Characterization of internal transcribed spacer-1 and apical membrane antigen-1 sequences provides insights into the genetic diversity of Eimeria tenella strains

@#Coccidiosis is a major recurring problem in the poultry industry and is caused by infection of one or more of the seven Eimeria species known to infect chickens, with Eimeria tenella among the best studied and economically important. Studies on the genetic diversity of E. tenella strains is essential for the development of universally acceptable diagnostic markers and vaccines against the disease. Eimeria tenella internal transcribed spacer-1 (ITS-1) and apical membrane antigen-1 (AMA-1) sequences from different parts of the world are available in the public domain and therefore provide suitable comparative markers for genetic diversity study. In this study, the ITS-1 and AMA-1 sequence diversity of two local E. tenella strains, namely EtNSN6 and EtSGR6 were characterized. Both ITS-1 and AMA-1 sequences for EtNSN6 and EtSGR6 were retrieved by mapping to their respective genome sequences generated using next generation sequencing. Multiple sequence alignment of the ITS-1 and AMA-1 sequences with selected homologous sequences revealed the presence of a total of five and 13 single nucleotide polymorphisms (SNPs) respectively. All SNPs appeared to occur at random and did not show any unique pattern based on geographical regions while no insertions and deletions (indels) was found to occur in the aligned sequences. However, unique bases that defined certain strains were detected. Phylogenetics analyses performed with Maximum Parsimony (MP) and Maximum Likelihood (ML) methods revealed similar topology for the internal groups with all the E. tenella ITS-1 and AMA-1 sequences grouped in the same clade supported by high bootstrap confidence. This confirmed that both EtNSN6 and EtSGR6 samples are E. tenella strains. Sequence comparison and phylogenetics analyses further suggest the possibility of low genetic diversity among E. tenella strains.

Imported case of Leishmania tropica cutaneous leishmaniasis in a 10-year-old child in Malaysia

@#The present paper reported a first imported case of cutaneous leishmaniasis in a 10-yearold child who returned from Saudi Arabia to Malaysia. Six weeks after his travel to Malaysia, two erythematous dermal nodules were developed over his right cheek and chin. Occurrence of intracellular amastigote of Leishmania was observed through examination of skin biopsy with hematoxylin and eosin stain. Furthermore, molecular analysis of ribosomal internal transcribed spacer 1 (ITS1) of Leishmania spp. confirmed the child was infected with Leishmania tropica. The child was given oral fluconazole and he had a 80% recovery before he went back to Saudi Arabia.

Morphological and molecular identification of Cladosporium sphaerospermum isolates collected from tomato plant residues / Identificação morfológica e molecular de isolados de Cladosporium sphaerospermum coletados de resíduos de plantas de tomate

This study was conducted at the Agriculture College University of Karbala, Iraq to isolate and morphologically and molecularly diagnose thirteen Cladosporium isolates collected from tomato plant residues present in desert regions of Najaf and Karbala provinces, Iraq. We diagnosed the obtained isolates by PCR amplification using the ITS1 and ITS4 universal primer pair followed by sequencing. PCR amplification and analysis of nucleotide sequences using the BLAST program showed that all isolated fungi belong to Cladosporium sphaerospermum. Analysis of the nucleotide sequences of the identified C. sphaerospermum isolates 2, 6, 9, and 10 showed a genetic similarity reached 99%, 98%, 99%, and 99%, respectively, with those previously registered at the National Center for Biotechnology Information (NCBl). By comparing the nucleotide sequences of the identified C. sphaerospermum isolates with the sequences belong to the same fungi and available at NCBI, it was revealed that the identified C. sphaerospermum isolates 2, 6, 9, and 10 have a genetic variation with those previously recorded at the National Center for Biotechnology Information (NCBl); therefore, the identified sequences of C. sphaerospermum isolates have been registered in GenBank database (NCBI) under the accession numbers MN896004, MN896107, MN896963, and MN896971, respectively.

