Isolation of infectious Zika virus from a urine sample cultured in SIRC cells from a patient suspected of having rubella virus

A great variety of viruses which cause exanthema share other clinical manifestations, making the etiologic identification a very difficult task, relying exclusively on the clinical examination. Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as congenital rubella syndrome (CRS). In the present report, we described the presence of Zika virus (ZIKV) particles in urine samples and also ZIKV isolation in SIRC cells from the urine of a patient in acute phase of suspected rubella disease. The 50-year-old unvaccinated woman living in Sao Paulo, Brazil, was admitted to the emergency room with fever, headache, rash, arthralgia and prostration. Urine samples were collected for virus isolation and RT-qPCR. SIRC and Vero cells were inoculated with urine samples during 7 days. RT-qPCR was performed using measles virus (MV) and RV primers and both were found to be negative. After this result, RT-qPCR was performed for parvovirus B19, herpes virus 6 and ZIKV. The urine sample and the isolate were positive by Real Time PCR for ZIKV and negative for all other viruses tested. The sequences isolated are from the Asiatic lineage

Ultrastructural Description of Sarcocystis Sp. in Cardiac Mus-cle of Naturally Infected Alpacas (Vicugna pacos)

Background: Recently, it was proposed the name of Sarcocystis masoni n. sp. for the Sarcocystis that causes microcyst in skeletal muscle of South American camelids. However, there are no ultrastructural reports of microcysts of Sarcocystis in cardiac muscle of alpacas. This study reports ultrastructural features of microcysts of Sarcocystis sp. from cardiac muscle of naturally infected alpacas. Methods: Thirty alpacas (age range: three to five years) from the province of Junin, Peruvian Central Andes, were included in this study in January 2015. Cardiac muscle samples were evaluated by histology and transmission electron microscopy. Results: Bradyzoites in cysts had typical characteristics of Apicomplexa including organelles, a large nucleus, micronemes, dense bodies, and polysaccharide granules. Moreover, cysts had a thin wall with numerous, short, finger-like shapes with rounded tip protrusions (0.51 x 0.17 µm). Conclusion: Sarcocystis sp. from the heart and S. masoni n. sp. from the skeletal muscle have similar ultrastructural characteristics

Immunocytochemical expression of p16 INK4A and Ki-67 in cytologically negative and equivocal pap smears positive for oncogenic human papillomavirus

This study was designed to analyze the cross-sectional comparison of the p16 INK4A and Ki-67 immunocytochemical expression in negative and equivocal (atyp-ical squamous cells of undetermined significance (ASC-US)) liquid-based cytology (LBC) samples testing positive for high-risk human papillomavirus (HPV) types with HC2 assay or polymerase-chain reaction (PCR). A series of 199 consecutive LBC speci-mens derived from the same number of women participating in the ongoing Latin American Screening Study at Leonor Mendes de Barros Hospital, São Paulo, were analyzed using immunocytochemistry for expression of p16 INK4A and Ki-67 in negative and equivocal LBC samples testing positive for high-risk HPV types with hybrid capture II test (HC2) or PCR. All patients with at least one test positive (cytology, PCR, and/or HC2) were followed each 6 months for 3 years. The follow-up procedure consisted of visual examination, colposcopic inspection, cytology, and HC2 assay. Among the neg-ative cytologic samples, 101 were HPV-positive and 55 HPV-negative. Of the HPV-pos-itive group, 59 of 101 cases (58.4%) were positive for both p16 and Ki67 immunostaining, and 17 of 101 (16.8%) were negative for both. The proportion of Ki-67-positivity increased almost in parallel with the increasing grade of p16-positivity (p = 0.0001 for linear trend). In the HPV-negative group, both markers were negative in 41 of 55 cases (74.5%), and no statistical relationship was observed between the two markers (Pearson, p = 0.595). HPV-positive ASC-US samples demonstrated a simulta-neous positive immunoreaction for p16 and Ki67 in 11 of 16 cases (68.7%), whereas 3 (18.7%) were concurrently negative. The relationship between the two markers was of borderline significance (Pearson, p = 0.053), but no linear relationship was found be-tween the graded p16 and Ki-67 expression (p = 0.065 for linear trend). In the HPV-negative ASC-US group, there was no statistical association between the graded p16 and Ki-67 positivity (Pearson, p = 0.281). After 36 months of follow-up of the ASC-US patients, 6 women still displayed ASC-US smear, of which 4 of 6 were HPV-positive and expressed both p16 and Ki-67 markers. Two of 43 ASC-US smears had high-grade squamous intraepithelial lesions diagnosed (4.6%), and 1 had low-grade squamous intraepithelial lesion (2.3%). All of those were positive for HPV, p16 and Ki-67. Patients with ASC-US diagnosis and positive high-risk HPV status and positive for p16 INK4A Ki67 should be carefully observed to exclude occurrence of a squamous intraepithelial lesion. The combination of these two markers can be a useful implement for manage-ment of women with equivocal cytology.European Commission (EC) - INCO-DEV Programme - Project #ICA4-CT-2001-10013

