Cross-genera amplification and identification of Colpodella sp. with Cryptosporidium primers in fecal samples of zoo felids from northeast China / Amplificação cruzada de gêneros e identificação de Colpodella sp. com iniciadores de Cryptosporidium em amostras fecais de zoofilia do nordeste da China

Abstract The protozoans include many intracellular human pathogens. Accurate detection of these pathogens is necessary to treat the diseases. In clinical epidemiology, molecular identification of protozoan is considered a more reliable and rapid method for identification than microscopy. Among these protozoans, Cryptosporidium considered being one of the important water-borne zoonotic pathogens and a major cause of a diarrheal disease named cryptosporidiosis in humans, domestic animals, and wild animals. This study was aimed to identify Cryptosporidium in zoo felids (N= 56) belonging to different zoo of China, but accidentlly Colpodella was encountered in the zoo felids sample and phylogenetic data confirmed this unexpected amplification from fecal samples using two-step nested-PCR. Phylogenetic analysis revealed the fact about the specific primers used previously by many researchers and cross-genera amplification. We came to know that genetically sequenced amplicon gives more accurate identification of species. This study suggests more investigation on Colpodella which has been neglected previously but gains the attention of researchers after identified from humans and animals and has been known to correlate with neurological symptoms in patients.

Resumo Os protozoários incluem muitos patógenos humanos intracelulares. A detecção acurada desses patógenos é necessária para tratar as doenças. Na epidemiologia clínica, a identificação molecular de protozoários é considerada o método de identificação mais confiável e rápido do que a microscopia. Entre esses protozoários, o Cryptosporidium é considerado um dos importantes patógenos zoonóticos transmitidos pela água e uma das principais causas de uma doença diarreica denominada criptosporidiose em humanos, animais domésticos e selvagens. Este estudo teve como objetivo identificar Cryptosporidium em zoofelídeos (N = 56) pertencentes a diferentes zoológicos da China, mas acidentalmente Colpodella foi encontrada na amostra de zoofelídeos e os dados filogenéticos confirmaram essa amplificação inesperada de amostras fecais usando nested-PCR em duas etapas. A análise filogenética revelou o fato sobre os primers específicos usados ​​anteriormente por muitos pesquisadores e a amplificação entre gêneros. Ficamos sabendo que o amplicon sequenciado geneticamente fornece uma identificação mais acurada das espécies. Este estudo sugere mais investigação sobre Colpodella, que foi negligenciada anteriormente, mas ganha a atenção dos pesquisadores depois de identificada em humanos e animais e é conhecida por se correlacionar com sintomas neurológicos em pacientes.

Evaluación de una prueba de reacción en cadena de la polimerasa múltiplex anidada para la detección de HTLV-1

El Perú es un área endémica al virus linfotrópico T humano tipo 1 (HTLV-1) y para su confirmación diagnóstica se usa pruebas serológicas que pueden dar resultados no concluyentes. Objetivos: evaluar una prueba de PCR múltiplex anidada para diagnosticar el HTLV-1. Métodos: la validación de la PCR se realizó con primers dirigidos a las regiones Pol y LTR del HTLV-1. Se empleó el gen ß-globina como control endógeno interno y el límite de detección se evaluó con células MT2. Los parámetros de precisión diagnóstica se evaluaron frente a 95 muestras sanguíneas de Referencia. Resultados: la prueba evaluada obtuvo un límite de detección de 0,5 ng/µL de ADN sensibilidad diagnóstica=97,1%, especificidad diagnóstica y analítica=100%, vpn=97,2%, vpp, repetibilidad y reproducibilidad=100%; Kappa, Índice Youden=0,97. Conclusiones: la prueba evaluada presenta un alto rendimiento diagnóstico y debido a su bajo costo se recomienda su implementación en el algoritmo del diagnóstico de HTLV-1 en Perú.

Peru is an endemic area for human T-lymphotropic virus type 1 (HTLV-1) and for its diagnostic confirmation serological tests are used, which can give inconclusive results. Objectives: to evaluate a nested multiplex PCR test to diagnose HTLV-1. Methods: PCR validation was performed with primers targeting the Pol and LTR regions of HTLV-1. The ß-globin gene was used as an internal endogenous control and the detection limit was evaluated with MT2 cells. Diagnostic accuracy parameters were evaluated against 95 Reference blood samples. Results: the evaluated test obtained a detection limit of 0.5 ng/µL of DNA; diagnostic sensitivity=97.1%, diagnostic and analytical specificity=100%, vpn=97.2%, vpp, repeatability and reproducibility=100%; Kappa, Youden Index=0.97. Conclusions: the evaluated test has a high diagnostic performance and due to its low cost, its implementation in the HTLV-1 diagnosis algorithm in Peru is recommended.