Este estudo foi conduzido na Agriculture College University of Karbala, Iraque, para isolar e diagnosticar morfológica e molecularmente treze isolados de Cladosporium coletados de resíduos de plantas de tomate presentes nas regiões desérticas das províncias de Najaf e Karbala, no Iraque. Diagnosticamos os isolados obtidos por amplificação por PCR usando o par de primers universais ITS1 e ITS4 seguido de sequenciamento. A amplificação por PCR e a análise de sequências de nucleotídeos usando o programa BLAST mostraram que todos os fungos isolados pertencem a Cladosporium sphaerospermum. A análise das sequências de nucleotídeos dos isolados 2, 6, 9 e 10 de C. sphaerospermum identificados mostrou similaridade genética de 99%, 98%, 99% e 99%, respectivamente, com aqueles previamente registrados no National Center for Biotechnology Informações (NCBl). Ao comparar as sequências de nucleotídeos dos isolados de C. sphaerospermum identificados com as sequências pertencentes aos mesmos fungos e disponíveis no NCBI, foi revelado que os isolados 2, 6, 9 e 10 de C. sphaerospermum identificados têm variação genética com aqueles anteriormente registrados no National Center for Biotechnology Information (NCBl). Portanto, as sequências identificadas de isolados de C. sphaerospermum foram registradas no banco de dados GenBank (NCBI) sob os números de acesso MN896004, MN896107, MN896963 e MN896971, respectivamente.

Morphological and molecular identification of Cladosporium sphaerospermum isolates collected from tomato plant residues

Abstract This study was conducted at the Agriculture College University of Karbala, Iraq to isolate and morphologically and molecularly diagnose thirteen Cladosporium isolates collected from tomato plant residues present in desert regions of Najaf and Karbala provinces, Iraq. We diagnosed the obtained isolates by PCR amplification using the ITS1 and ITS4 universal primer pair followed by sequencing. PCR amplification and analysis of nucleotide sequences using the BLAST program showed that all isolated fungi belong to Cladosporium sphaerospermum. Analysis of the nucleotide sequences of the identified C. sphaerospermum isolates 2, 6, 9, and 10 showed a genetic similarity reached 99%, 98%, 99%, and 99%, respectively, with those previously registered at the National Center for Biotechnology Information (NCBl). By comparing the nucleotide sequences of the identified C. sphaerospermum isolates with the sequences belong to the same fungi and available at NCBI, it was revealed that the identified C. sphaerospermum isolates 2, 6, 9, and 10 have a genetic variation with those previously recorded at the National Center for Biotechnology Information (NCBl); therefore, the identified sequences of C. sphaerospermum isolates have been registered in GenBank database (NCBI) under the accession numbers MN896004, MN896107, MN896963, and MN896971, respectively.

Resumo Este estudo foi conduzido na Agriculture College University of Karbala, Iraque, para isolar e diagnosticar morfológica e molecularmente treze isolados de Cladosporium coletados de resíduos de plantas de tomate presentes nas regiões desérticas das províncias de Najaf e Karbala, no Iraque. Diagnosticamos os isolados obtidos por amplificação por PCR usando o par de primers universais ITS1 e ITS4 seguido de sequenciamento. A amplificação por PCR e a análise de sequências de nucleotídeos usando o programa BLAST mostraram que todos os fungos isolados pertencem a Cladosporium sphaerospermum. A análise das sequências de nucleotídeos dos isolados 2, 6, 9 e 10 de C. sphaerospermum identificados mostrou similaridade genética de 99%, 98%, 99% e 99%, respectivamente, com aqueles previamente registrados no National Center for Biotechnology Informações (NCBl). Ao comparar as sequências de nucleotídeos dos isolados de C. sphaerospermum identificados com as sequências pertencentes aos mesmos fungos e disponíveis no NCBI, foi revelado que os isolados 2, 6, 9 e 10 de C. sphaerospermum identificados têm variação genética com aqueles anteriormente registrados no National Center for Biotechnology Information (NCBl). Portanto, as sequências identificadas de isolados de C. sphaerospermum foram registradas no banco de dados GenBank (NCBI) sob os números de acesso MN896004, MN896107, MN896963 e MN896971, respectivamente.

Morphological and molecular identification of Cladosporium sphaerospermum isolates collected from tomato plant residues / Identificação morfológica e molecular de isolados de Cladosporium sphaerospermum coletados de resíduos de plantas de tomate

This study was conducted at the Agriculture College University of Karbala, Iraq to isolate and morphologically and molecularly diagnose thirteen Cladosporium isolates collected from tomato plant residues present in desert regions of Najaf and Karbala provinces, Iraq. We diagnosed the obtained isolates by PCR amplification using the ITS1 and ITS4 universal primer pair followed by sequencing. PCR amplification and analysis of nucleotide sequences using the BLAST program showed that all isolated fungi belong to Cladosporium sphaerospermum. Analysis of the nucleotide sequences of the identified C. sphaerospermum isolates 2, 6, 9, and 10 showed a genetic similarity reached 99%, 98%, 99%, and 99%, respectively, with those previously registered at the National Center for Biotechnology Information (NCBl). By comparing the nucleotide sequences of the identified C. sphaerospermum isolates with the sequences belong to the same fungi and available at NCBI, it was revealed that the identified C. sphaerospermum isolates 2, 6, 9, and 10 have a genetic variation with those previously recorded at the National Center for Biotechnology Information (NCBl); therefore, the identified sequences of C. sphaerospermum isolates have been registered in GenBank database (NCBI) under the accession numbers MN896004, MN896107, MN896963, and MN896971, respectively.

