Swabs are used as a collecting device for many biological samples and its complete elution is a desired step for clinical and forensic diagnostics. Swabs are made of cotton, rayon, polyurethane, foam or polyester and come in a spun or flocked-tipped format. They are used to extract biological samples from a patient, which includes saliva, mucous, blood, semen or other body fluids. These body fluids then undergo the process of elution where the collected samples are extracted from the swabs into an elution fluid. Apart from biological samples, the importance of swabbing and elution also becomes more evident in forensics, where the concentration of available cells is very low. One such example is rape kit analysis. Another field of application is the capture and release of bacterial spores from environmental contact surfaces and food surfaces, which also indicate the use of swabs in non-biological areas. The recovery of the biological material from the fibre matrix of the swab has a significant influence on diagnostic sensitivity of any assay. The recovery of micro-organisms from a matrix of swab fibres depends on the nature of the body fluid, the type of the swab fibres and the process of elution. Various methods are used to elute samples from swab, including the use of chemicals to digest the cotton fibres to remove intact cells (~20% recovery), centrifugation (~58% recovery), piezoelectric vibration or pressurized fluid-flow (~60% recovery). These methods are either passive (chemical elution) or provides a gentle tangential shear force through associated flow (centrifugation, piezoelectric and pressurized flow), resulting in a low recovery. The success of all the downstream processes of elution, like lysis, DNA amplification and detection, depends on the number of cells eluted from the swab fibre matrix. Hence, the recovery efficiency is an important parameter for determining the performance of elution, and higher value of the same is desired for most diagnostic assays. This thesis reports a magnetically-actuated physical impingement method for elution and recovery of artificial sputum samples from cotton fibres. A device has been fabricated to induce a rotating magnetic field on smaller magnetic particles in a vial for striking the swab within a confined gap. Elution of samples from the swab using this device was demonstrated using artificial sputum prepared by mixing 2% methyl cellulose in deionised water, loaded with fluorescent-tagged polystyrene beads and Escherichia coli bacteria at various concentrations. The recovery efficiency was found to increase with both rotational speed and elution time, but plateaus after 400 RPM and 120s respectively. At higher concentration of polystyrene beads, a maximum recovery of ~85% was achieved at 5x108 particles/ml sample. With lower concentration (106 particles/ml), the maximum efficiency (~93%) was found to be almost twice of the static condition (46.7%), while using only 620µL of elution volume. Similar trends were found in experiments with artificial sputum loaded with E. coli cells, and the maximum recovery was found to be ~90% at 105 CFU/ml concentration. The robust design and smaller size allows the device to be used in different clinical, forensic and laboratory settings. Also, due to cheaper means of manufacturing and assembly, the vials and smaller magnets can be discarded after every experiment, thereby preventing contamination. The device is most suitable for recovering cells from different body fluids like saliva, mucous, semen or blood, absorbed by the swab fibres. Apart from body fluids samples, swabs holding biological agents from environmental surfaces can also be eluted. A higher recovery at lower concentration facilitates the use of this device where the available analyte concentration is low. / Thesis / Master of Applied Science (MASc)

Vårdrelaterade infektioner (VRI) är ett ökande problem inom hälso- och sjukvården. På neonatalavdelningen på Länssjukhuset Ryhov i Jönköping har det förekommit inkonsekventa odlingsresultat vid misstänkt VRI orsakad av Staphylococcus aureus. Att förebygga VRI samt värna om patientsäkerheten kräver känsliga och pålitliga laboratorieanalyser. Syftet med studien var att bestämma och jämföra lägsta detektionsintervall för metoderna odling och qPCR med och utan anrikning i MAMSA-buljong, av S. aureus. Seriespädningar av S. aureus tillreddes och koncentrationer för ursprungsrören uträknades via viable count (VC). Odling på blodagar samt qPCR med och utan anrikning i MAMSA-buljong utfördes. Resultaten användes för att bestämma ett lägsta detektionsintervall. Odling gav ett lägsta detektionsintervall mellan 0,5–62 CFU/ml, och qPCR mellan 6400–140 000 CFU/ml. Anrikning med MAMSA-buljong innan qPCR-analys gav ett lägsta detektionsintervall mellan 0,6–140 CFU/ml. För detektion via odling räcker enstaka till tiotals CFU/ml i analysprovet. Vid qPCR behövs tusentals till hundratusentals CFU/ml i analysprovet, men vid qPCR med MAMSA-anrikning kan detektionsintervallet sänkas till nivåer jämförbara med odling. För att kunna applicera studiens resultat på den laborativa verksamheten krävs vidare studier med fler bakteriearter och replikat. / Hospital-acquired infections (HAI) are an increasing problem in health care facilities. In the neonatal intensive care unit at Länssjukhuset Ryhov in Jönköping, inconsistent cultivation results have been observed for suspected HAI caused by Staphylococcus aureus. To prevent HAI and to maintain patient safety, sensitive and reliable laboratory tests are essential. The aim of the study was to determine and compare the lowest range of detection for cultivation and qPCR with and without enrichment in MAMSA broth, for S. aureus. Serial dilutions of S. aureus were made, and the original concentration was determined through viable count. Cultivation on blood agar and qPCR with and without enrichment in MAMSA broth was performed. The results were used to determine a minimum detection range. For cultivation, a minimum detection range of 0,5–62 CFU/ml was determined and for qPCR a range of 6400–140 000 CFU/ml. For qPCR after enrichment in MAMSA broth, a lowest detection range of 0,6–140 CFU/ml was determined. For detection with cultivation single to tenths of CFU/ml were needed in the sample. For qPCR thousands to tenths of thousands of CFU/ml were needed. For qPCR with enrichment in MAMSA broth the detection range could be lowered to levels comparable with cultivation. For clinical application, further studies are needed with more bacterial species and replicates.

