody response was weak and variable, although no significant differences were found between atopic and nonatopic patients. Three immunoreactive proteins of 74/80, 60, and 35 kDa were detected in almost all the analyzed sera. The 35 and 74/80-kDa proteins were virus specific, whereas the 60-kDa protein band was composed of a mix of human keratins. Immunoblotting of MCV lesions and vaccinia virus-infected cell extracts with either MCV patient serum or a rabbit antiserum against vaccinia virus showed no cross-reactivity of these two human poxviruses at the antigenic level.

Close relationship between equine and human molluscum contagiosum virus demonstrated by in situ hybridisation.

To determine whether the virus responsible for human molluscum contagiosum (MCV) is the causal agent of a similar disease in horses, in situ hybridisations using cloned fragments of human MCV DNA labelled with digoxigenin were carried out on formalin-fixed biopsy sections of lesions from two horses with molluscum contagiosum-like skin lesions. In both instances there was evidence of specific hybridisation of the labelled probe to target DNA in the sections under high stringency conditions, identified by the development of a deep blue-purple stain in the cytoplasm of cells in the stratum spinosum and stratum granulosum of the lesions and the absence of non-specific hybridisation in adjacent non-lesional areas of the epidermis. These results indicate that on the basis of very close homology of their viral DNA sequences, the causative virus of equine molluscum contagiosum is either identical with, or very closely related to, its human equivalent.

Identification and typing of molluscum contagiosum virus in clinical specimens by polymerase chain reaction.

A polymerase chain reaction (PCR) which enables the detection of molluscum contagiosum virus (MCV) genomes in either fresh or formalin-fixed clinical specimens is described. The primers used were designed to amplify a 167 bp region of the 3.8 kbp HindIII fragment K of the MCV 1 genome. The ability of this PCR to detect three common MCV types (1, 1v and 2) in clinical specimens was confirmed using frozen extracts from 75 molluscum lesions, and digests of single sections of 11 formalin-fixed, paraffin-embedded lesions; all of which had been previously typed by Southern hybridisation. In addition, 2 specimens previously negative by hybridisation were shown to be positive for MCV DNA by PCR. Confirmation of the identity of the PCR products and distinction between the two major MCV types (MCV 1/1v versus MCV 2) was achieved by comparison of the results of cleavage with the restriction endonucleases Hhal and Sacl. Sequencing of the PCR products revealed complete homology between MCV 1 and 1v, but minor nucleotide variations between MCV 1/1v and MCV 2 were identified. As well as providing a highly sensitive means of diagnosis, the technique may also prove useful for investigations into the pathogenesis, epidemiology and natural history of molluscum contagiosum infection.

The genome of molluscum contagiosum virus: analysis and comparison with other poxviruses.

Analysis of the molluscum contagiosum virus (MCV) genome revealed that it encodes approximately 182 proteins, 105 of which have direct counterparts in orthopoxviruses (OPV). The corresponding OPV proteins comprise those known to be essential for replication as well as many that are still uncharacterized, including 2 of less than 60 amino acids that had not been previously noted. The OPV proteins most highly conserved in MCV are involved in transcription; the least conserved include membrane glycoproteins. Twenty of the MCV proteins with OPV counterparts also have cellular homologs and additional MCV proteins have conserved functional motifs. Of the 77 predicted MCV proteins without OPV counterparts, 10 have similarity to other MCV proteins and/or distant similarity to proteins of other poxviruses and 16 have cellular homologs including some predicted to antagonize host defenses. Clustering poxvirus proteins by sequence similarity revealed 3 unique MCV gene families and 8 families that are conserved in MCV and OPV. Two unique families contain putative membrane receptors; the third includes 2 proteins, each containing 2 DED apoptosis signal transduction domains. Additional families with conserved patterns of cysteines and putative redox active centers were identified. Promoters, transcription termination signals, and DNA concatemer resolution sequences are highly conserved in MCV and OPV. Phylogenetic analysis suggested that MCV, OPV, and leporipoxviruses radiated from a common poxvirus ancestor after the divergence of avipoxviruses. Despite the acquisition of unique genes for host interactions and changes in GC content, the physical order and regulation of essential ancestral poxvirus genes have been largely conserved in MCV and OPV.