Este estudo foi conduzido na Agriculture College University of Karbala, Iraque, para isolar e diagnosticar morfológica e molecularmente treze isolados de Cladosporium coletados de resíduos de plantas de tomate presentes nas regiões desérticas das províncias de Najaf e Karbala, no Iraque. Diagnosticamos os isolados obtidos por amplificação por PCR usando o par de primers universais ITS1 e ITS4 seguido de sequenciamento. A amplificação por PCR e a análise de sequências de nucleotídeos usando o programa BLAST mostraram que todos os fungos isolados pertencem a Cladosporium sphaerospermum. A análise das sequências de nucleotídeos dos isolados 2, 6, 9 e 10 de C. sphaerospermum identificados mostrou similaridade genética de 99%, 98%, 99% e 99%, respectivamente, com aqueles previamente registrados no National Center for Biotechnology Informações (NCBl). Ao comparar as sequências de nucleotídeos dos isolados de C. sphaerospermum identificados com as sequências pertencentes aos mesmos fungos e disponíveis no NCBI, foi revelado que os isolados 2, 6, 9 e 10 de C. sphaerospermum identificados têm variação genética com aqueles anteriormente registrados no National Center for Biotechnology Information (NCBl). Portanto, as sequências identificadas de isolados de C. sphaerospermum foram registradas no banco de dados GenBank (NCBI) sob os números de acesso MN896004, MN896107, MN896963 e MN896971, respectivamente.

Identification of Fritillaria taibaiensis and its relatives by real-time PCR with a TaqMan-MGB probe / 药学学报

The molecular identification of Fritillaria taibaiensis and its relatives was studied by real-time PCR with a TaqMan-MGB probe. DNA was extracted from F. taibaiensis and its relatives. According to the sequence of ITS1 region, the mutation sites of F. taipaiensis and its related species were identified by MEGA7.0 software. The specific primers (a pair) and a TaqMan-MGB probe were designed by Primer Premier 6.0 software. In the Roche LightCycler 96 system, the lowest limit of detection for F. taipaiensis DNA template was 0.002 39 ng·μL-1, and the optimal Tm value range was 60 and 61 ℃. Specificity identification showed that the method had good specificity for F. taipaiensis, as it could be distinguished from other 13 different Fritillaria species including F. unibracteata. Since this method could accurately identify F. taipaiensis and its related species, it provides technical support for rational development of F. taipaiensis resources, management of Chinese medicinal market and supervision of raw materials in Chinese medicine manufacturing enterprises.

Fecal Fungal Community Structure of Newly Diagnosed Patients With Crohn's Disease in Suzhou, Jiangsu Province / 胃肠病学

Background: The pathogenic mechanism of Crohn's disease (CD) is currently unclear. Previous studies have shown that intestinal microorganism plays an important role in the pathogenesis of CD. Aims: To study the fecal fungal community structure of CD patients in Suzhou, Jiangsu Province and to analyze the relationship between alterations in fugal community structure and disease status. Methods: The feces of 23 newly diagnosed CD patients and 18 healthy subjects in Suzhou, Jiangsu Province were collected. Fecal genomic DNA was extracted, and the ITS1 fragments were amplified by PCR for clone library construction. After sequencing on HiSeq platform, the species diversity of fecal samples and the species differences between different groups were analyzed based on OTUs. Furthermore, the correlations between the fungi with up-regulated abundance and inflammatory indicators (CRP, ESR, and fecal calprotectin) and CD activity index (CDAI) were analyzed. Results: The fungal species diversity in fecal samples was significantly reduced in CD patients compared with healthy subjects. From the levels of phylum, class, order, family, genus, and species, the fungi with up-regulated abundance in fecal samples of CD patients were Saccharomycetes, Saccharomycetales, Incertae_sedis, Candida and Candida albicans; the fungi newly emerged were Trichosporonales and Trichosporonaceae; Wallemiomycetes was down-regulated in abundance and Glomeromycota, Glomeromycetes, Glomeraceae, Lodderomyces, Candida intermedia, and Candida sp were absent. The abundance of Candida albicans in feces was positively correlated with the fecal calprotectin in CD patients (r=0.557, P=0.031). Conclusions: Compared with healthy subjects, the structure of intestinal fungal community in CD patients changed significantly. The species diversity was reduced and the abundance of opportunistic pathogenic fungi such as Candida albicans was increased and might be involved in the disease progression.