Made available in DSpace on 2015-08-19T13:52:54Z (GMT). No. of bitstreams: 2 14.pdf: 5060838 bytes, checksum: 4a21bcf622237b8e6dbec3ff3d6c6b30 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Fármacos/Farmanguinhos. Rio de Janeiro, RJ, Brasil. / O presente trabalho teve por objetivo desenvolver e validar uma nova metodologia analítica para determinação de resíduos de Efavirenz por método de swab. Essa metodologia foi desenvolvida com base na técnica utilizada atualmente em Farmanguinhos (FIOCRUZRJ),que contempla os resultados solicitados pela Agência Nacional de Vigilância Sanitária(ANVISA) mas, não possui um desempenho considerado ideal.Os estudos foram baseados principalmente na comparação do poder extrativo de dois modelos de swab selecionados, o Coventry modelo 36060, e o Texwipe®, Alpha® Swab,modelo TX761. Cada um desses modelos foi testado com os seguintes solventes ou soluções extratoras: Acetonitrila, Acetonitrila / Água purificada (1:1), Metanol / Água purificada (1:1),Etanol, Etanol / Água purificada (1:1) e Isopropanol. Também foi objeto de estudo, o tempo de ultrassom utilizado para extrair o resíduo de Efavirenz dos swabs.Os resultados apontaram para o uso do modelo de swab Texwipe®, Alpha® Swab,modelo TX761, como sendo o de melhor desempenho para o presente resíduo. Quanto ao solvente extrator, o indicado foi o etanol, devido ao seu bom desempenho com relação à solubilização do Efavirenz e seu baixo caráter tóxico. O tempo de ultrassom selecionado foide 15 minutos.Todo o desenvolvimento elaborado no presente estudo também possibilitou uma abordagem que poderá ser utilizada em futuros desenvolvimentos de metodologias analíticas para determinação de resíduos de substâncias com conhecida dificuldade de solubilização. / This study aimed to develop and validate a new analytical methodology for the determination of residues of Efavirenz by swab method. This methodology was developed based on the technique currently used in Farmanguinhos (FIOCRUZ-RJ), which includes the results requested by the National Health Surveillance Agency (ANVISA) but does not have a performance considered ideal. The studies were based mainly on the comparison of the two models extractive swab selected, the Coventry ™ Model 36060, and Texwipe ® Alpha ® Swab, Model TX761. Each of these was tested with the following solvents or extraction solutions: acetonitrile, acetonitrile / purified water (1:1), methanol / purified water (1:1), ethanol, ethanol / purified water (1:1) and isopropanol. It was also the object of study, the duration of ultrasound used to extract the residue of Efavirenz swabs. The results point to the use of the model swab Texwipe ® Alpha ® Swab, Model TX761, as the best performance for this residue. As to the solvent extractor, the ethanol was indicated due to its good performance with respect to its solubilization of Efavirenz and low toxic character. The time ultrasound selected was 15 minutes. All development elaborated in this study also enabled an approach that could be used in future developments of analytical methods for determination of residues of substances with known difficulty of solubilization.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-11-13T10:36:11Z No. of bitstreams: 2 Dissertação - Thairiny Neres Silva - 2017.pdf: 2022674 bytes, checksum: a9ff46169fe46df4f2909e2719e18791 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-11-13T10:36:47Z (GMT) No. of bitstreams: 2 Dissertação - Thairiny Neres Silva - 2017.pdf: 2022674 bytes, checksum: a9ff46169fe46df4f2909e2719e18791 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-11-13T10:36:47Z (GMT). No. of bitstreams: 2 Dissertação - Thairiny Neres Silva - 2017.pdf: 2022674 bytes, checksum: a9ff46169fe46df4f2909e2719e18791 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-10-03 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Sapoviruses are important agents causing acute gastroenteritis worldwide, and are most often detected in children under five years of age. The present study aimed to evaluate the positivity and viral load index of sapovirus in faecal samples and nasopharyngeal swab of children, in association with the symptoms presented by these children. The study included 102 children hospitalized at the Hospital Materno Infantil, presenting symptoms of gastroenteritis, attended between May 2014 and May 2015, with a sample of faeces and a nasopharyngeal swab sample from each of them. Samples of faeces and nasopharyngeal swabs from all children were submitted to extraction of the genetic material from a commercial kit and screened for sapovirus by RT-qPCR. The viral load was determined by constructing a standard curve from recombinant plasmid. A faecal sample and a nasopharyngeal swab sample were obtained and 47% of the children were positive for sapovirus in at least one of the samples collected, being 10.7% positive only in faecal samples and 28.4% positive only in the samples of nasopharyngeal swab. Regarding viral load, a median of 7.77 x 108 CG / mL was found for stool samples. Regarding the viral load of the nasopharyngeal swab samples, a median 1.54 x 108 CG / mL was observed and a statistically significant result was observed (13/14 p = 0.01), for the samples with viral load above the median obtained in the rainy season. The median viral load of samples from positive children in both clinical specimens was 2.33 x 108 CG / mL in the