Detection and typing of molluscum contagiosum virus in skin lesions by using a simple lysis method and polymerase chain reaction.

A polymerase chain reaction (PCR) assay for the rapid detection and typing of molluscum contagiosum virus (MCV) was developed. The target DNA was a 393 base pair (bp) segment, which is present in the coding region of the MCV p43K gene product. Release of MCV DNA from skin lesions was performed by using a simple procedure that provided suitable template DNA for amplification, and allowed detection of MCV directly in clinical material. The PCR yielded a unique 393 bp product when MCV DNA was used as template. This product was not shown with DNA from other viruses and bacterial pathogens causing skin diseases. The specific PCR product was obtained with individual lesions from all patients clinically diagnosed with MCV infection, whereas no products were detected with skin samples from healthy individuals. Sequencing of this PCR product allowed determination of the virus subtype on the basis of previously described nucleotide differences between subtypes MCVI and MCVII. To avoid the sequencing process, a second PCR assay was developed, in which the target DNA sequence included a MCVI-specific recognition site for the restriction endonuclease BamHI. This PCR assay yielded a unique 575 bp product with lesions from either MCVI- or MCVII-infected patients. However, only the MCVI-derived product was susceptible to BamHI digestion, which generated two fragments of 291 and 284 bp, respectively. Amplification of specific MCV DNA sequences from single, individual lesions provides a sensitive and reliable method for laboratory diagnosis and molecular epidemiology studies of molluscum contagiosum.

Analysis of molluscum contagiosum virus genomes isolated in Japan.

The genomes of 477 Japanese strains of molluscum contagiosum virus (MCV) were analyzed using an in-gel digestion method with the restriction enzyme BamHI, and classified into four types, including a newly detected type (MCV type 4). All type 1 (MCV-1) genomes examined so far in Japan showed a common difference from the genome of the MCV-1 prototype (MCV-1p), the type reported to be most prevalent in Europe. The common markers of the variants of MCV-1 were 24-kbp fusion fragments generated by the loss of a BamHI site between the D2 and F fragments of MCV-1p. These variants of MCV-1 were classified into three groups (MCV-1va, MCV-1vb, MCV-1vc), with the variability among them being due to additions and losses of BamHI sites located in the right terminus and around the E and I fragments of MCV-1va. The restriction map of MCV-4 was generated and lined up with those of the other types. Cross-hybridization analysis revealed that the organization of all types of MCV genomes were essentially colinear. Considerable numbers of BamHI restriction sites were conserved between MCV-2 and 4, indicating a close analogy between them. The overall prevalence of MCV, as shown by the ratios of MCV-1 (MCV-1p):MCV-2:MCV-3:MCV-4, was 436(0):13:24:4. Thus, the molecular epidemiology of MCV in Japan is characterized by the absence of the European prototype of MCV-1, the exclusive occurrence and abundance of variants of MCV-1, a greater prevalence of MCV-3 over MCV-2, and the presence of MCV-4.

Enzyme-linked immunosorbent assay for measurement of IgG antibody to Molluscum contagiosum virus and investigation of the serological relationship of the molecular types.

An ELISA was developed for measuring levels of IgG antibodies to Molluscum contagiosum virus (MCV) using DNA typed purified virus as antigen. The assay was found to be specific and sensitive for antibodies to MCV as well as being economical in its use of antigen. A close relationship was found between antibody levels to the MCV molecular types 1, 1v and 2 by cross-testing sera on plates coated with the different molecular types of the virus as antigen (P < 0.001).

