Presence of Cryptosporidium spp. in calves from dairy herds in Northern Antioquia, Colombia / [Ocorrência de Cryptosporidium spp. em bezerros de rebanhos leiteiros do norte de Antioquia, Colômbia]

ABSTRACT Cryptosporidium spp. are important enteropathogen protozoan parasites that infect humans and other animals throughout the world. Cryptosporidiosis in cattle industry leads to considerable economic losses due to diarrhea, dehydration, growth retardation, weight loss, and possibly death, however, data on the occurrence ofCryptosporidiumspp. in cattle in Colombia are limited. Therefore, this study aimed to investigate the occurrence and possible factors associated to the excretion of Cryptosporidium spp. oocyst in pre-weaned calves from dairy farms in Northern Antioquia, Colombia. In addition, Sheather's sugar floatation (SSF), and Modified Ziehl-Neelsen staining (MZN) methods were compared. A total of 41 fecal samples were collected from calves between 1 and 92 days of age of which 23 were positive (56.1%). Crossbreed calves were nine times less susceptible than purebred dairy cattle to excretion of Cryptosporidium spp. oocysts (OR=0.10). MZN was the best technique for the detection of oocysts in fecal samples, however, the mean number of days to detect cryptosporidial oocysts was lower for the SSF method. Cryptosporidium is widespread among calves under 2 months of age in dairy herds of Northern Antioquia, although further investigations considering a greater number of farms and animals are necessary.

RESUMO Cryptosporidium spp. são importantes protozoários parasitas enteropatógenos que infectam humanos e outros animais em todo o mundo. A criptosporidiose na pecuária leiteira leva a perdas econômicas consideráveis devido à diarreia, à desidratação, ao retardo de crescimento, à perda de peso e possivelmente à morte, no entanto dados sobre a ocorrência de criptosporidiose em bovinos na Colômbia são limitados. Portanto, este estudo tem como objetivo investigar a ocorrência e possíveis fatores associados à excreção de oocistos de Cryptosporidium spp. em bezerros pré-desmamados de fazendas leiteiras no norte de Antioquia, Colômbia. Além disso, os métodos de flutuação de açúcar de Sheather (SSF) e de coloração de Ziehl-Neelsen modificado (MZN) foram comparados. Foram coletadas 41 amostras fecais de bezerros entre um e 92 dias de idade, das quais 23 foram positivas (56,1%). Bezerros mestiços foram nove vezes menos suscetíveis que bovinos leiteiros puros à excreção de oocistos de Cryptosporidium spp. (OR=0,10). MZN foi a melhor técnica para a detecção de oocistos em amostras fecais, entretanto o número médio de dias para detecção de oocistos foi menor para o método SSF. Cryptosporidium é amplamente difundido entre bezerros com menos de dois meses de idade em rebanhos leiteiros do norte de Antioquia, embora sejam necessárias mais investigações, considerando-se um maior número de fazendas e animais.

A controlled evaluation of filter paper use during staining of sputum smears for tuberculosis microscopy.

Background: Some sputum smear microscopy protocols recommend placing filter paper over sputum smears during staining for Mycobacterium tuberculosis (TB) . We found no published evidence assessing whether this is beneficial. We aimed to evaluate the effect of filter paper on sputum smear microscopy results. Methods: Sputum samples were collected from 30 patients with confirmed pulmonary TB and 4 healthy control participants. From each sputum sample, six smears (204 smears in total) were prepared for staining with Ziehl-Neelsen (ZN), auramine or viability staining with fluorescein diacetate (FDA). Half of the slides subjected to each staining protocol were randomly selected to have Whatman grade 3 filter paper placed over the dried smears prior to stain application and removed prior to stain washing. The counts of acid-fast bacilli (AFB) and precipitates per 100 high-power microscopy fields of view, and the proportion of smear that appeared to have been washed away were recorded. Statistical analysis used a linear regression model adjusted by staining technique with a random effects term to correct for between-sample variability.   Results: The inclusion of filter paper in the staining protocol significantly decreased microscopy positivity independent of staining with ZN, auramine or FDA (p=0.01). Consistent with this finding, there were lower smear grades in slides stained using filter paper versus without (p=0.04), and filter paper use reduced AFB counts by 0.28 logarithms (95% confidence intervals, CI=0.018, 0.54, p=0.04) independent of staining technique. In all analyses, auramine was consistently more sensitive with higher AFB counts versus ZN (p=0.001), whereas FDA had lower sensitivity and lower AFB counts (p<0.0001). Filter paper use was not associated with the presence of any precipitate (p=0.5) or the probability of any smear washing away (p=0.6) during the staining process. Conclusions: Filter paper reduced the sensitivity of AFB microscopy and had no detectable beneficial effects so is not recommended.

Pathological and molecular identification of Mycobacterium avium infection in a loft of domestic pigeons (Columba livia var. domestica) from India.

Mycobacterium avium is a zoonotic pathogen associated with a wide range of pulmonary and extrapulmonary manifestations in a range of host species like humans, animals, and birds. The disease is more common in the avian population, and opportunistic infections have been reported in immune-compromised or debilitated animals and humans. This study reports the pathological and molecular identification of Mycobacterium avium causing avian mycobacteriosis in a loft of domestic pigeons (Columba livia var. domestica). Out of 30 pigeons aged 2-3 years, ten adult racing pigeons revealed a severe chronic and debilitating disease followed by death. The clinical signs included chronic emaciation, dullness, ruffled feathers, lameness, and greenish, watery diarrhea. Post-mortem examination of birds revealed multifocal gray- to yellow-colored raised nodules in the liver parenchyma, spleen, lungs, intestines, bone marrow, and joints. Avian mycobacteriosis was suspected based on the tissue impression smears stained by Ziehl-Neelsen staining. Histopathological examination also revealed multifocal granulomatous lesions in affected organs, which is characteristic of avian mycobacteriosis. The PCR analysis based on 16S rRNA, IS1245, and IS901 regions suggested the presence of Mycobacterium avium infection belonging to either subspecies avium or sylvaticum. This is the first detailed report of avian mycobacteriosis in pigeons from India, warranting a strict surveillance program to identify the carrier status of these microorganisms in the pigeons, which may prove a fatal zoonotic infection in humans.