faeces and 2.67 x 108 CG / mL in the nasopharyngeal swab. Two of these children presented a considerably higher viral load when compared to the others. It is hoped that the data obtained may contribute to a better understanding of the molecular epidemiology of sapoviruses, as well as to the development of better preventive measures, in order to limit the risk of transmission of sapoviruses, especially in a hospital environment. This is the first study to carry out the research and detection of sapovirus in a respiratory tract sample, which could suggest a possible alternative route of viral transmission. / Sapovírus são importantes agentes causadores de gastroenterite aguda no mundo todo, sendo mais frequentemente detectados em crianças menores de cinco anos. O presente estudo teve como objetivo, avaliar o índice de positividade e carga viral de sapovírus em amostras de fezes e swab nasofaringeano de crianças, em associação com os sintomas apresentados por essas crianças. Participaram do estudo 102 crianças hospitalizadas no Hospital Materno Infantil, apresentando sintomas de gastroenteríte, atendidas entre o período de maio de 2014 a maio de 2015, sendo obtidas uma amostra de fezes e uma amostra de swab nasofaringeano de cada uma delas. As amostras de fezes e de swab nasofaringeano de todas as crianças, foram submetidas a extração do material genético a partir de kit comerciale triadas para sapovírus por RT-qPCR. A carga viral foi determinada através da construção de uma curva padrão, a partir de plasmídeo recombinante. Foram obtidas uma amostra de fezes e uma de amostra de swab nasofaringeano sendo 47% das crianças foram positivas para sapovírus em pelo menos uma das amostras coletadas, sendo 10,7% positivas apenas nas amostras de fezes e 28,4% positivas apenas nas amostras de swab nasofaringeano. Em relação a carga viral constatou-se uma mediana 7,77 x 10 8 CG/mL para as amostras de fezes. Em relação a carga viral das amostras de swab nasofaringeano, foi observada uma mediana 1,54 x 10 8 CG/mL e um resultado estatisticamente significativo foi observado (13/14 p= 0,01), para as amostras com carga viral acima da mediana obtidas no período chuvoso. A mediana da carga viral das amostras proveniente das crianças com positividade em ambos espécimes clínicos foi de 2,33 x 10 8 CG/mL nas fezes e de 2,67 x 10 8 CG/mL nos swab nasofaringeano. Duas dessas crianças apresentaram carga viral consideravelmente mais elevada, quando comparada com as demais. Espera-se que os dados obtidos possam contribuir para um melhor entendimento da epidemiologia molecular dos sapovírus, bem como para que melhores medidas preventivas sejam desenvolvidas, a fim de limitar os riscos de transmissão dos sapovírus, especialmente em ambiente hospitalar. Este é o primeiro estudo a realizar a pesquisa e detecção de sapovírus em amostra do trato respiratório, o que poderia sugerir uma possível rota alternativa de transmissão viral.

Submitted by Lara Oliveira (lara@ufersa.edu.br) on 2017-05-19T20:57:26Z No. of bitstreams: 1 Ismênia_DISSERT.pdf: 2286785 bytes, checksum: fe512f9cb09ab8ccaeca35cce30794cc (MD5) / Made available in DSpace on 2017-05-19T20:57:26Z (GMT). No. of bitstreams: 1 Ismênia_DISSERT.pdf: 2286785 bytes, checksum: fe512f9cb09ab8ccaeca35cce30794cc (MD5) Previous issue date: 2017-02-17 / Leprosy is a chronic infectious disease caused by Mycobacterium leprae and persists as a serious public health problem in Brazil. The fact that this microorganism is inculcable makes it difficult to diagnose and elucidate details of its transmissive chain. Aiming to clarify the role of house dust in the disease transmission route, this case-control study carried out in the Barrocas, Mossoró, RN, Brazil, the largest cluster in the state, compared clinical, epidemiological, slit skin smear and serological results of 22 newly diagnosed patients of the cluster, with molecular results of detection of the specific RLEP and 16S rRNA genomic regions of this bacillus in the nasal swab samples, saliva and house dust of these individuals and of the controls (44 household contacts and 44 endemic contacts) between november 2015 to november 2016. There was greater detection in women and school-age youth, with degree of disability, with predominance of paucibacillary forms. There was no statistical difference between the number of BCG vaccine scars from patients and endemic contacts, and 31.8% of the patients had no vaccine scar. The two rapid serological tests evaluated, ML-flow and OrangeLife® presented similar results, with a higher positivity among paucibacillary patients through the latter (54.5%). Both were superior to slit skin smear (9.1%). Regarding molecular research, the positivity in nasal swab and saliva of multibacillary patients with primer RLEP was 16.7% and 33.3%, respectively. There was no detection of bacterial DNA in domestic dust or between paucibacillary. Regarding the DNA analysis in saliva of endemic multibacillary contacts, there was 27.2% positivity. Associating molecular and serological results, it was possible to identify 4.5% of subclinicity between home contacts and 15% of asymptomatic carrier status. Regarding the risk variables, residing in dwellings with up to two windows offered 3.79 times more chance of progressing to this outcome, having a history of cases of leprosy in the family increased 2.89 times the risk of presenting seropositivity by the