Occult hepatitis B virus infection in patients with chronic liver disease of different etiology in a Brazilian referral center: comparison of two different hepatitis B virus deoxyribonucleic acid amplification protocols: a cross-sectional study

ABSTRACT BACKGROUND: Occult hepatitis B virus infection (OBI) is defined as the presence of hepatitis B virus (HBV) deoxyribonucleic acid (DNA) in the liver of individuals with undetectable hepatitis B virus surface antigen (HBsAg) in the serum. The actual prevalence of OBI and its clinical relevance are not yet fully understood. OBJECTIVE: To evaluate the prevalence of HBV DNA in liver biopsies of HBsAg-negative patients with chronic liver disease of different etiologies in a referral center in Brazil and compare two different HBV DNA amplification protocols to detect HBV. DESIGN AND SETTING: This cross-sectional observational study was conducted at the Liver Outpatient Clinic, Hospital das Clínicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil, between January 2016 and December 2019. METHODS: HBV DNA was investigated in 104 liver biopsy samples from individuals with chronic liver disease of different etiologies, in whom HBsAg was undetectable in serum by nested-polymerase chain reaction (nested-PCR), using two different protocols. RESULTS: OBI, diagnosed by detecting HBV DNA using both protocols, was detected in 6.7% of the 104 individuals investigated. Both protocols showed a good reliability. CONCLUSION: In addition to the differences in the prevalence of HBV infection in different regions, variations in the polymerase chain reaction technique used for HBV DNA amplification may be responsible for the large variations in the prevalence of OBI identified in different studies. There is a need for better standardization of the diagnostic methods used to diagnose this entity.

Cross-genera amplification and identification of Colpodella sp. with Cryptosporidium primers in fecal samples of zoo felids from northeast China / Amplificação cruzada de gêneros e identificação de Colpodella sp. com iniciadores de Cryptosporidium em amostras fecais de zoofilia do nordeste da China

The protozoans include many intracellular human pathogens. Accurate detection of these pathogens is necessary to treat the diseases. In clinical epidemiology, molecular identification of protozoan is considered a more reliable and rapid method for identification than microscopy. Among these protozoans, Cryptosporidium considered being one of the important water-borne zoonotic pathogens and a major cause of a diarrheal disease named cryptosporidiosis in humans, domestic animals, and wild animals. This study was aimed to identify Cryptosporidium in zoo felids (N= 56) belonging to different zoo of China, but accidentlly Colpodella was encountered in the zoo felids sample and phylogenetic data confirmed this unexpected amplification from fecal samples using two-step nested-PCR. Phylogenetic analysis revealed the fact about the specific primers used previously by many researchers and cross-genera amplification. We came to know that genetically sequenced amplicon gives more accurate identification of species. This study suggests more investigation on Colpodella which has been neglected previously but gains the attention of researchers after identified from humans and animals and has been known to correlate with neurological symptoms in patients.

Os protozoários incluem muitos patógenos humanos intracelulares. A detecção acurada desses patógenos é necessária para tratar as doenças. Na epidemiologia clínica, a identificação molecular de protozoários é considerada o método de identificação mais confiável e rápido do que a microscopia. Entre esses protozoários, o Cryptosporidium é considerado um dos importantes patógenos zoonóticos transmitidos pela água e uma das principais causas de uma doença diarreica denominada criptosporidiose em humanos, animais domésticos e selvagens. Este estudo teve como objetivo identificar Cryptosporidium em zoofelídeos (N = 56) pertencentes a diferentes zoológicos da China, mas acidentalmente Colpodella foi encontrada na amostra de zoofelídeos e os dados filogenéticos confirmaram essa amplificação inesperada de amostras fecais usando nested-PCR em duas etapas. A análise filogenética revelou o fato sobre os primers específicos usados anteriormente por muitos pesquisadores e a amplificação entre gêneros. Ficamos sabendo que o amplicon sequenciado geneticamente fornece uma identificação mais acurada das espécies. Este estudo sugere mais investigação sobre Colpodella, que foi negligenciada anteriormente, mas ganha a atenção dos pesquisadores depois de identificada em humanos e animais e é conhecida por se correlacionar com sintomas neurológicos em pacientes.

Cross-genera amplification and identification of Colpodella sp. with Cryptosporidium primers in fecal samples of zoo felids from northeast China

Abstract The protozoans include many intracellular human pathogens. Accurate detection of these pathogens is necessary to treat the diseases. In clinical epidemiology, molecular identification of protozoan is considered a more reliable and rapid method for identification than microscopy. Among these protozoans, Cryptosporidium considered being one of the important water-borne zoonotic pathogens and a major cause of a diarrheal disease named cryptosporidiosis in humans, domestic animals, and wild animals. This study was aimed to identify Cryptosporidium in zoo felids (N= 56) belonging to different zoo of China, but accidentlly Colpodella was encountered in the zoo felids sample and phylogenetic data confirmed this unexpected amplification from fecal samples using two-step nested-PCR. Phylogenetic analysis revealed the fact about the specific primers used previously by many researchers and cross-genera amplification. We came to know that genetically sequenced amplicon gives more accurate identification of species. This study suggests more investigation on Colpodella which has been neglected previously but gains the attention of researchers after identified from humans and animals and has been known to correlate with neurological symptoms in patients.

Resumo Os protozoários incluem muitos patógenos humanos intracelulares. A detecção acurada desses patógenos é necessária para tratar as doenças. Na epidemiologia clínica, a identificação molecular de protozoários é considerada o método de identificação mais confiável e rápido do que a microscopia. Entre esses protozoários, o Cryptosporidium é considerado um dos importantes patógenos zoonóticos transmitidos pela água e uma das principais causas de uma doença diarreica denominada criptosporidiose em humanos, animais domésticos e selvagens. Este estudo teve como objetivo identificar Cryptosporidium em zoofelídeos (N = 56) pertencentes a diferentes zoológicos da China, mas acidentalmente Colpodella foi encontrada na amostra de zoofelídeos e os dados filogenéticos confirmaram essa amplificação inesperada de amostras fecais usando nested-PCR em duas etapas. A análise filogenética revelou o fato sobre os primers específicos usados anteriormente por muitos pesquisadores e a amplificação entre gêneros. Ficamos sabendo que o amplicon sequenciado geneticamente fornece uma identificação mais acurada das espécies. Este estudo sugere mais investigação sobre Colpodella, que foi negligenciada anteriormente, mas ganha a atenção dos pesquisadores depois de identificada em humanos e animais e é conhecida por se correlacionar com sintomas neurológicos em pacientes.