Evidence of a sylvatic enzootic cycle of Leishmania infantum in the State of Amapá, Brazil

Abstract INTRODUCTION: Leishmania infantum was considered to be absent from Amapá until 2017 when canine infection was detected. However, there is a lack of knowledge about which reservoir species are involved in transmission in this region. METHODS: Between 2014 and 2016, 86 samples from wild mammals and 74 from domestic dogs were collected in Wajãpi Indigenous Territory and were tested for the presence of deoxyribonucleic acid (DNA) of Leishmania. RESULTS: The DNA of Le. infantum was detected in two rodent samples, Dasyprocta sp. and Proechimys cuvieri. CONCLUSIONS: This is the first evidence characterizing a sylvatic transmission cycle of Le. infantum in the State of Amapá.

Postpartum histoplasmosis in an HIV-negative woman: a case report and phylogenetic characterization by internal transcribed spacer region analysis

Abstract The present report describes the first case of postpartum disseminated histoplasmosis in a 24-year-old HIV-negative woman. On the tenth day after vaginal delivery, the patient presented with dyspnea, fever, hypotension, tachycardia, and painful hepatomegaly. Yeast-like Histoplasma capsulatum features were isolated in the buffy coat. The phylogenetic analysis demonstrated that the fungal isolate was similar to other H. capsulatum isolates identified in HIV patients from Ceará and Latin America. Thus, histoplasmosis development in individuals with transitory immunosuppression or during the period of immunological recovery should be carefully examined.

Molecular detection of Leishmania spp. in cattle from Brazil by means of PCR using internal transcribed spacer 1 / Detecção molecular de Leishmania spp. em gado no Brasil por PCR com espaçador interno transcrito 1

Abstract Leishmania spp. are important agents of human and animal leishmaniases that have an important impact on public health. In this study, we aimed to detect the circulation of Leishmania spp. in cattle from a visceral leishmaniasis non-endemic area of the state of São Paulo, Brazil. DNA was extracted from blood samples from 100 heifers in the municipality of Pirassununga and was amplified using primers specific for the first internal transcriber spacer (ITS1), to assess the presence of trypanosomatids. The assays revealed that one sample presented bands of between 300 and 350 base pairs. In GenBank, this sample matched 100% with Leishmania infantum (314 base pairs). The results suggest that cattle can be infected by Leishmania infantum in Brazil.

Resumo Leishmania spp. são agentes causadores das leishmanioses em humanos e em animais, gerando grande impacto à saúde pública. Este estudo objetivou detectar a circulação de Leishmania spp. em área não endêmica para leishmaniose visceral de São Paulo, Brasil. Foram extraídas amostras de DNA de 100 novilhas da cidade de Pirassununga. Estas amostras foram amplificadas com os iniciadores específicos para tripanosomatídeos Internal Transcriber Spacer 1 (ITS1). Os ensaios revelaram uma amostra com bandas entre 300 e 350 pares de base (pb). A amostra demonstrou 100% de identidade com Leishmania infantum (314 pb). Os resultados sugerem que o gado pode ser infectado por L. infantum no Brasil.

Establishment of a T(m)-shift Method for Detection of Cat-Derived Hookworms

Melting temperature shift (T(m)-shift) is a new detection method that analyze the melting curve on real-time PCR thermocycler using SYBR Green I fluorescent dye. To establish a T(m)-shift method for the detection of Ancylostoma ceylanicum and A. tubaeforme in cats, specific primers, with GC tail of unequal length attached to their 5′ end, were designed based on 2 SNP loci (ITS101 and ITS296) of the internal transcribed spacer 1 (ITS1) sequences. The standard curve of T(m)-shift was established using the standard plasmids of A. ceylanicum (AceP) and A. tubaeforme (AtuP). The T(m)-shift method stability, sensitivity, and accuracy were tested with reference to the standard curve, and clinical fecal samples were also examined. The results demonstrated that the 2 sets of primers based on the 2 SNPs could accurately distinguish between A. ceylanicum and A. tubaeforme. The coefficient of variation (CV) of T(m)-values of AceP and AtuP was 0.07% and 0.06% in ITS101 and was 0.06% and 0.08% in ITS296, respectively. The minimum detectable DNA concentration was 5.22×10⁻⁶ and 5.28×10⁻⁶ ng/μl samples of AceP and AtuP, respectively. The accuracy of T(m)-shift method reached 100% based on examination of 10 hookworm DNA samples with known species. In the clinical detection of hookworm in 69 stray cat fecal sample, the T(m)-shift detection results were consistent with the microscopic examination and successfully differentiated between the 2-hookworm species. In conclusion, the developed method is a rapid, sensitive and accurate technique and can provide a promising tool for clinical detection and epidemiological investigation of cat-derived hookworms.