Characterisation by restriction mapping of three subtypes of molluscum contagiosum virus.

DNA from Molluscum contagiosum virus (MCV) isolates was analysed by restriction endonuclease digestion, identifying three virus subtypes. The structural features of MCV DNA are typical of poxviral DNA. Physical maps of cleavage sites for BamHI, CIaI, and HindIII were constructed for single isolates of each subtype. These differ extensively, indicating the independence of the three subtypes. However, they are closely related, as determined by molecular hybridisation and nucleotide sequence analysis, and their genomes are essentially colinear. There is marked geographical variation in the relative incidence of MCV I and II, whilst MCV III is uniformly rare.

Stability of molluscum contagiosum virus DNA among 184 patient isolates: evidence for variability of sequences in the terminal inverted repeats.

The stability of the Molluscum contagiosum virus Type 1 genome (188 kbp) was studied in 184 DNA isolates from 131 patients. Variability of up to 1.5 kbp at both ends of the genome symmetrically was observed using restriction analysis of the DNA isolates and by Southern Blot experiments using cloned and labeled HindIII terminal DNA fragments of MCV-1 prototype DNA. The variable sequences were mainly confined to the terminal fragments and parts of the MCV-1 terminal repeats. Labeled probes did not detect terminal sequences of MCV Type 2 under the applied stringency. A less marked instability of the central MCV-1 BamHI DNA fragment F was observed within the genome coordinates 0.431 to 0.454 mu. Reiteration of tandem repeats similar to those described for vaccinia virus might explain the variability of the terminal sequences and might be involved in viral replication.

[Molluscum contagiosum virus types found in Japan].

Total DNAs obtained from Japanese patients with molluscum contagiosum (MC) were analyzed by agarose gel electrophoresis after digestion with Bam HI, Hind III or Cla I restriction enzymes, which revealed the presence of four different cleavage patterns of MC virus (MCV) DNAs. The comparison with previously reported MCV types clarified that two of them were identical with MCV-1 and -2, respectively. The other two isolates were considered as yet unrecognized types and named MCV-3 and -4, respectively.

Molecular epidemiology of Australian isolates of molluscum contagiosum.

Molluscum contagiosum lesions obtained from 75 Australian patients, 22 (29%) of whom were HIV positive, were examined by restriction endonuclease analysis and Southern blot hybridisation using radiolabelled and digoxigenin-labelled MCV DNA probes. The isolates were classified (MCV 1, 1v, 2, and 2v) on the basis of these results, which were in turn correlated with the clinical features of each lesion. A total of 44 (59%) of the lesions contained MCV 1 or 1v, 24 (32%) contained MCV 2 or 2v, three (4%) contained multiple MCV types, whereas four (5%) of lesions submitted contained no detectable MCV DNA. The ratio of MCV 1/1v to MCV 2/2v was determined to be approximately 1.75:1. There were substantial differences between the distribution of MCV types 1 and 2 among patients of different age groups, but no significant relationship between MCV type and the sex of the patient was found. MCV 2 was more frequently detected in lesions from anogenital areas and in immunosuppressed (HIV-positive) patients, and MCV 1 was more commonly isolated from skin rather than genital lesions, but neither association was statistically significant. Fragment F (10 kbp) obtained from the genome of MCV 1 was capable of differentiating MCV 1 from MCV 2 when used as a probe in hybridisation experiments with BamHI cut samples and may be useful when small amounts of lesion prevents differentiation by direct visualisation of restriction endonuclease fragments.

Molluscum contagiosum virus types in genital and non-genital lesions.

The endonuclease digest patterns of viral DNA from 48 genital and 45 non-genital molluscum contagiosum virus (MCV) lesions were examined. The overall ratio of MCVI to MCVII was 3.23:1. There was no predominance of either MCV type in genital lesions. No obvious morphological differences were seen between MCVI and MCVII lesions. MCVII was not found in any patient under 15 years old.