Cryptosporidium infection in diarrheal bovine dairy calves: occurrence and risk factors in Santa Catarina, Brazil / Infecção por Cryptosporidium em bezerros com diarreia: ocorrência e fatores de risco em Santa Catarina, Brasil

Cryptosporidium protozoa genus are parasites that cause acute enteric disease in young and immunocompromised animals, resulting in anorexia, loss and decrease in weight gain, and, in severe cases, death. Therefore, this study aimed: i) to determine the occurrence of Cryptosporidium spp. in calves with clinical diarrhea in different regions of Santa Catarina, Brazil; ii) to evaluate the risk factors involved with the frequency of infection. iii) to determine the species most involved with the disease in the region. For this, 425 samples were collected in 141 dairy farms, from animals with ages ranging from 0 to 150 days. For this purpose, the samples were submitted to the modified Ziehl-Neelsen technique, with molecular analysis of the positive samples being performed. It was observed 62.1% occurrence of Cryptosporidium spp. in this sampling, especially between 8 to 15 days. Regarding the risk factors evaluated, such as age, management, facilities, water source and Koppen climate (CFA and CFB), none showed statistical significance. Samples positive by the Ziehl-Neelsen technique (32 samples) were randomly selected for molecular diagnosis. Of these, 10 were sequenced, allowing the identification of Crypstosporidium parvum in 6 samples. However, this study proves the existence and high occurrence of the protozoan in different regions of the state of Santa Catarina, Brazil.

Os protozoários do gênero Cryptosporidium são parasitas que causam doença entérica aguda em animais jovens e imunocomprometidos, resultando em anorexia, perda e diminuição do ganho de peso e, em casos graves, morte. Portanto, este estudo teve como objetivo determinar a ocorrência de Cryptosporidium spp. em bezerros com diarreia clínica em diferentes regiões de Santa Catarina, Brasil; bem como avaliar os fatores de risco envolvidos com a frequência de infecção. Além disso, com um número seleto de amostras, buscou-se determinar as espécies mais envolvidas com a doença na região por meio de técnicas moleculares. Para isso, foram coletadas 425 amostras em 141 fazendas leiteiras, de animais com idade variando de 0 a 150 dias. Observou-se 62,1% de ocorrência de Cryptosporidium spp. nesta amostragem, principalmente entre 8 a 15 dias. Em relação aos fatores de risco avaliados, como idade, manejo, instalações, fonte hídrica e clima de Koppen (CFA e CFB), nenhum apresentou significância estatística. No entanto, este estudo comprova a existência e alta ocorrência do protozoário em diferentes regiões do estado de Santa Catarina, Brasil.

Occurrence of Cryptosporidium in Anglonubian goats in the municipality of Teresina, state of Piauí, Brazil / Ocorrência de Cryptosporidium em cabras da raça Anglonubiana no município de Teresina, estado do Piauí, Brasil

This research aimed to investigate the occurrence of Cryptosporidium and correlate it with types of housing, feces consistency, and physiological parameters related to the reproductive status of Anglo-Nubian goats reared in the State of Piauí, Brazil. A total of 180 fecal samples were collected from 60 non-pregnant and lactating goats with a mean weight of 35 kg, a body condition score of 3.5, and a mean age of three years from an experimental herd at the Federal University of Piauí (UFPI). Oocysts of protozoa of the genus Cryptosporidium could be found in the studied animals using the modified Ziehl-Neelsen technique in fecal smears and the image analysis system to perform morphometry. Each independent variable in the quantitative and qualitative analyses, that is, weight, body condition score (BCS), physiological status (non-pregnant or lactating), feces consistency (normal, pasty, or diarrheal), and floor types (concrete and slatted), was tested with the dependent variable (positive samples, i.e., the presence of Cryptosporidium oocysts). Twenty-four out of the total number of fecal samples were considered positive for the presence of the protozoan, which means that 13.3% of the animals were parasitized. Moreover, 100% of the positive feces samples had normal consistency (firm) and all parasitized animals were reared in pens with a concrete floor. A statistical variation was observed in the BCS of parasitized animals compared to non-parasitized ones (p > 0.0253). The results showed that the occurrence of Cryptosporidium in experimental goats located in the municipality of Teresina, State of Piauí, Brazil, was considered low, requiring sanitary management measures to prevent infection in animals and humans. This is the first report of Cryptosporidium infection in goats in the State of Piauí.