Ten Cases of Taenia saginata Infection Confirmed by Analysis of the Internal Transcribed Spacer 1 rDNA Region in the Republic of Korea

From October 2015 to August 2018, tapeworm proglottids were obtained from 10 patients who were residents of Daegu and Gyeongbuk provinces and had a history of raw beef consumption. Most of them had no overseas travel experience. The gravid proglottids obtained from the 10 cases had 15–20 lateral uterine branches. A part of internal transcribed spacer 1 (ITS1) DNA of the 10 cases, amplified by polymerase chain reaction (PCR) and digested with AleI restriction enzyme, produced the same band pattern of Taenia saginata, which differentiated from T. asiatica and T. solium. Sequences of ITS1 and cytochrome c oxidase subunit 1 (cox1) showed higher homology to T. saginata than to T. asiatica and T. solium. Collectively, these 10 cases were identified as T. saginata human infections. As taeniasis is one of the important parasitic diseases in humans, it is necessary to maintain hygienic conditions during livestock farming to avoid public health concerns.

Identificación y evaluación de actividad celulolítica en aislamientos nativos de Trichoderma spp obtenidos de biomasa de palma de aceite / Identification and screening of lignocellulosic activity in native isolates of Trichoderma spp., obtained from palm oil biomass

RESUMEN Se aislaron previamente ocho cepas nativas de racimos de palma de aceite en descomposición de Trichoderma sp. provenientes de la región de Cumaral, Meta, Colombia. Se utilizó la región de los ITS1-ITS4 para la identificación molecular y se determinó la actividad celulolítica (actividad sobre papel filtro) del complejo producido por las cepas utilizando residuos de palma como sustrato. Siete aislamientos nativos presentarón 100% de similaridad con hongos del género Trichoderma. Se observó para siete cepas, la presencia de las cinco anclas que identifican hongos del género Trichoderma, identificándose cuatro de los hongos nativos como Trichoderma koningiopsis (HR-04-89; HR-11-89; HR-19-89; y HR-06-89) y cuatro como Trichoderma asperellum (HR-01-89; HR-03-89; HR-16-89; HR-18-89). El bioensayo mostró que las cepas evaluadas de Trichoderma son estadísticamente significativas sobre la actividad enzimática de celulasas sobre papel filtro (p<0.05). Además, las cepas HR-01-89, HR-03-89, HR-11-89, HR-04-89 y HR-18-89 no presentaron diferencias en la actividad enzimática. La cepa Trichoderma reesei utilizada como referencia, presentó un comportamiento superior y diferente comparado con las cepas nativas. La cepa nativa HR-18-89 (Trichoderma asperellum) presentó mayores niveles de actividad enzimática, 78% del valor de la cepa referencia. Es importante identificar y evaluar cepas nativas de Trichoderma sp. con novedosas actividades biológicas que permitan degradar la celulosa recalcitrante de los racimos de palma africana.

ABSTRACT Previously, there were isolated eight native strains of Trichoderma sp. from a cluster of decomposing oil palm from Cumaral, Meta, Colombia. The ITS1-ITS4"s region was used for the molecular identification and the cellulase activity (filter paper activity) of the complex produced by strains was determined using palm waste as substrate. Seven native isolations showed between 97-100% similarity with fungi of the genus Trichoderma. It was observed for seven of the eight strains the presence of the five anchors which identify fungi of the genus Trichoderma, finding five of the native fungi such as Trichoderma koningiopsis (HR-04-89; HR-11-89; HR-19-89; y HR-06-89) four as Trichoderma asperellum (HR-01-89; HR-03-89; HR-16-89; HR-18-89). The bioassay showed that Trichoderma strains tested are statistically significant on the enzymatic activity of cellulases on filter paper (p <0.05). In addition, strains HR-01-89, HR-03-89, HR-11-89, HR-04-89 and HR-18-89 showed no differences in enzymatic activity. The reference strain used, Trichoderma reesei produce a superior and different behavior compared with the native strains. The native strain HR-18-89 (Trichoderma asperellum) had higher levels of enzyme activity, 78% of the value of the reference strain. It is important to identify and evaluate native strains of Trichoderma sp. with innovative biological activities that allow to degrade the recalcitrant cellulose of the African palm clusters.