OrangeLife® serological test In this community and being over 60 years of age gave 3.6 times more chances of having positive serological reaction for leprosy with the test evaluated, which denotes prolonged exposure to the bacillus. Although not statistically significant, it was noted that the pattern of low schooling, low income, environmental insalubrity, population agglomeration and inadequate living habits made up a sociodemographic and epidemiological profile that attests to the context of social vulnerability of the affected population, requiring integrated actions of rehabilitation that go beyond the physical aspect of their dwellings, since they need to contemplate the human and social development of these people in the broad sense / A hanseníase é uma doença infecciosa crônica causada pelo Mycobacterium leprae e persiste como grave problema de saúde pública no Brasil. O fato deste micro-organismo ser incultivável dificulta o diagnóstico e elucidação de detalhes da sua cadeia transmissiva. Objetivando analisar a dinâmica de transmissão ambiental da hanseníase, este estudo caso-controle realizado no Bairro Barrocas, Mossoró/RN, maior cluster do estado, confrontou resultados de avaliação clínica, epidemiológica, baciloscópica e sorológica de 22 doentes recém-diagnosticados nas Barrocas, com resultados moleculares de detecção das regiões genômicas específicas RLEP e 16S rRNA deste bacilo nas amostras de swab nasal, saliva e poeira domiciliar destes indivíduos e dos seus controles (44 contactantes domiciliares e 44 contactantes endêmicos), entre novembro de 2015 a novembro de 2016. Os indivíduos foram diagnosticados através de busca ativa nos domicílios, nas escolas e na sede da unidade básica de saúde local. Houve maior detecção da doença em mulheres, nos menores de 15 anos, com grau 0 de incapacidade e predominância de formas paucibacilares. Não houve diferença estatística entre o número de cicatrizes vacinais de BCG de doentes e de contatos endêmicos, e 31,8% dos hansenianos não apresentaram cicatriz vacinal. Os dois testes rápidos sorológicos avaliados, ML-flow (IgM ND-O-BSA) e OrangeLife® (IgM e IgG anti NDO-LID 1) apresentaram resultados semelhantes, com maior positividade entre os paucibacilares através deste último (54,5%). Ambos foram superiores à baciloscopia (9,1%). Quanto à pesquisa molecular, a positividade em swab nasal e saliva de doentes multibacilares com primer RLEP foi de 16,7% e 33,3%, respectivamente. Não houve detecção de DNA bacteriano na poeira doméstica nem entre os paucibacilares. Já em relação à pesquisa de DNA em saliva de contatos endêmicos de multibacilares, houve 27,2% de positividade. Associando-se resultados moleculares e sorológicos, foi possível identificar 4,5% de subclinicidade e 15% de status portador assintomático entre contatos domiciliares. Em relação às variáveis de risco de soropositivar com o teste OrangeLife®, este estudo revelou que residir em moradias com até duas janelas ofereceu 3,79 vezes mais chance de evoluir para este desfecho, ter histórico de casos de hanseníase na família aumentou 2,89 vezes o risco e ter mais de 60 anos de idade conferiu 3,6 vezes mais chances, o que denota exposição prolongada destes indivíduos ao bacilo. Embora sem significância estatística, fez-se notório que o padrão de baixa escolaridade, baixa renda, insalubridade ambiental, aglomeração populacional e hábitos de vida inadequados compuseram um perfil sociodemográfico e epidemiológico que atestou o contexto de vulnerabilidade social da população afetada, exigindo ações integradas de reabilitação que vão além do aspecto físico de suas moradias, pois necessitam contemplar o desenvolvimento humano e social dessas pessoas no seu sentido amplo / 2017-05-18

Leprosy is an important health problem in Brazil, with a high detection rate despite the application of the multidrug therapy. The nasal mucosa is considered the preferential site of entry and exit of the Mycobacterium leprae, although some lesions have been found in the buccal mucosa. However, the buccal mucosa involvement in bacilli transmission has never been investigated. We have shown the presence of the M. leprae DNA in buccal swabs of leprosy patients (334) and household contacts (1288) through conventional polymerase chain reaction (PCR), and results were correlated with clinical and other laboratorial evaluations. The overall positivity for patients was 18.26%, divided into 12.03% and 21.23% for paucibacillary and multibacillary forms, respectively. Among contacts, the positivity reached 6.83%, which were considered either as healthy carriers or sub-clinically infected, when the ELISA test presented a positive anti-PGL-1 result. This study showed important evidences that the buccal mucosa may be a secondary site of M. leprae transmission and infection. Furthermore, contacts with positive PCR may be actively involved in the transmission. Our findings have great epidemiological relevance, especially for the leprosy control programs and for the dentistry clinics, and must be considered in the new strategies of control and prevention. / A hanseníase é um importante problema de saúde pública no Brasil, com elevada taxa de detecção, apesar da aplicação da poliquimioterapia. A mucosa nasal é considerada o local preferencial de entrada e saída do Mycobacterium leprae, embora algumas lesões tenham sido encontradas na mucosa bucal. No entanto, o envolvimento