O objetivo desta pesquisa foi investigar a ocorrência de Crypstoporidium e correlacionar com tipos de alojamento, consistência das fezes e parâmetros fisiológicos ligados ao estado reprodutivo de cabras da raça Anglonubiana criadas no estado do Piauí, Brasil. Foram utilizadas 180 amostras de fezes de 60 cabra, com peso médio de 35kg, escore de condição corporal de 3,5, com idade em média de três anos, e cabras vazias e lactantes, de um rebanho experimental da Universidade Federal do Piauí (UFPI). Utilizando-se a técnica de Ziehl-Neelsen modificada em esfregaço fecal e sistema de análise de imagens para a realização da morfometria, foi possível encontrar oocistos de protozoários do gênero Crypstoporidium nos animais estudados. Nas análises quantitativa e qualitativa, cada variável independente: peso, escore de condição corporal (ECC), estado fisiológico (vazia ou lactante), consistência das fezes (normal, pastosa ou diarreica) e tipos de piso (concreto e ripado), foi testada com a variável dependente (amostras positivas, ou seja, presença de oocistos de Cryptosporidium). Do total de amostras fecais analisadas, 24 delas foram consideradas positivas à presença do protozoário, o que significa que 13,3% dos animais estavam parasitados na ocasião da pesquisa. Foi observado que 100% das amostras de fezes positivas apresentaram consistência normal (firme) e que todos os animais parasitados eram criados em aprisco com piso de concreto. Houve uma variação estatística no ECC dos animais parasitados comparados aos não parasitados (p > 0,0253). Os resultados evidenciaram que a ocorrência de Cryptosporidium em caprinos experimentais localizados no município de Teresina, no estado do Piauí, foi considerada baixa, sendo necessária medidas de manejo sanitário para prevenir a infecção nos animais e no homem. Este é o primeiro relato da infecção por Cryptosporidium em cabras no estado do Piauí.

Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients / Comparação de diferentes modalidades de diagnóstico para isolamento de Mycobacterium tuberculosis entre pacientes com suspeita de linfadenite tuberculosa

Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.(AU)

A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados para o isolamento completo do Mycobacterium tuberculosis.(AU)

Investigação da presença do bacilo Mycobacterium tuberculosis em amostras sugestivas de tuberculose oral / Investigation of the presence of Mycobacterium tuberculosis bacillus in samples suggestive of oral tuberculosis

A Tuberculose (TB) é um considerável problema de saúde pública mundial. Em 2021, de acordo com a Organização Mundial de Saúde (OMS) estimou-se que, no mundo, cerca de 10,6 milhões de pessoas desenvolveram TB e 1,4 milhão morreu devido à doença. Com isso, tornou-se a principal causa de morte por infecção em todo o mundo e uma das dez principais causas de morte em geral. A TB tem o pulmão como o principal sítio de acometimento, sendo denominada de TB Pulmonar (TBP). Porém pode ser diagnosticada em muitos órgãos do corpo de maneira Extrapulmonar (TBEP), sendo o linfonodo o local mais comum. Porém, o envolvimento pleural, neurológico, sinovial, pericárdico, abdominal, geniturinário e oral tem sido descrito, o que mostra a potencial capacidade de disseminação do Mycobacterium tuberculosis (MTB). A detecção do Bacilos Álcool-Ácido Resistentes (BAAR), geralmente ocorre pela observação das características microscópicas da morfologia dos tecidos, presença de granulomas com necrose caseosa, histiócitos epitelióides e células gigantes do tipo Langhans, associada à coloração para BAAR, pela técnica de Ziehl Neelsen (ZN). Ademais, investigação por imuno-histoquímica (IHQ), testes de amplificação de ácido nucleico pela Reação em Cadeia da Polimerase Hemianinhada (nested-PCR) e pelo sistema de detecção automatizado GeneXpert® MTB/RIF são métodos aplicados para o diagnóstico da infecção. Com isso, este estudo teve como objetivo investigar a presença do bacilo Mycobacterium tuberculosis em amostras orais em parafina que continham granulomas com necrose caseosa. Ao todo, como critério de inclusão, foram selecionadas biópsias que apresentaram granulomas com necrose caseosa, sugerindo o diagnóstico de TB. Foram excluídas aquelas que após a revisão das fichas e histológicas, não apresentavam os granulomas exibindo necrose caseosa e aquelas que foram de biópsias intraósseas. O M. tuberculosis foi procurado por meio da coloração de ZN, IHC, nested-PCR e ensaios GeneXpert® MTB/RIF. Foram então selecionadas nove amostras com granulomas com necrose caseosa. Houve predominância de indivíduos do sexo masculino (2,5:1), com idade média de 50 anos (±23,08; 19-89), sendo a língua o local anatômico mais afetado (n=4). O bacilo não foi identificado pela técnica de ZN em nenhuma amostra, e a coloração por IHC mostrou um padrão granular grosseiro, sugerindo M. tuberculosis, em três delas. Nested-PCR e os ensaios GeneXpert® MTB/RIF foram positivos em duas e três das amostras, respectivamente. Conclui-se que testes moleculares e IHC podem ser métodos auxiliares úteis para casos suspeitos de tuberculose.