Molecular detection of Leishmania species in human and animals from cutaneous leishmaniasis endemic areas of Waziristan, Khyber Pakhtunkhwa, Pakistan

Objectives: To detect Leishmania species in human patients, animal reservoirs and Phlebotomus sandflies in Waziristan, Pakistan. Methods: Tissue smears and aspirates from 448 cutaneous leishmaniasis (CL) suspected patients were analyzed. To sort out role of the reservoir hosts, skin scrapings, spleen and liver samples from 104 rodents were collected. Furthermore, buffy coat samples were obtained from 60 domestic animals. Sandflies were also trapped. All human, animals and sandfly samples were tested by microscopy, kinetoplastic PCR and internal transcribed spacer 1 (ITS1) PCR followed by restriction fragment length polymorphism for detection of Leishmania species. Results: An overall prevalence of 3.83% and 5.21% through microscopy and ITS1 PCR respectively was found. However, the statistically non-significant correlation was found between area, gender, and number of lesions. The presence of rodents, sandflies, domestic animals and internally displaced people increased the risk of CL. Using ITS1-PCR-RFLP, Leishmania tropica (L. tropica) was confirmed in 106 samples while 25 of the isolates were diagnosed as Leishmania major (L. major). Similarly, 3/104 rodents were positive for L. major and 14 pools of DNA samples containing Phlebotomus sergenti sandflies were positive for L. tropica. None of samples from domestic animals were positive for leishmaniasis. Conclusions: In the present study, L. tropica and L. major are found to be the main causative agents of CL in study area. Movement of internally displaced people from CL endemic areas presents a risk for nearby CL free areas. To the best of our knowledge, we report for the first time L. major infection in rodents (Rattus rattus) and L. tropica in Phlebotomus sergenti sandflies trapped in Waziristan, Pakistan.

Molecular detection of Leishmania species in human and animals from cutaneous leishmaniasis endemic areas of Waziristan, Khyber Pakhtunkhwa, Pakistan

Objectives:To detect Leishmania species in human patients, animal reservoirs and Phlebotomus sandflies in Waziristan, Pakistan.Methods:Tissue smears and aspirates from 448 cutaneous leishmaniasis (CL) suspected patients were analyzed. To sort out role of the reservoir hosts, skin scrapings, spleen and liver samples from 104 rodents were collected. Furthermore, buffy coat samples were obtained from 60 domestic animals. Sandflies were also trapped. All human, animals and sandfly samples were tested by microscopy, kinetoplastic PCR and internal transcribed spacer 1 (ITS1) PCR followed by restriction fragment length polymorphism for detection of Leishmania species.Results:An overall prevalence of 3.83% and 5.21% through microscopy and ITS1 PCR respectively was found. However, the statistically non-significant correlation was found between area, gender, and number of lesions. The presence of rodents, sandflies, domestic animals and internally displaced people increased the risk of CL. Using ITS1-PCR-RFLP, Leishmania tropica (L. tropica) was confirmed in 106 samples while 25 of the isolates were diagnosed as Leishmania major (L. major). Similarly, 3/104 rodents were positive for L. major and 14 pools of DNA samples containing Phlebotomus sergenti sandflies were positive for L. tropica. None of samples from domestic animals were positive for leishmaniasis.Conclusions:In the present study, L. tropica and L. major are found to be the main causative agents of CL in study area. Movement of internally displaced people from CL endemic areas presents a risk for nearby CL free areas. To the best of our knowledge, we report for the first time L. major infection in rodents (Rattus rattus) and L. tropica in Phlebotomus sergenti sandflies trapped in Waziristan, Pakistan.

Secondary structure of nrDNA Internal Transcribed Spacers as a useful tool to align highly divergent species in phylogenetic studies

Abstract Recently, it has been suggested that internal transcribed spacer (ITS) sequences are under selective constraints to preserve their secondary structure. Here, we investigate the patterns of the ITS nucleotide and secondary structure conservation across the Passiflora L. genus to evaluate the potential use of secondary structure data as a helpful tool for the alignment in taxonomically complex genera. Considering the frequent use of ITS, this study also presents a perspective on future analyses in other plant groups. The ITS1 and ITS2 sequences presented significant differences for mean values of the lowest energy state (LES) and for number of hairpins in different Passiflora subgenera. Statistical analyses for the subgenera separately support significant differences between the LES values and the total number of secondary structures for ITS. In order to evaluate whether the LES values of ITS secondary structures were related to selective constraints, we compared these results among 120 ITS sequences from Passiflora species and 120 randomly generated sequences. These analyses indicated that Passiflora ITS sequences present characteristics of a region under selective constraint to maintain the secondary structure showing to be a promising tool to improve the alignments and identify sites with non-neutral substitutions or those correlated evolutionary steps.