Development and preliminary evaluation of a fluorescence RPA assay for the rapid detection of Necator americanus / 中国热带医学

@#Abstract: Objective To establish a rapid detection assay based on fluorescence recombinase polymerase amplification (RPA) targeting Necator americanus eggs, and to evaluate its efficacy, providing technical support for rapid detection of Necator americanus in fecal samples. Methods The fluorescence RPA primers and probe were designed based on the cox1 gene of Necator americanus and then screened the optimal combination to develop the assay. The genomic DNA of Necator americanus eggs was diluted to 7 concentration gradients including 100 pg/µL, 10 pg/µL, 1 pg/µL, 100 fg/µL, 10 fg/µL, 1 fg/µL, 0.1 fg/µL, to determine the detection limit of the assay. The specificity of the assay was demonstrated by detected genomic DNA from Schistosoma japonicum, Ascaris lumbricoides, Clonorchis sinensis and Fasciola hepatica. A total of 44 fecal samples were collected and DNA extraction was performed, and the modified Kato-Katz method, semi-nest PCR method, and fluorescent RPA method were simultaneously used for detection to evaluate the sensitivity and specificity. Results The established fluorescence RPA assay can specifically amplify a fragment of 194 bp of the Necator americanus cox1 gene within 20 min, with a detection limit of 10 fg/µL. There was no cross-reactivity with Schistosoma japonicum, Ascaris lumbricoides, Clonorchis sinensis, Fasciola hepatica after specificity validation. In 44 fecal samples, 27 positive samples were detected by the fluorescence RPA assay, and 26 positive samples were detected by both the Kato-Katz and the semi-nested PCR. The fluorescence curve of sample number 1 was slightly higher than the negative control in the later stage of the reaction, but did not show a similar trend to the positive control, and was therefore judged to be a suspected negative sample. Compared with the Kato-Katz method and the semi-nest PCR method, The sensitivity of the fluorescent RPA method were 100.00% and the specificity were 94.44%, and the consistency of the detection results was good (Kappa=0.953>0.75). Conclusions The assay based on the fluorescence RPA is an efficient, sensitive and specific technique for detecting Necator americanus and it can be applied for surveillance and early warning of hookworm infection.

A nested-PCR for detection and identification of Brucella vaccine A19 strain / 中华地方病学杂志

Objective:A nested-PCR assay is developed to detect and identify the genomic DNA of Brucella vaccine A19 strain. Methods:The whole genomic sequences of Brucella vaccine A19 strain and other Brucella spp. strains were compared and analyzed. The primers were designed by nucleotide difference sites. The nested-PCR assay was established to detect and identify Brucella vaccine A19 strain. The genomic DNA of Brucella vaccine A19 strain was extracted and diluted. The diluted template DNA was tested for sensitivity of using nested-PCR assay. And the specificity of nested-PCR assay was tested for the genomic DNA of other Brucella spp. strains and non- Brucella spp. strains. Results:The minimum detection limit of the nested-PCR assay was 3.43 fg. The nested-PCR assay established for amplification of Brucella vaccine A19 strain showed 246 bp electrophoresis bands, while other Brucella spp. strains showed 314 bp electrophoresis bands, and non- Brucella spp. strains did not produce electrophoresis bands. Conclusions:The nested-PCR assay established has the characteristics of high sensitivity and specificity. It can be detected when there is one copy of Brucella vaccine A19 strain genomic DNA in the reaction system. This method is particularly suitable for the detection and identification of trace genomic DNA of Brucella vaccine A19 strain in sample.

HBV serologic and virus characteristics of HBsAg negative and NAT non-discriminated samples from blood donors / 中国输血杂志

【Objective】 To investigate the HBV infection of TMA initially reactive but discriminatory test non-reactive samples(NDR) after the individual donation nucleic acid detection(ID-NAT)of TMA, and analyze its serological and molecular biological characteristics, so as to improve the safety of blood transfusion. 【Methods】 121 970 samples of blood donors in the center from January 1, 2021 to December 31, 2021 were routinely tested by serology and nucleic acid of ID NAT, and 21 HBsAg(-)/ NDR samples were random collected. After the plasma samples were concentrated by ultra-high speed centrifugation, the gene sequences of BCP/PC, pre-S/S and S region were amplified by Nested PCR. The S region sequence was also sequenced to analyze the viral genotype and amino acid variation. At the same time, the original TMA retest discriminatory test was adopted, and Roche MPX 2.0 was used for ID-NAT, and the samples was not virus-concentrated.NDR samples were supplemented with electrochemiluminescence for anti-HBc and anti-HBs quantitative detection. 【Results】 Of the 121 970 samples screened, 117(0.096%) were found to be HBsAg(-)/NDR samples, of which 21 samples underwent a confirmation test. Sixteen(76.2%) cases were positive for HBV DNA by TMA retest, 7(33.3%) positive for HBV DNA by Roche MPX 2.0 ID-NAT, 9(42.9%) confirmed by Nested PCR, and 8(38.1%) positive by any two methods. Test results of serological markers were as follows: 17(80.9%) positive anti-HBc and 8(38.1%) positive anti-HBs. Eight infected cases were confirmed to have occult hepatitis B infection(OBI). The gene sequence of S region was successfully amplified and sequenced in 3 cases, all of which belonged to C type. Two mutations occurred in specimen S-2, all of which were outside MHR. There were 13 mutations in sample S-6, 6 mutations outside MHR and 7 mutations inside MHR. 【Conclusion】 Nearly 40% of NDR samples can still be detected as HBV DNA positive after virus concentration. Anti-HBc has a high detection rate, and there may be a potential risk of HBV transmission. The current NAT detection sensitivity should be improved. The amino acid mutation of S gene sequence may be related to OBI formation.