Tuberculosis (TB) is a significant global public health issue. In 2021, according to the World Health Organization (WHO), it was estimated that approximately 10.6 million people developed TB worldwide, and 1.4 million died from the disease. Consequently, it became the leading cause of death due to infection worldwide and one of the top ten overall causes of death. TB primarily affects the lungs and is referred to as Pulmonary TB (PTB). However, it can be diagnosed in various organs of the body as Extrapulmonary TB (EPTB), with lymph nodes being the most common site of involvement. Moreover, pleural, neurological, synovial, pericardial, abdominal, genitourinary, and oral involvement have been described, demonstrating the potential for Mycobacterium tuberculosis (MTB) dissemination. The detection of Acid-Fast Bacilli (AFB) typically involves the observation of microscopic tissue characteristics, the presence of granulomas with caseous necrosis, epithelioid histiocytes, and Langhans giant cells, along with AFB staining using the Ziehl-Neelsen (ZN) technique. Furthermore, immunohistochemistry (IHC), nucleic acid amplification tests by Nested Polymerase Chain Reaction (nested-PCR), and the automated detection system GeneXpert® MTB/RIF are methods employed for diagnosing the infection. Therefore, the aim of this study was to investigate the presence of Mycobacterium tuberculosis in paraffin-embedded oral samples containing granulomas with caseous necrosis. Inclusion criteria were based on the selection of biopsies displaying granulomas with caseous necrosis, suggesting a diagnosis of TB. Biopsies without these features upon review of records and histological findings, as well as intraosseous biopsies, were excluded. M. tuberculosis was sought using ZN staining, IHC, nested-PCR, and GeneXpert® MTB/RIF assays. Nine samples with granulomas and caseous necrosis were selected. The majority of individuals were male (2.5:1 ratio), with an average age of 50 years (±23.08; range 19-89), and the tongue was the most affected anatomical site (n=4). AFB was not identified by the ZN technique in any of the samples, and IHC staining exhibited a coarse granular pattern, suggestive of M. tuberculosis, in three of them. Nested-PCR and GeneXpert® MTB/RIF assays yielded positive results in two and three of the samples, respectively. In conclusion, molecular tests and IHC can be valuable auxiliary methods for suspected cases of tuberculosis.

Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients / Comparação de diferentes modalidades de diagnóstico para isolamento de Mycobacterium tuberculosis entre pacientes com suspeita de linfadenite tuberculosa

Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados para o isolamento completo do Mycobacterium tuberculosis.

Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients

Abstract Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

Resumo A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados para o isolamento completo do Mycobacterium tuberculosis.

Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients / Comparação de diferentes modalidades de diagnóstico para isolamento de Mycobacterium tuberculosis entre pacientes com suspeita de linfadenite tuberculosa

Abstract Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

Resumo A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado ​​para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados ​​para o isolamento completo do Mycobacterium tuberculosis.

Acid-fast bacteria as causative agents of skin and soft tissue infections: case presentations and literature review

ABSTRACT Acid-fast bacteria can be implicated in skin and soft tissue infections. Diagnostic identification can be challenging or not feasible by routine laboratory techniques, especially if there is no access to the Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) technology. Here, we present two cases of skin and soft tissue infections caused by two different acid-fast bacteria, Nocardia brasiliensis and Mycobacterium marinum. They both grew on Löwenstein-Jensen medium, Sabouraud agar medium and blood agar medium. Both bacteria appeared acid-fast by Ziehl-Neelsen stain and Gram-positive by Gram stain. The identification was performed by MALDI-TOF MS and gene analysis. N. brasiliensis and nontuberculous mycobacterium M. marinum represent rare pathogens that cause severe skin and soft tissue infections. Failure to identify the causative agent and subsequent inappropriate or inadequate treatment may lead to severe complications or even disseminated disease, especially in immunocompromised individuals.

The Re-Identification of Previously Unidentifiable Clinical Non-Tuberculous Mycobacterial Isolates Shows Great Species Diversity and the Presence of Other Acid-Fast Genera.

Non-tuberculous mycobacteria that cannot be identified at the species level represent a challenge for clinical laboratories, as proper species assignment is key to implementing successful treatments or epidemiological studies. We re-identified forty-eight isolates of Ziehl-Neelsen (ZN)-staining-positive "acid-fast bacilli" (AFB), which were isolated in a clinical laboratory and previously identified as Mycobacterium species but were unidentifiable at the species level with the hsp65 PCR restriction fragment length polymorphism analysis (PRA). As most isolates also could not be identified confidently via 16S, hsp65, or rpoB DNA sequencing and a nBLAST search analysis, we employed a phylogenetic method for their identification using the sequences of the 16S rDNA, which resulted in the identification of most AFB and a Mycobacterium species diversity not found before in our laboratory. Most were rare species with only a few clinical reports. Moreover, although selected with the ZN staining as AFB, not all isolates belonged to the genus Mycobacterium, and we report for the first time in Latin America the isolation of Nocardia puris, Tsukamurella pulmosis, and Gordonia sputi from sputum samples of symptomatic patients. We conclude that ZN staining does not differentiate between the genus Mycobacterium and other genera of AFB. Moreover, there is a need for a simple and more accurate tree-based identification method for mycobacterial species. For this purpose, and in development in our lab, is a web-based identification system using a phylogenetic analysis (including all AFB genera) based on 16S rDNA sequences (and in the future multigene datasets) and the closest relatives.

Evaluation of fecal smear methods for research on Cryptosporidium spp. oocysts in the feces of dairy calves / Avaliação dos métodos de esfregaço fecal para a pesquisa de oocistos de Cryptosporidium spp. nas fezes de bezerros da bovinocultura de leite

The objective of this study is to compare the direct fecal smear (DFS) and centrifugal sedimentation (CS) methods in the detection of Cryptosporidium spp. oocysts in fecal samples of dairy calves. One hundred and fourteen fecal samples were collected from calves aged up to six months from 10 dairy farms located in Palotina and Francisco Alves, Paraná, Brazil. The microscopic analysis revealed the presence of Cryptosporidium spp. oocysts in 51.75% (59/114) of the samples in both methods. In CS, 48.25% (55/114) of the samples were positive, while in DFS slides, only 6.14% (7/114) were positive. Only 4 samples were positive exclusively in DFS. To ensure that there were no false-negative results in the microscopic analysis, the 55 samples that were negative in both DFS and CS were selected for molecular analysis using the nested PCR (nPCR). Of these 55 samples, 24% (13/55) were positive and forwarded for sequencing part of the genome, which made it possible to identify C. parvum, C. bovis and C. ryanae. Besides the characterization of the Cryptosporidium species, it was possible to identify bacteria of the genus Acinetobacter interfering directly in the analyzed samples. The microscopic analysis also revealed higher sensitivity when CS was used to make the fecal smears. However, some samples that were negative in this technique had positive PCR results. Thus, molecular analysis is indicated to confirm cases of Cryptosporidium spp. Further studies are necessary to prove the specificities of the used primers since the results obtained in nPCR were positive for the protozoan but, when genetic sequencing was performed, Acinetobacter spp. was identified.(AU)