Estandarización de la técnica de PCR-RFLP de la región ITS1, para la caracterización molecular de leishmania (L) infantum, en muestras caninas / Standardization of PCR-RFLP technique of the ITS-1 region for leishmania (L) infantum molecular characterization in canine samples

La leishmaniasis es una enfermedad producida por protozoarios parásitos del género Leishmania. Estos parásitos infectan a hospedadores mamíferos, entre los cuales los perros han sido implicados como reservorios del parásito. Este trabajo planteó estandarizar la técnica de la PCR-RFLP luego de la amplificación de la región ITS1 de Leishmania spp, como herramienta útil para la detección y caracterización molecular. Se utilizaron promastigotes de cultivo y muestras de biopsias procedentes de perros con leishmaniasis visceral previamente diagnosticados en el Centro Antirrábico Nacional. La región ITS1 del ADN genómico nuclear de Leishmania spp. fue amplificada utilizando los cebadores LITSR y L5,8S. La técnica ITS1 PCR-RFLP aplicada, permitió la detección de Leishmania (L) infantum en 10/10 aislados de parásitos mantenidos en medio NNN, en 10/18 muestras de bazo y 10/18 muestras de ganglio linfático poplíteo. Las condiciones óptimas de reacción fueron 0,2 mM de dNTPs, 0,1 pmol de cada cebador y 1U de Taq polimerasa. La sensibilidad de la PCR fue de 3 ng/µL de ADN en aislados de cultivo NNN y 60 ng/µL de ADN en muestras de biopsias, mientras que la especificidad fue de 100% para la detección de Leishmania sp. La enzima de restricción Hae III, determinó fragmentos de 184, 72 y 55 pb., que resultaron específicos para la especie Leishmania (L.) infantum. El marcador utilizado resultó confiable para la detección y caracterización de Leishmania sp. en perros procedentes de zonas endémicas, lo cual podría ser útil para verificar las especies de parásitos circulantes entre los perros.

Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania. The separasites infect to mammalian hosts, including canines that have been implicated as reservoirs of the parasite. The aim of this research was to standardize the technique of PCR RFLP after amplification of the ITS1 region of Leishmania (Leishmania) infantum, as a useful tool for detection and molecular characterization. Promastigotes from culture and biopsies from dogs with visceral Leishmaniasis previously diagnosed by the Centro Antirrábico Nacional. The ITS1 region of the genomic DNA of Leishmania sp. was amplified using LITSR and L5,8S primers. The technique ITS1 PCR-RFLP applied, allowed the detection of Leishmania (L.) infantum in 10/10 of the isolates from parasites maintained in NNN culture medium, in 10/18 samples from spleen and 10/18 samples from popliteal lymph node. Optimal reaction conditions were 0.2 mM dNTPs, 0.1 pmol of each primer and 1U of Taq polymerase. The sensitivity of PCR was 3 ng/µL DNA in isolates of parasites from NNNculture medium and 60 ng/µL DNA in biopsy samples while the specificity was 100% for the detection of DNA of Leishmania sp. The restriction enzyme Hae III determined fragments of184, 72 and 55 bp., which were specific to Leishmania (L.) infantum. The marker used isreliable for the detection and characterization of Leishmania sp. in dogs from endemic areas, which could be useful to verify the species of parasites circulating among animals.

Analysis of the genetic diversity of Candida isolates obtained from diabetic patients and kidney transplant recipients

Yeasts of the genus Candida have high genetic variability and are the most common opportunistic pathogenic fungi in humans. In this study, we evaluated the genetic diversity among 120 isolates of Candida spp. obtained from diabetic patients, kidney transplant recipients and patients without any immune deficiencies from Paraná state, Brazil. The analysis was performed using the ITS1-5.8S-ITS2 region and a partial sequence of 28S rDNA. In the phylogenetic analysis, we observed a consistent separation of the species C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. parapsilosis, C. metapsilosis and C. orthopsilosis, however with low intraspecific variability. In the analysis of the C. albicans species, two clades were formed. Clade A included the largest number of isolates (91.2%) and the majority of isolates from GenBank (71.4%). The phylogenetic analysis showed low intraspecific genetic diversity, and the genetic polymorphisms between C. albicans isolates were similar to genetic divergence found in other studies performed with isolates from Brazil. This low genetic diversity of isolates can be explained by the geographic proximity of the patients evaluated. It was observed that yeast colonisation was highest in renal transplant recipients and diabetic patients and that C. albicans was the species most frequently isolated.