Microscopio and PCR-based detection of microsporidia spores in human stool samples

Abstract Microsporidia are obligate intracellular fungi with a remarkable ability to infect a wide range of invertebrate and vertebrate hosts. Namely, Enterocytozoon bieneusi is the most frequently microsporidia reported worldwide, and mainly associated with chronic diarrea and wasting syndrome in AIDS patients. Microscopy and PCR-based detection techniques are effective for diagnosis and identification of species and genotypes; however, these methods should be standardized in each laboratory. In this study, we performed microscopy and nested PCR techniques with PCR product sequencing to detect E. bieneusi in human stool samples. These techniques, if applied together, might prove useful for diagnosis and future epidemiological studies of intestinal microsporidiosis in Argentina.

Resumen Los microsporidios son hongos intracelulares obligados con una notable capacidad para infectar una amplia gama de hospedadores invertebrados y vertebrados. Enterocytozoon bieneusi es el microsporidio más frecuentemente reportado en todo el mundo, principalmente tricrómicaasociado con diarrea crónica y síndrome debilitante en pacientes con sida. Las técnicas dedetección basadas en microscopía y PCR son útiles para el diagnóstico y la identificación deespecies y genotipos, pero estos métodos deben estar estandarizados en cada laboratorio.En este estudio evaluamos técnicas de microscopía y PCR anidada, con secuenciación de losproductos, para detectar E. bieneusi en muestras de heces humanas. Estas técnicas, usadas con-juntamente, podrían ser útiles para su aplicación en el diagnóstico de microsporidiosis intestinaly para realizar estudios epidemiológicos de esta afección en Argentina.

Pathological, molecular and phylogenic study of fowlpox virus in domesticated chickens of Tikrit City, Iraq / Estudo patológico, molecular e filogenético do vírus da varíola aviária em frangos de corte domesticados da cidade de Tikrit, Iraque

Fowlpox virus (FPV) is one of the viruses affecting chickens worldwide, causing pathological and economic losses in the poultry industry. Viral lesions are easily recognizable by the eye and usually appear in the featherless areas, especially the head. Moreover, the virus could lead to blindness and mortality in some cases. This study diagnosed the suspected fowlpox cases, identified and classified the causative agent. We also analyzed the differences and similarities of closely related viruses at the neighboring and regional countries. Fifty samples were collected from three locations of Tikrit city from the domesticated chickens, which showed cutaneous lesions. Virus DNA was extracted directly from tissue samples before the nested PCR technique was performed. The virion core protein (P4b) gene is partially sequenced and analyzed with routine histological sectioning. Results showed that the virus causes pock lesions of dermal hyperplasia and hyperkeratosis. Hyperplasia and congestion of the chorioallantoic membrane were also recorded. The study also showed that the DNA of FPV could be extracted directly from animal tissue without further purification. The sequence analysis showed that the FPV was confirmed in all samples clustered in clade A identical with Iranian and Egyptian isolates. In conclusion, this study approved that the virus belongs to the classical dermal type of poxviruses and the short genetic distances between viruses related to closely neighboring countries. We also concluded that the conservative P4b gene included mutation sites that make this gene practical for diagnosing the virus and phylogenetic analysis.(AU)

O vírus da varíola aviária (VVA) é um dos vírus que acometem os frangos de corte em todo o mundo, causando perdas patológicas e econômicas na indústria aviária. As lesões causadas pelo vírus são facilmente reconhecidas pela observação visual e usualmente aparecem nas áreas do corpo das aves livres de penas, especialmente na cabeça. Além disso, em alguns casos a doença pode provocar a cegueira e a mortalidade de animais acometidos. O presente trabalho foi delineado para diagnosticar casos suspeitos de varíola aviária, identificar o agente causal e classificá-lo. Adicionalmente foram analisadas diferenças e similaridades com outros vírus estreitamente relacionados em localidades vizinhas e regionais. Cinquenta amostras foram colhidas em três localidades da cidade de Tikrit de frangos de corte, domesticados, que apresentavam lesões cutâneas. O DNA do vírus foi extraído diretamente das amostras de tecidos antes que a técnica de PCR fosse realizada. As proteínas do core do vírus, gene (P4b), foram parcialmente sequenciadas de analisadas em secções da rotina histológica. Os resultados obtidos revelaram que o vírus causa lesões variólicas com hiperplasia dermal e hiperqueratose. A hiperplasia e a congestão da membrana corioalantóica também foram registradas. O estudo também revelou que o DNA do VVA pode ser extraído diretamente de tecidos animais sem a realização de uma pré-purificação. A análise sequencial revelou que o VVA foi confirmado em todas as amostras agrupando-se em uma classe A, idêntica com isolados iranianos e egípcios. A conclusão obtida foi que o presente trabalho confirmou que o vírus pertence ao tipo dérmico clássico dos poxvirus e que as curtas distâncias genéticas entre os vírus relacionados são encontrados em países vizinhos. Também foi concluído que o gene conservador P4b inclui pontos de mutação que o tornam um gene prático para diagnosticar o vírus em análises filogenéticas.(AU)