O objetivo deste trabalho foi comparar os métodos de esfregaço fecal direto (DFS) e centrífugo sedimentação (CS) para a pesquisa de oocistos de Cryptosporidium spp. em amostras fecais de bezerros leiteiros. Foram coletadas 114 amostras fecais de bezerros com até seis meses de idade, provenientes de dez propriedades leiteiras localizadas nos municípios de Palotina e Francisco Alves, Paraná. Por meio da análise microscópica, foi possível observar oocistos de Cryptosporidium spp. em 51,75% (59/114) das amostras. Pelo método da CS foi identificada positividade em 48,25% (55/114) das amostras, enquanto nas lâminas pelo método do esfregaço fecal direto (DFS), apenas 6,14% (7/114) foram positivas. Somente 4 amostras foram positivas exclusivamente no método do DFS. Para assegurar que na análise microscópica não houvesse resultados falso-negativos, as 55 amostras negativas para os dois métodos de confecção de lâminas (DFS e CS) foram selecionadas para análise molecular por meio da técnica de Nested PCR. Das 55 amostras submetidas a nPCR, 24% (13/55) apresentaram-se positivas e foram encaminhadas para o sequenciamento genético de uma porção do genoma, o qual possibilitou identificar as espécies de C. parvum, C. bovis e C. ryanae. Além da caracterização das espécies de Cryptosporidium, foi possível identificar a presença de bactérias do gênero Acinetobacter interferindo diretamente nas amostras analisadas. A análise microscópica revelou maior sensibilidade quando o método de CS foi usado para a confecção dos esfregaços fecais, entretanto, amostras negativas por meio dessa metodologia apresentaram resultados positivos na nPCR. Desta forma, para a confirmação dos casos de Cryptosporidium spp., é indicado a realização da análise molecular. Novos estudos são necessários para se comprovar a especificidade dos primers utilizados, uma vez que na nPCR o resultado obtido foi positivo, porém ao realizar o sequenciamento genético houve a identificação de Acinetobacter spp.(AU)

Tuberculosis of the central nervous system in cattle in Paraíba, Brazil / Tuberculose no sistema nervoso central de bovinos na Paraíba

This paper describes six cases of tuberculosis in the central nervous system (CNS) of cattle in the state of Paraíba in northeastern Brazil. We reviewed the autopsy reports of 851 bovine necropsies performed from 2003 to 2016. Seventy-three (8.6%) cattle were diagnosed with tuberculosis and six showed lesions in the CNS. Three cases affected cattle up to two-year-old and other three affected adults. Three cattle presented exclusively nervous signs, two had respiratory signs and weight loss and one did not present any clinical signs. At necropsy, five cattle had thickening of the leptomeninges of the cerebellum, pons, obex, spinal cord and cortex, mainly, in the region near the brain basilar Willis´ circle. Another animal, presented a single focal lesion in the cerebellum. Microscopically we observed moderate to severe granulomatous meningitis and encephalitis. Five cattle presented lesions in the lungs and mediastinal lymph nodes and three of them had disseminated lesions in other organs. In all cattle acid-fast bacilli were observed in the lesions and marked positive for immunohistochemistry with polyclonal antibody anti-Mycobacterium tuberculosis. It is concluded that bovine tuberculosis of central nervous system occurs sporadically in Paraíba, in cattle of different ages, most of them with disseminate lesions in other organs. The location of the lesions suggests that the agent invaded the brain by hematogenous route through the circle of Willis.(AU)

Descrevem-se seis casos de tuberculose no sistema nervoso central (SNC) em bovinos, no semiárido da Paraíba. Foram revisados os laudos de um total de 851 necropsias de bovinos realizadas no período de 2003 a 2016. Destes, 73 (8,6%) foram diagnosticados com tuberculose e seis apresentavam lesões no SNC. Três casos ocorreram em bovinos de até dois anos de idade e três em bovinos adultos. Três bovinos apresentaram exclusivamente sinais nervosos, dois tinham sinais respiratórios e perda de peso e um não apresentava nenhum sinal clínico. Macroscopicamente, em cinco bovinos, havia espessamento das leptomeninges do cerebelo, medula espinhal, ponte, obex, colículos e córtex, principalmente, na região basilar encefálica próxima ao polígono de Willis. Em apenas um bovino houve a presença de tubérculo único no cerebelo. Microscopicamente observou-se moderada a acentuada meningite e encefalite granulomatosa. Cinco bovinos apresentaram lesões pulmonares e nos gânglios mediastínicos e três deles tinham lesões disseminadas em outros órgãos. Em todos os bovinos foram encontrados bacilos ácido-álcool resistentes intralesionais e todos tiveram marcação positiva na técnica de imuno-histoquímica com anticorpo policlonal anti-Mycobacterium tuberculosis. Conclui-se que a tuberculose do sistema nervoso central de bovinos ocorre de forma esporádica na Paraíba, principalmente em bovinos com lesões disseminadas em outros órgãos. Sugere-se que a disseminação do agente ocorre pela via hematógena, possivelmente através do polígono de Willis.(AU)