Molecular Description of Macroorchis spinulosus (Digenea: Nanophyetidae) Based on ITS1 Sequences

We performed a molecular genetic study on the sequences of 18S ribosomal RNA (ITS1 region) gene in 4-day-old adult worms of Macroorchis spinulosus recovered in mice experimentally infected with metacercariae from crayfish in Jeollanam-do Province, Korea. The metacercariae were round, 180 µm in average diameter, encysted with 2 layers of thick walls, but the stylet on the oral sucker was not clearly seen. The adult flukes were oval shape, and 760-820 µm long and 320-450 µm wide, with anterolateral location of 2 large testes. The phylogenetic tree based on ITS1 sequences of 6 M. spinulosus samples showed their distinguished position from other trematode species in GenBank. The most closely resembled group was Paragonimus spp. which also take crayfish or crabs as the second intermediate host. The present study is the first molecular characterization of M. spinulosus and provided a basis for further phylogenetic studies to compare with other trematode fauna in Korea.

Comparação do sistema de identificação automatizado Vitek 2 e PCR-ITS para caracterização das espécies dos isolados clínicos de Candida spp / Comparison of Vitek-2 automated identification system and PCR-ITS for species characterization of clinical isolates of Candida spp

As infecções graves causadas pelo gênero Candida têm se tornado um desafio na questão diagnóstica, no intuito de se detectar e identificar o agente etiológico de forma ágil, precisa e padronizada nos laboratórios clínicos. A predição da susceptibilidade aos antifúngicos, bem como a necessidade da geração de dados epidemiológicos reforçam a importância da identificação rotineira adequada das espécies de leveduras envolvidas em infecções. Dentre as 200 espécies de Candida já descritas, C. albicans, C. parapsilosis, C. tropicalis, C. glabrata, C. guilliermondii, C. krusei e C. lusitaniae são mais frequentemente relacionadas a infecções em humanos. Todos os métodos fenotípicos de identificação de Candida apresentam limitações,em especial na caracterização de espécies não C. albicans, porém, a aplicação de métodos moleculares pode refletir no aumento de custo e tempo despendido para a obtenção de resultados laboratoriais. A fim de avaliara aplicação do sistema automatizado Vitek 2-YST ID (bio Merieux) aliado ao uso de agar cromogênico na identificação rotineira de espécies de Candida, foram testados 44 isolados de infecção invasiva por inoculação em agar cromogênico e no painel automatizado e realização de amplificação do DNA relativo às regiões do espaçador interno transcrito 1 e 2 do rRNA (PCR-ITS). Oligo nucleotídeos espécie específicos foram utilizados e o tamanho do produto amplificado foi correlacionado aos demais resultados. O sistema automatizado identificou 95,4% dos isolados quando em associação com as características coloniais observadas no meio cromogênico, porém, o uso de PCR-ITS ou metodologias mais sensíveis seria necessário para solucionar os demais resultados, ambíguos e errôneos.

Serious infections caused by genus Candida have become a challenge in the diagnostic question, in order todetect and identify the etiologic agent of agile, precise and standardized form in manner clinical laboratories. The prediction of susceptibility to antifungal agents, and the need to generate epidemiological data highlight the importance of routine identification of yeast species involved in infections. Among the 200 Candida species already described, C. albicans, C. parapsilosis, C. tropicalis, C. glabrata, C. guilliermondii, C. krusei and C. lusitaniae are most often related with infections in humans. All of the phenotypic methods of identification of Candida have limitations, especially the characterization of the species not C. albicans, however, the application of molecular methods may reflect the increased cost and spent time for obtain results on laboratory. In order to evaluate the implementation of the automated system Vitek 2 ID - YST(bioMerieux) combined with the use of chromogenic agar in the routine identification of Candida species were tested 44 isolates from invasive infection by inoculation of chromogenic agar and automated panel and realization DNA amplification for the internal transcribed spacer regions of rRNA 1 and 2 (ITS - PCR). Oligonucleotides specific species were used and the size of the amplified product was correlated to other results. The automated system identified 95.4 % of the isolates when in association with colonial features observed in chromogenic medium, however, the use of PCR -ITS or more sensitive methodologies would be needed to solve the other results, ambiguous and erroneous.