Occurrence and molecular characterization of Giardia duodenalis from naturally infected dogs in the municipality of Santa Maria, Rio Grande do Sul, Brazil

ABSTRACT: Giardiasis is an important and prevalent zoonosis in dogs and humans caused by Giardia spp. The close relationship between pets and humans has physical, emotional and social benefits. The dogs have an important role in Giardia duodenalis cycle and transmission. This study aimed to verify the occurrence of the parasite in dogs from Central Region, in Santa Maria, Rio Grande do Sul State, Brazil, from April to October 2018. Dog feces (230) were submitted to Faust coproparasitological and molecular analyses. The positive samples in the nested-PCR (-giardin gene) were sent for DNA sequencing and phylogenetic analyses (Neighbor-Joining). The occurrence of G. duodenalis, was 5.6% (13/230) and 4.3% (10/230) detected by coproparasitological technique and nested-PCR, respectively. There was no difference in the sensitivity of the tests used. From the faecal samples analyzed, there were no differences among the variables: diagnostic techniques, local, sex, and age of the animals (p>0.05). Only in the stool examination methodology a difference was observed between the ages (p 0.05). G. duodenalis assemblages were C and D, frequently reported in dogs. The close relationship between dogs and people may allow co-infections of circulating parasites in the population, including Giardia spp. and increasing the risk of transmission of zoonotic agents.

RESUMO: A giardíase é uma zoonose importante e prevalente em cães e humanos, sendo causada por Giardia spp. A estreita relação entre animais de estimação e seres humanos traz benefícios físicos, emocionais e sociais. Os cães têm um papel importante no ciclo e transmissão de Giardia duodenalis. Este estudo teve como objetivo verificar a ocorrência do parasita em cães da Região Central, em Santa Maria, RS, Brasil, de abril a outubro de 2018. As fezes de cães (230) foram submetidas a técnica coproparasitológica de Faust e análises moleculares. As amostras positivas no nested-PCR (gene -giardin) foram enviadas para sequenciamento de DNA e posterior análise filogenética (Neighbor-Joining). A ocorrência de G. duodenalis foi de 5,6% (13/230) e 4,3% (10/230) detectados pela técnica coproparasitológica e nested-PCR, respectivamente. Não houve diferença na sensibilidade dos testes utilizados. Das amostras fecais analisadas, não houve diferenças entre as variáveis: técnicas de diagnóstico, local, sexo e idade dos animais (p>0,05). Somente na metodologia de exame de fezes observou-se diferença entre as idades (p 0,05). As assemblages de G. duodenalis encontradas foram C e D, frequentemente relatadas em cães. A estreita relação entre cães e pessoas pode permitir co-infecções de parasitas circulantes na população, incluindo Giardia spp. e aumentando o risco de transmissão de agentes zoonóticos.

Sarcocystidae in wild birds of southeastern Brazil / Sarcocystidae em aves silvestres do sudeste do Brasil

Abstract This study aimed to identify members of the Sarcocystidae family in naturally infected wild birds at a rescue center in the state of Minas Gerais, southeastern Brazil. The heart and brain of 44 wild birds were evaluated by bioassay in mice to detect T. gondii, and extracted DNA was used for nested PCR of the 18S ribosomal DNA gene to detect members of the Sarcocystidae family. The positive samples were sequenced, assembled, edited and compared with sequences deposited in GenBank. Toxoplasma gondii was isolated from six (13.6%) out of 44 birds. Toxoplasma gondii DNA was identified in 10/44 (22.7%) of the birds. The amplified sequences exhibited 100% similarity with the DNA of the ME49 strain of T. gondii. Sarcocystis DNA (99% similarity) was identified in 5/44 (11.4%) of the birds. T. gondii and Sarcocystis spp. are common in wild birds in Minas Gerais, Brazil.

Resumo O objetivo deste estudo foi identificar membros da família Sarcocystidae em aves silvestres de vida livre naturalmente infectadas e resgatadas no estado de Minas Gerais, Brasil. Coração e cérebro de 44 aves silvestres foram avaliados por bioensaio em camundongos para detecção de T. gondii e extração de DNA para Nested-PCR do gene 18S do DNA ribossomal de membros da família Sarcocystidae. As amostras positivas foram sequenciadas, analisadas, editadas e comparadas com sequências depositadas no GenBank. Toxoplasma gondii foi isolado de seis (13,6%) das 44 aves. DNA de T. gondii foi identificado em 10/44 (22,7%) das 44 aves. As sequências amplificadas exibiram 100% de similaridade com o DNA da cepa ME49 de T. gondii. DNA de Sarcocystis (99% de similaridade) foi identificado em 5/44 (11,4%) das 44 aves. T. gondii e Sarcocystis spp. são encontrados, comumente, em aves silvestres no estado de Minas Gerais, Brasil.