Tuberculosis of the central nervous system in cattle in Paraíba, Brazil / Tuberculose no sistema nervoso central de bovinos na Paraíba

This paper describes six cases of tuberculosis in the central nervous system (CNS) of cattle in the state of Paraíba in northeastern Brazil. We reviewed the autopsy reports of 851 bovine necropsies performed from 2003 to 2016. Seventy-three (8.6%) cattle were diagnosed with tuberculosis and six showed lesions in the CNS. Three cases affected cattle up to two-year-old and other three affected adults. Three cattle presented exclusively nervous signs, two had respiratory signs and weight loss and one did not present any clinical signs. At necropsy, five cattle had thickening of the leptomeninges of the cerebellum, pons, obex, spinal cord and cortex, mainly, in the region near the brain basilar Willis´ circle. Another animal, presented a single focal lesion in the cerebellum. Microscopically we observed moderate to severe granulomatous meningitis and encephalitis. Five cattle presented lesions in the lungs and mediastinal lymph nodes and three of them had disseminated lesions in other organs. In all cattle acid-fast bacilli were observed in the lesions and marked positive for immunohistochemistry with polyclonal antibody anti-Mycobacterium tuberculosis. It is concluded that bovine tuberculosis of central nervous system occurs sporadically in Paraíba, in cattle of different ages, most of them with disseminate lesions in other organs. The location of the lesions suggests that the agent invaded the brain by hematogenous route through the circle of Willis.(AU)

Descrevem-se seis casos de tuberculose no sistema nervoso central (SNC) em bovinos, no semiárido da Paraíba. Foram revisados os laudos de um total de 851 necropsias de bovinos realizadas no período de 2003 a 2016. Destes, 73 (8,6%) foram diagnosticados com tuberculose e seis apresentavam lesões no SNC. Três casos ocorreram em bovinos de até dois anos de idade e três em bovinos adultos. Três bovinos apresentaram exclusivamente sinais nervosos, dois tinham sinais respiratórios e perda de peso e um não apresentava nenhum sinal clínico. Macroscopicamente, em cinco bovinos, havia espessamento das leptomeninges do cerebelo, medula espinhal, ponte, obex, colículos e córtex, principalmente, na região basilar encefálica próxima ao polígono de Willis. Em apenas um bovino houve a presença de tubérculo único no cerebelo. Microscopicamente observou-se moderada a acentuada meningite e encefalite granulomatosa. Cinco bovinos apresentaram lesões pulmonares e nos gânglios mediastínicos e três deles tinham lesões disseminadas em outros órgãos. Em todos os bovinos foram encontrados bacilos ácido-álcool resistentes intralesionais e todos tiveram marcação positiva na técnica de imuno-histoquímica com anticorpo policlonal anti-Mycobacterium tuberculosis. Conclui-se que a tuberculose do sistema nervoso central de bovinos ocorre de forma esporádica na Paraíba, principalmente em bovinos com lesões disseminadas em outros órgãos. Sugere-se que a disseminação do agente ocorre pela via hematógena, possivelmente através do polígono de Willis.(AU)

Disseminated tuberculosis in a dog: a putative zooanthroponosis

Background: Zooantroponoses that affect pets are rare. Microorganisms involved in human tuberculosis has great importance to public health and has potentiality to produce reverse zoonoses. Infection by Mycobacterium tuberculosis is in top of the most important infectious diseases related to public health. In dogs, the infection occurs through prolonged exposure to aerosols and respiratory secretions from humans. Canine tuberculosis is little reported and known in Brazil, this work aims to describe the anatomopathological findings of the first case of canine tuberculosis in a 5-year-old dog in the Federal District and alert to the presence of this disease in this region.Case: The dog had history of close contact with its owner, who has been treated for pulmonary tuberculosis six months before. At necropsy scattered multifocal, firm, white to yellow-gray nodules, varying in size (3 mm to 10 mm) involving lungs, kidneys, heart, liver and spleen was observed. In frontal and occipital cerebral cortex, pale-gray circular lesions were detected with the same characteristics. The histological findings were, multifocal to coalescent granulomas, well organized with a central core of necrosis with foci of mineralization, surrounded by histiocytes, epithelioid macrophage, rare Langhans giant cells, some intact and degenerate plasma cells and neutrophils, and a rim of reactive fibroblasts producing fibrous connective tissue. Histological staining with ZN revealed free acid-fast bacilli and in the cytoplasm of macrophages and granulomas in all affected organs. The PCR allowed identification of DNA was extracted from formalin fixed paraffin embedded (FFPE) tissues using the ReliaPrep™ FFPE gDNA Miniprep System®, according to the manufacturers instructions (Promega, EUA). The Speed-Oligo Mycobacteria assay was used to identify the presence of M. tuberculosis complex DNA in tissue samples.[...](AU)