da mucosa oral na transmissão do bacilo nunca foi investigado. Nós mostramos a presença do DNA do M. leprae em swab bucal de pacientes com hanseníase (334) e contatos domiciliares (1288) por meio da reação em cadeia da polimerase (PCR) convencional, e os resultados foram correlacionados com outras avaliações clínica e laboratorial. A positividade geral de pacientes foi de 18,26%, dividida em 12,03% e 21,23% para as formas paucibacilares e multibacilares, respectivamente. Entre os contatos, a positividade alcançou 6,83%, que foram considerados como portadores sadios ou infectados subclínicos, quando o teste ELISA anti-PGL-1 apresentou resultado positivo. Este estudo mostrou evidências importantes de que a mucosa bucal pode ser um sítio secundário de infecção e transmissão do M. leprae. Além disso, contatos com PCR positivo podem estar envolvidos ativamente na transmissão. Nossos resultados têm grande relevância epidemiológica, especialmente para os programas de controle da hanseníase e para as clínicas de odontologia, e devem ser considerados em novas estratégias de controle e prevenção. / Mestre em Ciências da Saúde

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-25T13:37:26Z No. of bitstreams: 2 Dissertação - Nathânia Dábilla Alves Silva - 2016.pdf: 1926312 bytes, checksum: 08f15d1c8a44d1ed6118f432a6917f2d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-25T13:38:53Z (GMT) No. of bitstreams: 2 Dissertação - Nathânia Dábilla Alves Silva - 2016.pdf: 1926312 bytes, checksum: 08f15d1c8a44d1ed6118f432a6917f2d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-25T13:38:53Z (GMT). No. of bitstreams: 2 Dissertação - Nathânia Dábilla Alves Silva - 2016.pdf: 1926312 bytes, checksum: 08f15d1c8a44d1ed6118f432a6917f2d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-13 / The norovirus (NoVs) are important viral causative agents of acute gastroenteritis (AGE), affecting individuals of all ages in distinct parts of the word; however, the highest morbi-mortality rates occur mainly in children under five years of age and the elderly. The aim of this study was to to screening NoV by Polymerase Chain Reaction Post Reverse Transcription (RT-qPCR) and real-time (RT-qPCR) in fecal and nasal swab of children up to six years of age, with and without AGE symptoms. Samples were obtained at the Materno Infantil Hospital, from May/2014 to May/2015. Secretor status of children was also determined by enzyme immunoassay and genotyping (FUT2 gene) from the sediment of nasal swab epithelial cells. A global positivity index of 17% (37/219) for NoV in feces, and from these positive children, 48.6% (18/37) had AGE symptoms. Mean viral load in fecal samples was 2.59 x 1010 CG/g from symptomatic and 1.37 x 109 CG/g in asymptomatic. A higher positivity rate (70%) was observed GII NoV, compared to GI NoV (30%) and a higher positivity in children up to 24 month old (67.5%), although not statistically significant. As for the secretor status of children positive for NoV in fecal samples, 94.6% positive secretory status. The NoV were detected in practically every month of the study, and no particular pattern of circulation in relation to dry or rainy seasons was observed. Most children positive for NoV (70%) had the record they have received at least the first dose of the vaccine against Rotavirus, being the highest viral load detected among vaccinated children. The NoV RNA was detected in 8.7% of nasal swab samples of the children and of these, 58% had AGE symptoms. The mean viral load in swab samples from symptomatic children was 2.10 x 108 and in the asymptomatic children was 2.41 x 107 CG/mL. A high NoV genotype variability was found in the study (GI.2, GI.3, GI.5, GII.3, GII.4 and GII.6), with a predominance of GII.4 (28.6%), with this being the first report of NoV GI.5 in Brazil. The data obtained in this study reveal a high frequency, viral load, and genetic variability of NoVs among children attended in a hospital of Goiânia, Goiás. The results are important for a better understanding of NoV epidemiology in nosocomial environment, and may constitute useful information on the advent of the development of an effective vaccine. The viral load in nasal swab samples is a novel data that may contribute for the elucidation of a possible alternative rout of NoV transmission. / Os norovírus (NoVs) são importantes agentes virais causadores de gastroenterite aguda (GEA), atingindo indivíduos de todas as idades em diversas partes do mundo, porém os maiores índices de morbimortalidade ocorrem principalmente nas crianças menores de cinco anos e idosos. O objetivo do presente estudo foi realizar a pesquisa de NoV, através da Reação em Cadeia pela Polimerase Pós-Transcrição Reversa (RT-PCR) e por tempo real (RT-qPCR) em amostras fecais e swab nasal de crianças com até seis anos de idade, com e sem sintomas de GEA. As amostras foram coletadas no Hospital Materno Infantil, entre maio/2014 e maio/2015. Procedeu-se ainda à determinação do status secretor das crianças, por Ensaio Imunoenzimático e genotipagem (gene FUT2), em sedimento de células epiteliais do swab nasal. Foi observado um índice de positividade global de 17% (37/219) para NoV nas fezes, sendo que destas crianças positivas, 48,6% (18/37) apresentavam sintomas de GEA. A carga viral média nas amostras de fezes de crianças com sintomas foi 2,59 x 1010 CG/g e 1,37 x 109 CG/g nas assintomáticas. Observou-se maior