Occurrence and molecular characterization of Giardia duodenalis from naturally infected dogs in the municipality of Santa Maria, Rio Grande do Sul, Brazil / Ocorrência e caracterização molecular de Giardia duodenalis em cães naturalmente infectados no município de Santa Maria, Rio Grande do Sul, Brasil

Giardiasis is an important and prevalent zoonosis in dogs and humans caused by Giardia spp. The close relationship between pets and humans has physical, emotional and social benefits. The dogs have an important role in Giardia duodenalis cycle and transmission. This study aimed to verify the occurrence of the parasite in dogs from Central Region, in Santa Maria, Rio Grande do Sul State, Brazil, from April to October 2018. Dog feces (230) were submitted to Faust coproparasitological and molecular analyses. The positive samples in the nested-PCR (β-giardin gene) were sent for DNA sequencing and phylogenetic analyses (Neighbor-Joining). The occurrence of G. duodenalis, was 5.6% (13/230) and 4.3% (10/230) detected by coproparasitological technique and nested-PCR, respectively. There was no difference in the sensitivity of the tests used. From the faecal samples analyzed, there were no differences among the variables: diagnostic techniques, local, sex, and age of the animals (p>0.05). Only in the stool examination methodology a difference was observed between the ages (p<0.05). G. duodenalis assemblages were C and D, frequently reported in dogs. The close relationship between dogs and people may allow co-infections of circulating parasites in the population, including Giardia spp. and increasing the risk of transmission of zoonotic agents.(AU)

A giardíase é uma zoonose importante e prevalente em cães e humanos, sendo causada por Giardia spp. A estreita relação entre animais de estimação e seres humanos traz benefícios físicos, emocionais e sociais. Os cães têm um papel importante no ciclo e transmissão de Giardia duodenalis. Este estudo teve como objetivo verificar a ocorrência do parasita em cães da Região Central, em Santa Maria, RS, Brasil, de abril a outubro de 2018. As fezes de cães (230) foram submetidas a técnica coproparasitológica de Faust e análises moleculares. As amostras positivas no nested-PCR (gene β-giardin) foram enviadas para sequenciamento de DNA e posterior análise filogenética (Neighbor-Joining). A ocorrência de G. duodenalis foi de 5,6% (13/230) e 4,3% (10/230) detectados pela técnica coproparasitológica e nested-PCR, respectivamente. Não houve diferença na sensibilidade dos testes utilizados. Das amostras fecais analisadas, não houve diferenças entre as variáveis: técnicas de diagnóstico, local, sexo e idade dos animais (p>0,05). Somente na metodologia de exame de fezes observou-se diferença entre as idades (p<0,05). As assemblages de G. duodenalis encontradas foram C e D, frequentemente relatadas em cães. A estreita relação entre cães e pessoas pode permitir co-infecções de parasitas circulantes na população, incluindo Giardia spp. e aumentando o risco de transmissão de agentes zoonóticos.(AU)

Molecular detection and clinical aspects of porcine circovirus 3 infection in pigs from Brazil / [Detecção molecular e aspectos clínicos da infecção por circovírus suíno 3 em suínos do Brasil]

Porcine circovirus 3 (PCV-3) DNA has been detected in serum samples from apparently healthy pigs as well as pigs with different clinical conditions. Molecular detection of PCV-3 was observed in swine serum samples from Southeastern - Brazil using a nested PCR designed specifically for this study. The epidemiology and clinical aspects of PCV-3 infection were evaluated. The samples originated from 154 pigs of both genders from different production phases and with different clinical presentations, sampled from 31 pig farms visited between 2013 and 2018. In this study, PCV-3 was detected in 26.7% of samples from all populations across varying ages. Statistical association (P=0.0285) was observed only between animals with respiratory signs and PCV-3; no PCV-3-positive animal had diarrhea. No statistical association was observed between PCV-3 and age, or gender of the pigs. Because PCV-3 is a newly discovered virus, there is very little information about its epidemiology. We hope that these data can help in future studies investigating PCV-3 epidemiology.(AU)

O DNA do circovírus suíno 3 (PCV-3) foi detectado em amostras de soro de suínos aparentemente saudáveis, bem como em suínos com diferentes condições clínicas. A detecção molecular do PCV-3 foi observada em amostras de soro de suínos da região Sudeste do Brasil, com uma nested PCR desenhada especificamente para este estudo. A epidemiologia e os aspectos clínicos da infecção por PCV-3 foram avaliados. As amostras foram coletadas de 154 suínos de ambos os sexos, de diferentes fases de produção e com diferentes sinais clínicos. Os animais pertenciam a 31 granjas visitadas entre 2013 e 2018. Neste estudo, o PCV-3 foi detectado em 26,7% das amostras de animais saudáveis e de animais com variados sinais clínicos, de ambos os sexos e de idades variadas. Associação estatística (P=0,0285) foi observada apenas entre animais com sinais respiratórios e PCV-3; nenhum animal positivo para PCV-3 apresentava diarreia. Não foi observada associação estatística entre o PCV-3 e a idade ou o sexo dos suínos. Por se tratar de um vírus recém-descoberto, existem poucas informações sobre sua epidemiologia. Espera-se que os dados deste trabalho possam contribuir para futuros estudos sobre a epidemiologia do PCV-3.(AU)

Detection of Treponema spp. in bovine digital dermatitis in the Amazon biome, Brazil / Detecção de Treponema spp. em dermatite digital bovina no bioma amazônico, Brasil