DNA-Citoliq Sstem (DCS) : a new liquid base cytology system : technical aspects

Introduction: DCS is a new liquid-base cytology system for cell collected in UCM. Objective: assess technical aspects of DCS under different pro-tocols. Methods: the first experiment was performed with 144 samples. After cervical scraping with Ayre spatula and endocervical brushing, the sample was immediately smeared on the slide and alcohol-fixed. The same brush with residual cells was used to scrap the ectocervical surface and conditioned in a UCM tube. For each case, 4 slides were prepared according to DCS procedures, except the vortexing time (5, 10, 15 and 20 seconds) from protocols 1 to 4. The second experiment was made with 50 samples, with a vortexing time of 30 seconds. Cytological criteria for sample quality were: cell distribution (empty areas and clumping), fixation artifacts, cellularity and presence of TZC. Results: on the experiment 1, significant dif-ferences were detected between protocols 1 and 4, but not between protocols 2 and 3. Compared to conventional smears, DCS preparations showed lesser empty areas in slides 1 and 4 (p = 0.024 and p = 0.003). No fixation artifacts and high TZC representation were found in protocols 3 and 4. In the experiment 2, although not statistically significant, more cases with TZC were seen. No adverse effects were found on Pap staining, cytomorphol-ogy or on fixation cells properties. Conclusion: these preliminary results with 20 or 30 seconds vortexing yielded well-preserved cytomorphology, with high representation of endocervical cells and better cells distribution, thus leading to a favorable expectative regarding its use in large trials for assessing diagnostic performance.Introdução: DCS é um novo sistema de citologia em base líquida para células coletadas no UCM. Objetivo: avaliar aspectos técnicos do DCS, com diferentes protocolos, em um primeiro experimento com 144 amostras. Em segundo experimento, com 50 amostras, estudar os achados com 30 segundos de vórtex. Métodos: colheu-se material para o preparado convencional com espátula de Ayre e escova endocervical. Usou-se a mesma escova, com células endocervicais residuais, para escovar a ectocérvice, acondicionando-a em tubo com UCM. De cada amostra obteve-se quatro lâminas, com tempo de vórtex de 5, 10, 15 e 20 segundos, protocolos 1 a 4. Utilizaram-se os seguintes critérios citológicos para definir a qualidade da amostra: distribuição celular (áreas vazias e amassamento), artefatos de fixação, celularidade e presença de células da ZT. Resultado: no primeiro experimento observaram-se diferenças significativas apenas entre os protocolos 1 e 4. Comparados aos esfregaços convencionais, os preparados DCS mostraram menos áreas vazias nas lâminas 1 e 4 (p = 0,024 e p = 0,003). Nenhum artefato de fixação e alta representação da ZT foi encontrado nos protocolos 3 e 4. No experimento 2, embora sem significância estatística, observou-se maior celularidade da ZT. Não se verificou qualquer efeito adverso na coloração de Papanicolaou, citomorfologia ou propriedades de fixação. Conclusão: os resultados com 20 ou 30 segundos de vórtex proporcionam citomorfologia bem preservada, alta representação de células endocervicais e melhor distribuição celular, sugerindo expectativa favorável quanto à performance desse sistema para fins diagnósticos.Digene do Brasil Ltda

Extracellular Vesicles from <i>Leishmania (Leishmania) infantum</i> Contribute in Stimulating Immune Response and Immunosuppression in Hosts with Visceral Leishmaniasis