positividade para NoV GII (70%) quando comparado ao GI (30%) e maior positividade nas crianças de até 24 meses de idade (67,5%), entretanto este dado não foi estatisticamente significativo. Quanto ao status secretor das crianças positivas para NoV nas fezes, 94,6% status secretor positivo. Os NoV foram detectados em praticamente todos meses do estudo, não sendo observado padrão de circulação definido em relação às estações seca e chuvosa. A maioria das crianças positivas para NoV (70%) tinham o registro de terem recebido pelo menos a primeira dose da vacina contra Rotavírus, sendo a carga viral mais elevada detectada entre crianças vacinadas. O RNA de NoV foi ainda detectado em 8,7% (19/219) das amostras de swab nasal das crianças e destas, 58% apresentavam sintomas de GEA. A carga viral média nas amostras de swab das crianças sintomáticas foi 2,10 x 108 CG/mL e nas assintomáticas 2,41 x 107 CG/mL. Foi observada elevada variabilidade de genótipos de NoV no estudo (GI.2, GI.3, GI.5, GII.3, GII.4 e GII.6), com maior predominância de GII.4 (28,6%), sendo este o primeiro relato de NoV GI.5 no Brasil. Os dados obtidos neste estudo revelam elevada frequência, carga viral e variabilidade genética de NoVs entre crianças atendidas em um hospital de Goiânia, Goiás. Os resultados são importantes para o melhor entendimento da epidemiologia dos NoVs em ambiente nosocomial, e poderão ser uteis como informação no advento do desenvolvimento de uma vacina eficaz. A determinação da carga viral de NoV em xii amostras de swab nasal é um dado novo, podendo este contribuir para a elucidação de uma possível rota alternativa de transmissão dos NoV.

Panther Fusion® system är ett fullständigt automatiserat in vitro diagnostiksystem som har högt flöde av prov-till-resultat och tillåter utförandet av in vitro diagnostiska tester genom användning av realtids Polymerase Chain Reaction (realtids-PCR). Syftet med detta projekt är att validera om det finns möjlighet att använda ett kit från Amplidiag på Panther fusion open access för att diagnostisera vankomycin resistenta enterokocker (VRE) samt att utvärdera analys direkt på E-swab prover istället för anrikningsbuljong. Det är viktigt att kunna detektera vanA-och vanB-generna i prover för att VRE hyser dessa gener. Sensitivitet och effektivitet av Panther fusion open access undersöktes genom att analysera proverna parallellt med Bio-Rad CFX384/96-PCR som är rutinmetoden för VRE-diagnostik på Klinisk mikrobiologi i Lund. Specificitet av en tidigare publicerad PCR-mix (PCR-mix) som kan detektera vanA och vanB i prover jämfördes med Amplidiag assay mix 2 som används i rutinmetoden. För att kunna utvärdera skillnaden av specificiteten och känsligheten mellan E-swab och anrikningsbuljong utfördes analysen på både E-swab och anrikningsbulong med både Panther fusion open access och Bio-Rad CFX384/96-PCR. MgCl2- koncentration optimerades till 3,0 mM. Sensitivitet och effektivitet av Panther fusion open access var högre med PCR-mix än Amplidiag assay mix 2 och uppvisade optimalt linjäritet. Specificitetstest av PCR-mixen visade att den bara kunde detektera vanA och vanB. Resultaten av spikade VRE-negativa patientprover (E-swab) visade sig vara orimliga då flera av proverna och kontrollerna gav resultat för båda generna. För att ersätta VRE-diagnostik på Bio-Rad CFX384/96 med Panther fusion open access behöver ytterligare experiment genomföras direkt på E-swab och vankomycine variabla enterokocker (VVE)-prover på Panther fusion open access. / Panther Fusion® system is a fully automated in vitro diagnostic system that has a high flow of sample-to-results and allows the performance of in vitro diagnostics tests using real-time Polymerase Chain Reaction (real-time PCR). The purpose of this project is to validate whether it is possible to use a kit from Amplidiag on Panther fusion open access to diagnose vancomycin resistant enterococcus (VRE). In addition, this project aims to evaluate and analyze E-swab samples directly instead of enrichment broth. It is important to be able to detect VanA and VanB genes in samples because VRE harbors these genes. Sensitivity and efficiency of Panther fusion open access were tested by analyzing the samples simultaneously with Bio-Rad CFX384/96 which is the routine method for VRE diagnostic at Clinical Microbiology in Lund. Specificity of a previously published PCR-mix (PCR-mix) that can detect VanA and VanB genes in samples was compared with Amplidiag assay mix 2 used in the routine method. In order to evaluate the difference in specificity and sensitivity between E-swab and enrichment broth, the analysis was performed on both E-swab and enrichment broth with both Panther fusion open access and Bio-Rad CFX384/96-PCR. MgCl2 concentration was optimized to 3,0 mM. Both sensitivity and efficiency of Panther fusion open access were higher with PCR-mix than those with Amplidiag assay mix 2 and it showed optimal linearity. Specificity test of PCR-mix detected only VanA and VanB genes. The results of inoculated the negative VRE-patient samples (E-swab) indicated that they were unreasonable because some of the samples and controls gave results of both genes. Replacement of the VRE-diagnostic method on Bio-Rad CFX384/96 with Panther fusion open access requires performing additional experiments directly on E-swab and vancomycin variable enterococcus (VVE) samples using Panther fusion open access.