Bovine digital dermatitis (BDD) is a polybacterial claw disease that is endemic to dairy cattle kept in loose house systems, and treponemas are the main bacteria implicated in this disease. The objective of this study was to report the occurrence of Treponema spp. in BDD from crossbred dairy cattle (Holstein x Zebu) kept in a pasture in the Brazilian Amazon biome. The diagnostic of BDD was performed by inspecting the distal extremities of cattle during milking in one or more visits comprising 15 farms. In total, it could be inspected 1,847 cows from August 2016 to July 2017, and 25 lesions of BDD were diagnosed. The feet were scored (System M: M0 = no lesion, M1 = ulcer stage <2cm, M2 = ulcer stage >2cm, M3 = healing stage, M4 = chronic stage, M4.1 = chronic stage with ulcer area). Twenty four biopsy samples were taken from feet with BDD and five biopsy samples from feet with no lesions. The histopathology of stained tissues was performed by hematoxylin and eosin and Warthin-Starry method. The samples were also tested by nested PCR for the three previously isolated BDD Treponema phylogroups (T. medium/T. vincentii-like, T. phagedenis-like and T. putidum/T. denticola-like). Spirochetes were observed in 54.2% (13/24) of the lesions, and in 91.7% (22/24) of the samples were detected the DNA of this spirochete belonging to the treponema phylogroups implicated in BDD. In 25% (6/24) of the lesions were detected all the phylogroups. Forty percent (40%, 2/5) of the M0 samples were also positive for the nested Polymerase Chain Reaction (nested-PCR), as 8.3% (2/24) of the lesions were negative in both techniques employed. Treponema putidum/T. denticola-like was the most detected bacterial in all the stages, and active lesions (M2 and M4.1) presented a greater proportion of T. medium/T. vincentii-like and T. phagedenis-like, but no statistical differences were observed (p>0.05). It could be concluded that BDD lesions in crossbred dairy cattle kept to pasture in the Amazon biome were classified as "polytreponemal" infections and the phylogroup T. putidum/T. denticola-like was the most frequent in the lesions.(AU)

Dermatite digital bovina (DDB) é uma enfermidade polibacteriana dos dígitos endêmica em vacas leiteiras criadas em estábulos e as treponemas são as principais bactérias envolvidas. Este estudo teve como objetivo relatar a ocorrência de Treponema spp. em DDB em bovinos leiteiros mestiços (Holandês x Zebu) criados a pasto no bioma amazônico brasileiro. O diagnóstico da DDB foi realizado pela inspeção, em uma ou mais visitas, das extremidades distais das vacas durante a ordenha em 15 propriedades. No total, foram inspecionadas 1.847 vacas de agosto de 2016 a julho de 2017 e diagnosticou-se 25 lesões de DDB. As extremidades distais inspecionadas foram classificadas em escores (M0 = sem lesão, M1 = estágio ulcerado <2cm, M2 = estágio ulcerado >2cm, M3 = estágio em cicatrização, M4 = estágio crônico, M4.1 = estágio crônico com área ulcerada) e realizada 24 biópsias de dígitos com DDB e cinco biópsias de dígitos em estágio M0. Foram realizadas a histopatologia pelas colorações de hematoxilina e eosina e pelo método de Warthin-Starry, e a nested de reação em cadeia de polimerase (nested-PCR) para os três filogrupos de treponemas previamente isolados de DDB (Treponema medium/T. vincentii-like, T. phagedenis-like e T. putidum/T. denticola-like). Espiroquetas foram observadas em 54,2% (13/24) das lesões e em 91,7% (22/24) detectou-se o DNA de, pelo menos, um dos filogrupos de treponemas pesquisados. Em 25% (6/24) das lesões foram detectados o DNA dos três filogrupos. Em 40% (2/5) das amostras em estágio M0 também foram positivas na nested-PCR, assim como 8,3% (2/24) das lesões foram negativas em ambas as técnicas empregadas. T. putidum/T. denticola-like foi o filogrupo mais detectado em todos os estágios e lesões ativas (M2 e M4.1) apresentaram uma maior proporção para Treponema medium/T. vincentii-like e T. phagedenis-like, mas não se obteve diferença estatística na ocorrência dos filogrupos entre os estágios das lesões (P>0,05). Conclui-se que lesões de DDB em rebanhos leiteiros mestiços criados a pasto no bioma amazônico brasileiro são "politreponemais" e o filogrupo T. putidum/T. denticola-like é o mais frequente nas lesões.(AU)

Molecular Epidemiology of Plasmodium falciparum Chloroquine Resistance Transporter Genes among School Children in Kwara State, Southwestern Nigeria

Background: Plasmodium falciparum existence continues to develop resistance to conventional antimalaria drugs in malaria endemic areas. Plasmodiaoften prevent drugs from interacting with the target site, hence, developing resistance to antimalaria drugs. Mutations in the Plasmodium falciparum chloroquine resistance transporter (Pfcrt), are the major determinant of chloroquine resistance in human malaria parasite.Methodology:Malaria infection, Pfcrtand Pfmdr1 genes of isolates among school students within the age range of 11-22 years from four selected rural communities of Kwara state were studied.One hundred and eighty seven subjects (187) wereselected for the study. Blood samples were collected by finger prick method for malaria screening. Nested PCR and restriction fragment length polymorphism (RFLP) were done to detect alleles of pfcrtat codon 76 and pfmdr1at codon 86. DNA of isolates wasappropriately extracted from the filter paper blots using the methanol fixation method. Logistic regression was performed on the binary observations obtained while linear regression was conducted on the fifty (50) subjects that tested positive to malaria.Results:Out of 187 subjects screened, 26.7% (50) were positive to P. falciparum. Highest malaria parasite count of 36.4% was recorded in 14-16years age group while 20-22 years age group had the least malaria parasite count (15.4%). The result of the studied isolates indicated that out of 50 isolates analyzed for Pfcrtgene, wild type alleles accounted for 32% (16) while mutant alleles accounted for 68% (34). Alakuko Community accounted for the least number of T76 mutant alleles 10% (5) while Apado community recorded the highest number of T76 mutant gene 22% (11). For Pfmdr1gene analysis at codon 86, isolates from Apado community showed the highest mutant type alleles (Y86) of 22% (11), while Igbonla community in Ifelodun local government had the least mutant alleles, 6% (3).Conclusion:The overall result revealed existence of mutant alleles in both the Pfcrtand Pfmdr1genes which was higher than the wild type gene in both cases. The presence of chloroquine resistance genes among the studied population implies that alternative antimalaria drugs should be designed by pharmaceutical industry.