Visceral leishmaniasis (VL) is a chronic systemic disease. In Brazil this infection is caused by Leishmania (Leishmania) infantum. Extracellular vesicles (EVs) released by Leishmania species have different functions like the modulation of host immune systems and inflammatory responses, among others. This study evaluated the participation of EVs from L. (L.) infantum (Leish-EVs) in recognition of the humoral and cellular immune response of hosts with VL. Promastigotes were cultivated in 199 medium and, in the log phase of growth, they were centrifuged, washed, resus-pended in RPMI medium, and incubated for 2 to 24 h, at 25 °C or 37 °C to release Leish-EVs. This dynamic was evaluated using transmission (TEM) and scanning (SEM) electron microscopies, as well as nanoparticle tracking analysis (NTA). The results suggested that parasite penetration in mammal macrophages requires more Leish-EVs than those living in insect vectors, since promastigotes incubated at 37 °C released more Leish-EVs than those incubated at 25 °C. Infected THP-1 cells produced high EV concentration (THP-1 cells-EVs) when compared with those from the control group. The same results were obtained when THP-1 cells were treated with Leish-EVs or a crude Leishmania antigen. These data indicated that host–EV concentrations could be used to distinguish infected from uninfected hosts. THP-1 cells treated with Leish-EVs expressed more IL-12 than control THP-1 cells, but were unable to express IFN-γ. These same cells highly expressed IL-10, which inhibited TNF-α and IL-6. Equally, THP-1 cells treated with Leish-EVs up-expressed miR-21-5p and miR-146a-5p. In conclusion, THP-1 cells treated with Leish-EVs highly expressed miR-21-5p and miR-146a-5p and caused the dysregulation of IL-10. Indirectly, these results suggest that high expression of these miRNAs species is caused by Leish-EVs. Consequently, this molecular via can contribute to immunosuppression causing enhanced immunopathology in infected hosts

Human extracellular vesicles and correlation with two clinical forms of toxoplasmosis.

AIM:This study analyzed microvesicles and exosomes, called as extracellular vesicles (EVs) excreted in serum and cerebrospinal fluid (CSF) from patients with cerebral or gestational toxoplasmosis. METHODS:Clinical samples from 83 individuals were divided into four groups. Group I, 20 sera from healthy individuals and pregnant women (seronegative for toxoplasmosis); group II, 21 sera from seropositive patients for toxoplasmosis (cerebral or gestational forms); group III, 26 CSF samples from patients with cerebral toxoplasmosis/HIV co-infection (CT/HIV) (seropositive for toxoplasmosis); and group IV, 16 CSF samples from seronegative patients for toxoplasmosis, but with HIV infection and other opportunistic infections (OI/HIV). Serum and CSF samples were ultracentrifuged to recover EVs. Next, vesicle size and concentration were characterized by Nanoparticle Tracking Analysis (NTA). RESULTS:Concentrations of serum-derived EVs from toxoplasmosis patients (mean: 2.4 x 1010 EVs/mL) were statically higher than of non-infected individuals (mean: 5.9 x 109 EVs/mL). Concentrations of CSF-derived EVs were almost similar in both groups. CT/HIV (mean: 2.9 x 109 EVs/mL) and OI/HIV (mean: 4.8 x 109 EVs/mL). Analyses by NTA confirmed that CSF-derived EVs and serum-derived EVs had size and shape similar to microvesicles and exosomes. The mean size of EVs was similar in serum and CSF. Thus, the concentration, and not size was able distinguish patients with toxoplasmosis than healthy individuals. Presence of exosomes was also confirmed by transmission electron microscopy and evidence of tetraspanins CD63 and CD9 in immunoblotting. Relative expressions of miR-146a-5p, miR-155-5p, miR-21-5p, miR-29c-3p and miR-125b-5p were estimated in exosomal miRNA extracted of EVs. Serum-derived EVs from group II (cerebral and gestational toxoplasmosis) up-expressed miR-125b-5p and miR-146a-5p. CSF-derived EVs from CT/HIV patients) up-expressed miR-155-5p and miR-21-5p and were unable to express miR-29c-3p. CONCLUSION:These data suggest the participation of EVs and exosomal miRNAs in unbalance of immune response as elevation of TNF-α, IL-6; and downregulation of IFN-γ in cerebral and gestational forms of toxoplasmosis