A leishmaniose visceral canina é uma zoonose, no Brasil causado pelo protozoário Leishmania chagasi (syn. L. infantum). É endêmica em 88 países, os quais compreendem regiões tropicais e subtropicais do Velho e do Novo Mundo, com incidência estimada em 2 milhões de casos por ano. No ambiente urbano, o cão doméstico é considerado o principal reservatório do parasito. A transmissão da doença ocorre através da picada do vetor, díptero flebotomíneo, da espécie Lutzomyia longipalpis. O diagnóstico pode ser feito através de métodos diretos, como esfregaço preparado com os diferentes órgãos linfoides, Reação em Cadeia da Polimerase (PCR), cultivo in vitro do parasito, ou por métodos sorológicos, como ELISA (Ensaio Imunoenzimático) e RIFI (Reação de Imunofluorescência Indireta). O controle epidemiológico da doença humana envolve o tratamento sistemático dos casos humanos, borrifação de inseticida em região domiciliar e peridomiciliar e eliminação de cães soropositivos, ponto mais controverso do programa de controle. O objetivo do presente trabalho foi avaliar a técnica não invasiva do suabe conjuntival na identificação por PCR de leishmaniose canina. As amostras, de sangue e suabe conjuntival, foram coletadas durante inquérito epidemiológico realizado na cidade de Ilha Solteira - SP. A prevalência de animais positivos em pelo menos um dos três testes (RIFI, PCR de sangue, e PCR de suabe conjuntival) foi de 28,17% (60/213). No teste sorológico RIFI, um total de 13,6% (29/213) dos cães reagiram positivamente. Na PCR do suabe conjuntival, 13,1% (28/213) dos cães foram positivos, e na PCR de sangue total também obtivemos 13,1% (28/213) positivos. Comparando os animais testados pela RIFI e pela PCR de suabe conjuntival, 7,9% (17/213) animais foram positivos em ambos os testes, enquanto 81,2% (173/213) animais foram negativos em ambos os testes (PCR-SC e RIFI). Dos animais testados para PCR-SC, 5,1% (11/213) foram positivos apenas neste teste. Na sorologia, 5,6% (12/213) reagiram positivamente apenas pela RIFI. A sensibilidade e especificidade da PCR-SC foram respectivamente 58,6% e 94,0%, valor preditivo positivo de 61,0%, valor preditivo negativo de 94,0%, e o índice kappa 0,53, o qual demonstra moderada concordância entre os testes. Ao se comparar a RIFI com a PCR-SG, dos animais testados, 3,3% (7/213) foram positivos nos dois testes simultaneamente, 13,6% (29/213) foram positivos apenas na RIFI, logo, 9,9% (21/213) foram positivos apenas na PCR-SG. Foram negativos nos dois testes 76,5% (163/231). A PCR-SG demonstrou sensibilidade de 24,1%, especificidade de 88,5%, valor preditivo positivo de 25,0% e valor preditivo negativo de 88,0% e índice kappa de 0,13, quando comparado à RIFI, indicando uma fraca concordância. Além disso, foram positivos em todos os três testes 2,35% (5/213), e apenas 0,47% (1/213) foi positivo nos testes de PCR-SG e PCR-SC. Os resultados demonstraram que o suabe conjuntival é uma boa alternativa, fácil e pouco invasiva, que pode ser utilizada para auxiliar inquéritos e estudos epidemiológicos da Leishmaniose Visceral Canina. / Canine visceral leishmaniasis is a zoonosis in Brazil caused by the protozoan Leishmania chagasi (syn. L. infantum). It is endemic in 88 countries, which include tropical and subtropical regions of the Old and New World, with an estimated incidence of 2 million cases per year. In the urban environment, the domestic dog is the main reservoir of the parasite. Disease transmission occurs through the bite of the vector, sandfly, Lutzomyia longipalpis species. The diagnosis by direct methods can be made, such as smear prepared with different lymphoid organs, Polymerase Chain Reaction (PCR) in vitro culture of the parasite, or by serological methods such as ELISA (enzyme immunoassay) and IFAT (Indirect Immunofluorescence Test). The epidemiological control of human disease involves the systematic treatment of human cases, insecticide spraying in domiciliary area and elimination of seropositive dogs, most controversial point of the control program. The objective of this study was evaluate the noninvasive technique of conjunctival swab for canine leishmaniasis PCR identification. Were collected Samples of blood and conjunctival swab during an epidemiological survey conducted in the city of Ilha Solteira SP. The prevalence of positive animals in at least one of the three test (IFAT, blood PCR, and swab PCR) was 28.17% (60/213). In IFAT serological test, 13.6% (29/213) of the dogs responded positively. In conjunctival swabs PCR, 13.1% (28/213) dogs were positive, and PCR of whole blood obtained 13.1% (28/213) positive. Comparing the animals tested by IFAT and conjunctival swab PCR, 7.9% (17/213) were positive in both tests, while 81.2% (173/213) animals were negative in both tests. Of the tested animals for swab PCR, 5.1% (11/213) were positive only in this test. In serology, 5.6% (12/213) reacted positively only by IFAT. The sensitivity and specificity of swab PCR were respectively 58.6% and 94.0%, positive predictive value 61.0%, negative predictive value of 94.0%, and kappa 0.53, which demonstrates moderate agreement between tests. Comparing the IFAT with blood PCR, the animals tested, 3.3% (7/213) were positive in both tests simultaneously, 13.6% (29/213) were positive only by IFAT, so, 9, 86% (21/213) were positive only by blood PCR. Were negative in both tests 76.5% (163/213). The blood PCR demonstrated a sensitivity of 24.1%, specificity 88.5%, positive predictive value 25% and negative predictive value of 88% and coefficient of agreement (kappa) of 0.13, compared to IFAT, indicating poor agreement. Furthermore, were positive in all three tests 2.35% (5/213), and only 0.47% (1/213) were positive in the PCR-SC and PCR-SG test. The results showed that conjunctival swab is a good alternative, easier and less invasive that can be used to aid investigations and epidemiological studies of Canine Visceral Leishmaniasis.