Detection of parvovirus B19 viral DNA in serum of women of childbearing age in Xiangyang City / 公共卫生与预防医学

Objective To analyze the infection of human parvovirus B19 among women of childbearing age in Xiangyang City, and to provide a reference for pregnant women's health care. Methods A total of 303 women of childbearing age in Xiangyang City from 2018 to 2019 were selected as the research subjects. B19 virus DNA in serum of the subjects was detected by nested PCR technology. The differences in the detection rate of B19 viral DNA among normal pregnancy, abnormal pregnancy, and infertility serum were statistically analyzed. The differences in the detection rate of B19 virus DNA among women of childbearing age at different ages were compared. Results The detection rate of B19 viral DNA in all 303 women of child-bearing age was 27.72%. The detection rate of B19 virus DNA in 26-35 year old women was higher than that in other age groups. The detection rate of B19 virus DNA in abnormal pregnancy group was significantly higher than that in normal pregnancy group (P <0.05). Conclusion The detection rate of B19 virus DNA in abnormal pregnancy and infertility group was significantly higher than that in normal pregnancy group, with the detection rate of B19 virus in 26-35 year old women of childbearing age being the highest among all age groups. It is necessary to strengthen the screening of B19 virus in pregnant women of childbearing age in this region to reduce its impact on fetal abortion.

Novel SNP markers on ginsenosides biosynthesis functional gene for authentication of ginseng herbs and commercial products / 中国天然药物

Panax ginseng and Panax quinquefolius have similar bioactive components and morphological characteristics, but they are known to have different medicinal values, high-sensitive and accurate method is expected to identify the sources of ginseng products and evaluate the quality, but with a huge challenge. Our established UHPLC-TOF/MS method coupled with orthogonal partial least squares discriminant analysis (OPLS-DA) model based on 18 ginsenosides was applied to discriminate the sources of raw medicinal materials in ginseng products, and nested PCR strategy was used to discover 6 novel single nucleotide polymorphism (SNP) sites in functional dammarenediol synthase (DS) gene for genetic authentication of P. ginseng and P. quinquefolius for the first time. OPLS-DA model could identify the sources of raw ginseng materials are real or not. SNP markers were applied to identify ginseng fresh samples as well as commercial products, and proved to be successful. This established molecular method can tell exact source information of adulterants, and it was highly sensitive and specific even when total DNA amount was only 0.1 ng and the adulteration was as low as 1%. Therefore, this study made an attempt at the exploration of new type SNP marker for variety authentication and function regulation at the same time, and the combination of chemical and molecular discrimination methods provided the comprehensive evaluation and authentication for the sources of ginseng herbs and products.

Study on the application of Wondfo Rapid Diagnostic Kit in the detection of imported malaria in Hubei Province / 公共卫生与预防医学

Objective To evaluate the effectiveness of the application of Wondfo Rapid Diagnostic Kit (RDTs) in the diagnosis of imported malaria cases in the Malaria Reference Laboratory of Hubei Provence. Methods The complete blood samples of malaria cases and negative card deletion cases reported in Hubei Province from January 2015 to June 2018 were collected and retrospectively analyzed. The results of the provincial malaria reference laboratory were used as the standard, and were compared with those results detected by RDTs, microscopic examination and nested PCR. The differences were statistically analyzed. Results A total of 440 complete samples were collected by the Malaria Reference Laboratory of Hubei Provence, of which 418 samples were confirmed as positive, and 22 samples were confirmed as negative. In terms of the identification ability of P. falciparum, RDTs performed the best, with a coincidence rate of 100.00%, and the coincidence rates nested PCR and microscopic examination were 97.49% and 91.40%, respectively. In terms of the identification specificity for another 3 species of Plasmodium (P. vivax, P. ovarian and P. vivax), nested PCR was the best, the microscopy method was the second best, and RDTs was the lowest. Based on the comprehensive analysis of 12 individual indicators, RDTs had the highest score (32), while the microscopic examination and nested PCR scored 24 and 19, respectively. Conclusion RDTs had certain advantages in the detection of malaria, but they had a low identification specificity for different species. Thus, they can be used as auxiliary tools for microscopic examination and widely used in surveillance work after malaria elimination in Hubei Province.

Assessment of Incidence of Enterocytozoon hepatopenaei (EHP) in Penaeus vannamei in Maharashtra and Gujarat

The occurrence of Enterocytozoon hepatopenaei in Penaeus vannamei samples were collected from Maharashtra and Gujarat farms. In the present study, shrimp samples from various shrimp ponds from two districts of Maharashtra and two districts of Gujarat were collected over a period of one year (February 2016 to April 2017). A total of 4513 shrimp samples were assessed for the presence of EHP by molecular characterization. Out of shrimp samples analysed, 31.2% samples were positive for EHP. The screening of EHP was done by single step and nested PCR targeting spore wall protein gene (SWP) of EHP resulting in product size of 514 bp and 148 bp for EHP respectively