Disseminated tuberculosis in a dog: a putative zooanthroponosis

Background: Zooantroponoses that affect pets are rare. Microorganisms involved in human tuberculosis has great importance to public health and has potentiality to produce reverse zoonoses. Infection by Mycobacterium tuberculosis is in top of the most important infectious diseases related to public health. In dogs, the infection occurs through prolonged exposure to aerosols and respiratory secretions from humans. Canine tuberculosis is little reported and known in Brazil, this work aims to describe the anatomopathological findings of the first case of canine tuberculosis in a 5-year-old dog in the Federal District and alert to the presence of this disease in this region.Case: The dog had history of close contact with its owner, who has been treated for pulmonary tuberculosis six months before. At necropsy scattered multifocal, firm, white to yellow-gray nodules, varying in size (3 mm to 10 mm) involving lungs, kidneys, heart, liver and spleen was observed. In frontal and occipital cerebral cortex, pale-gray circular lesions were detected with the same characteristics. The histological findings were, multifocal to coalescent granulomas, well organized with a central core of necrosis with foci of mineralization, surrounded by histiocytes, epithelioid macrophage, rare Langhans giant cells, some intact and degenerate plasma cells and neutrophils, and a rim of reactive fibroblasts producing fibrous connective tissue. Histological staining with ZN revealed free acid-fast bacilli and in the cytoplasm of macrophages and granulomas in all affected organs. The PCR allowed identification of DNA was extracted from formalin fixed paraffin embedded (FFPE) tissues using the ReliaPrep™ FFPE gDNA Miniprep System®, according to the manufacturer’s instructions (Promega, EUA). The Speed-Oligo Mycobacteria assay was used to identify the presence of M. tuberculosis complex DNA in tissue samples.[...]

Real-time PCR assay for the diagnosis of pleural tuberculosis.

INTRODUCTION: The diagnosis of pleural tuberculosis requires an invasive and time-consuming reference method. Polymerase chain reaction (PCR) is rapid, but validation in pleural tuberculosis is still weak. OBJECTIVE: To establish the operating characteristics of real-time polymerase chain reaction (RT-PCR) hybridization probes for the diagnosis of pleural tuberculosis. METHODS: The validity of the RT-PCR hybridization probes was evaluated compared to a composite reference method by a cross-sectional study at the Hospital Universitario de la Samaritana. 40 adults with lymphocytic pleural effusion were included. Pleural tuberculosis was confirmed (in 9 patients) if the patient had at least one of three tests using the positive reference method: Ziehl-Neelsen or Mycobacterium tuberculosis culture in fluid or pleural tissue, or pleural biopsy with granulomas. Pleural tuberculosis was ruled out (in 31 patients) if all three tests were negative. The operating characteristics of the RT-PCR, using the Mid-P Exact Test, were determined using the OpenEpi 2.3 Software (2009). RESULTS: The RT-PCR hybridization probes showed a sensitivity of 66.7% (95% CI: 33.2%-90.7%) and a specificity of 93.5% (95% CI: 80.3%-98.9%). The PPV was 75.0% (95% CI: 38.8%-95.6%) and a NPV of 90.6% (95% CI: 76.6%-97.6%). Two false positives were found for the test, one with pleural mesothelioma and the other with chronic pleuritis with mesothelial hyperplasia. CONCLUSIONS: The RT-PCR hybridization probes had good specificity and acceptable sensitivity, but a negative value cannot rule out pleural tuberculosis.

INTRODUCCIÓN: El diagnóstico de tuberculosis pleural requiere un método de referencia invasivo y demorado. La reacción en cadena de la polimerasa es rápida, pero su validación en tuberculosis pleural aún es débil. OBJETIVO: Establecer las características operativas de la reacción en cadena de la polimerasa en tiempo real (RT-PCR) sondas de hibridación para el diagnóstico de tuberculosis pleural. MÉTODOS: Se evaluó la validez de la RT-PCR sondas de hibridación comparada con un método de referencia compuesto mediante un estudio transversal en el Hospital Universitario de la Samaritana. Se incluyeron 40 adultos con derrame pleural linfocitario. Tuberculosis pleural fue confirmada (en 9 pacientes) si el paciente tenía mínimo una de tres pruebas del método de referencia positiva: Ziehl-Neelsen o cultivo para Mycobacterium tuberculosis en líquido o tejido pleural, o biopsia pleural con granulomas; se descartó tuberculosis pleural (en 31 pacientes) si las tres pruebas eran negativas. Se determinaron las características operativas de la RT-PCR, mediante la Prueba Mid-P Exact, con el Software OpenEpi 2.3 (2009). RESULTADOS: La RT-PCR sondas de hibridación mostró una sensibilidad del 66.7% (IC 95%: 33.2%-90.7%) y una especificidad del 93.5% (IC 95%: 80.3%-98.9%). El VPP fue de 75.0% (IC 95%: 38.8%-95.6%) y un VPN de 90.6% (IC 95%: 76.6%-97.6%). Se encontraron dos falsos positivos para la prueba, uno con mesotelioma pleural y otro con pleuritis crónica con hiperplasia mesotelial. CONCLUSIONES: La RT-PCR sondas de hibridación tuvo una buena especificidad y una aceptable sensibilidad, pero un valor negativo no puede descartar tuberculosis pleural.

Occurrence and molecular characterization of Cryptosporidium sp. in sheep / Ocorrência e caracterização molecular de Cryptosporidium sp. em ovinos

Considered a zoonosis of utmost importance, cryptosporidiosis has a worldwide distribution and can infect mammals, birds, reptiles, and amphibians. It is caused by a highly resistant protozoan present in the environment and can cause death in immunosuppressed individuals and pups, as well as in farm animals such as cattle and sheep, generating losses. The aim of this study was to investigate the presence of Cryptosporidium spp. in sheep feces from the farms of Western Paraná, which have different management styles, and compare the results with their respective management methods. One hundred and forty-four stool samples were collected (69 from Property 1 and 75 from Property 2) and analyzed using a fecal smear on slides after staining by the modified Ziehl-Neelsen method. Samples tested positive by this method were subjected to nested PCR and the products obtained were sent for sequencing to determine the species. While 82.60% of the samples from Property 1 were tested positive, only 36% of the samples from Property 2 were tested positive. On analyzing the sequencing data, it was observed that the Cryptosporidium species of samples from Property 1 showed high similarity to Cryptosporidium xiaoi and those from Property 2, to Cryptosporidium ubiquitum. The reason for divergence in results can be attributed to differences in management systems adopted by each property, thus showing the importance of detecting carrier animals, as they can contaminate the environment,especially the water sources, and spread the disease to humans and other animals.(AU)