Cães, gatos e equinos domésticos podem ser importantes reservatórios de agentes infecciosos potencialmente zoonóticos, podendo-se destacar entre eles as leishmanioses. Por esse motivo, torna-se necessário aprimorar o conhecimento sobre essas enfermidades, estudando mais detalhadamente sua ecoepidemiologia, importante fonte de informações para a tomada de decisões com relação ao controle das mesmas. O presente estudo objetivou verificar a ocorrência de Leishmania spp. em cães, gatos e equinos procedentes de regiões endêmicas e não endêmicas do estado de São Paulo, por meio dos testes diagnósticos Imunofluorescência Indireta (RIFI) e Reação em cadeia pela Polimerase (PCR) e objetivou também comprovar a eficácia da PCR realizada com DNA extraído de amostras de suabe da conjuntiva ocular de gatos e equinos. Foram encontrados cães infectados por Leishmania spp. em Itapecerica da Serra, São Lourenço da Serra e São Paulo, com freqüências de 3,12% (1/32) (PCR de suabe), 10% (1/10) (PCR de suabe) e 1,72% (02/116) (PCR de suabe) / 6,89% (08/116) (PCR de sangue), respectivamente. As amostras analisadas dos gatos provenientes dos municípios de Pirassununga e São Lourenço da Serra, foram positivas para Leishmania spp., com frequências de 28,57% (02/07) (PCR de suabe) / 28,57% (02/07) (RIFI) e 33,33% (1/3) (RIFI) respectivamente. Equinos infectados por Leishmania spp. foram verificados nas cidades de Bragança Paulista e Ilha Solteira, com frequências de 100% (14/14) (PCR de sangue) e 90% (36/40) (PCR de suabe)/ 100% (40/40) (PCR de sangue)/ RIFI 2,5% (01/40) respectivamente. Os resultados demonstram a presença de Leishmania spp infectando cães, gatos e equinos no Estado de São Paulo e que o suabe de conjuntiva ocular foi capaz de detectar gatos e equinos infectados. A coleta de amostras da conjuntiva ocular mostrou-se de fácil execução em felinos, mas nem tanto em equinos, quando comparado à coleta de sangue. / Domestic dogs, cats and horses can be important reservoirs of potential zoonotic agents, as leishmaniasis diseases. Therefore it is necessary improving knowledge on these diseases by studying the ecoepidemiology of leishmaniasis, which is an important source to make decisions on the disease control. The present study aimed to verify the occurrence of Leishmania spp. in dogs, cats and horses from endemic and non-endemic regions from São Paulo State using diagnostics tests as Indirect Immunofluorescence Test (RIFI) and Polymerase Chain Reaction (PCR), and to study the efficacy of the PCR protocol on DNA extracted from ocular conjunctival swab samples from cats, dogs and horses. Leishmania spp. was found in dogs from Itapecerica da Serra, São Lourenço da Serra and São Paulo with frequencies of 3,12% (1/32) (swab PCR), 10%(1/10) (swab PCR) and 1,72% (02/116) (swab PC)/ 6,89% (08/116) (blood PCR) respectively. The cats samples analyzed from Pirassununga and São Lourenço da Serra were positive for Leishmania spp. showing frequencies of 28,57%(02/07) (swab PCR)/ 28,57%(02/07) (RIFI) and 33,33%(1/3) (RIFI) respectively. Leishmania spp. infected horses were detected in Bragança Paulista and Ilha Solteira cities with frequencies of 100% (14/14) (blood PCR) and 90% (36/40) (swab PCR)/ 100% (40/40) (blood PCR)/ RIFI 2,5% (01/40) respectively. Results demonstrated the presence of Leishmania spp infection in dogs, cats and horses from São Paulo and that is possible detect Leishmania spp. DNA in ocular conjunctival swab from infected cats and horses. Colect samples from ocular conjuntiva is more easily made in felines than horses, when compared to blood sample collection.