Considerada uma zoonose de extrema importância, a cryptosporidiose possui distribuição mundial e pode infectar mamíferos, aves, répteis e anfíbios. É causada por um protozoário muito resistente no ambiente, podendo gerar mortes em indivíduos imunossuprimidos e filhotes, principalmente em animais de produção como bovinos e ovinos, gerando prejuízos. O objetivo desse trabalho foi pesquisar a presença de Cryptosporidium spp. em fezes de ovinos de propriedades rurais do Oeste do Paraná, que possuem manejos diferenciados e comparar os resultados encontrados com o respectivo manejo. Foram coletadas 144 amostras de fezes, sendo 69 da Propriedade 1 e 75 da Propriedade 2, analisadas pelo esfregaço de fezes em lâminas após a coloração pelo método de Ziehl-Neelsen modificado. As amostras positivas por esse método foram submetidas à Nested-PCR sendo os produtos obtidos enviados ao sequenciamento para a determinação da espécie. A Propriedade 1 apresentou positividade em 82,60% das amostras, enquanto a Propriedade 2 obteve 36% de amostras positivas. Foi encontrada alta similaridade para Cryptosporidium xiaoi na primeira propriedade e Cryptosporidium ubiquitum na segunda propriedade. A divergência nos resultados pode ser comprovada pela diferente forma de manejo adotada em cada propriedade, mostrando a importância de detectar os animais portadores, uma vez que estes podem contaminar o meio ambiente, especialmente os cursos dágua, e promover a disseminaçãoda doença para outros animais e para o homem.(AU)

Real-time PCR assay for the diagnosis of pleural tuberculosis / Reacción en cadena de la polimerasa en tiempo real para el diagnóstico de tuberculosis pleural

Abstract Introduction: The diagnosis of pleural tuberculosis requires an invasive and time-consuming reference method. Polymerase chain reaction (PCR) is rapid, but validation in pleural tuberculosis is still weak. Objective: To establish the operating characteristics of real-time polymerase chain reaction (RT-PCR) hybridization probes for the diagnosis of pleural tuberculosis. Methods: The validity of the RT-PCR hybridization probes was evaluated compared to a composite reference method by a cross-sectional study at the Hospital Universitario de la Samaritana. 40 adults with lymphocytic pleural effusion were included. Pleural tuberculosis was confirmed (in 9 patients) if the patient had at least one of three tests using the positive reference method: Ziehl-Neelsen or Mycobacterium tuberculosis culture in fluid or pleural tissue, or pleural biopsy with granulomas. Pleural tuberculosis was ruled out (in 31 patients) if all three tests were negative. The operating characteristics of the RT-PCR, using the Mid-P Exact Test, were determined using the OpenEpi 2.3 Software (2009). Results: The RT-PCR hybridization probes showed a sensitivity of 66.7% (95% CI: 33.2%-90.7%) and a specificity of 93.5% (95% CI: 80.3%-98.9%). The PPV was 75.0% (95% CI: 38.8%-95.6%) and a NPV of 90.6% (95% CI: 76.6%-97.6%). Two false positives were found for the test, one with pleural mesothelioma and the other with chronic pleuritis with mesothelial hyperplasia. Conclusions: The RT-PCR hybridization probes had good specificity and acceptable sensitivity, but a negative value cannot rule out pleural tuberculosis.

Resumen Introducción: El diagnóstico de tuberculosis pleural requiere un método de referencia invasivo y demorado. La reacción en cadena de la polimerasa es rápida, pero su validación en tuberculosis pleural aún es débil. Objetivo: Establecer las características operativas de la reacción en cadena de la polimerasa en tiempo real (RT-PCR) sondas de hibridación para el diagnóstico de tuberculosis pleural. Métodos: Se evaluó la validez de la RT-PCR sondas de hibridación comparada con un método de referencia compuesto mediante un estudio transversal en el Hospital Universitario de la Samaritana. Se incluyeron 40 adultos con derrame pleural linfocitario. Tuberculosis pleural fue confirmada (en 9 pacientes) si el paciente tenía mínimo una de tres pruebas del método de referencia positiva: Ziehl-Neelsen o cultivo para Mycobacterium tuberculosis en líquido o tejido pleural, o biopsia pleural con granulomas; se descartó tuberculosis pleural (en 31 pacientes) si las tres pruebas eran negativas. Se determinaron las características operativas de la RT-PCR, mediante la Prueba Mid-P Exact, con el Software OpenEpi 2.3 (2009). Resultados: La RT-PCR sondas de hibridación mostró una sensibilidad del 66.7% (IC 95%: 33.2%-90.7%) y una especificidad del 93.5% (IC 95%: 80.3%-98.9%). El VPP fue de 75.0% (IC 95%: 38.8%-95.6%) y un VPN de 90.6% (IC 95%: 76.6%-97.6%). Se encontraron dos falsos positivos para la prueba, uno con mesotelioma pleural y otro con pleuritis crónica con hiperplasia mesotelial. Conclusiones: La RT-PCR sondas de hibridación tuvo una buena especificidad y una aceptable sensibilidad, pero un valor negativo no puede descartar tuberculosis pleural.