Does each bead count? A reduced-cost approach for recovering waterborne protozoa from challenge water using immunomagnetic separation

Giardia duodenalis and Cryptosporidium spp. are two of the most prominent aetiological agents of waterborne diseases. Therefore, efficient and affordable methodologies for identifying and quantifying these parasites in water are increasingly necessary. USEPA Method 1623.1 is a widely used and validated protocol for detecting these parasites in water samples. It consists of a concentration step, followed by parasite purification and visualization by immunofluorescence microscopy. Although efficient, this method has a high cost particularly due to the immunomagnetic separation (IMS) step, which is most needed with complex and highly contaminated samples. Based on this, the present study aimed to determine whether it is possible to maintain the efficiency of Method 1623.1 while reducing the amount of beads per reaction, using as a matrix the challenge water recommended by the World Health Organization. As for Giardia cysts, a satisfactory recovery efficiency (RE) was obtained using 50% less IMS beads. This was evaluated both with a commercial cyst suspension (56.1% recovery) and an analytical quality assessment (47.5% recovery). Although RE rates obtained for Cryptosporidium parvum did not meet Method 1623.1 criteria in any of the experimental conditions tested, results presented in this paper indicated the relevance of the described adaptations, even in challenge water. HIGHLIGHTS The high cost of current protozoa detection methods limits their widespread use in limited settings.; Immunomagnetic separation improves detection by cleaning the sample.; Recovery efficiency is maintained for Giardia duodenalis with 50% less beads.; Organisms adhering to beads after dissociation may impact recovery levels.

Exploring challenges in Giardia cyst visualisation by common microscopy methods

Giardia spp. is an intestinal parasite responsible for worldwide disease outbreaks. Guiding researchers and practitioners to choose among current methods for microscopy detection of the infectious forms may be directly beneficial to public health and the environment. This study provides an overall comparison of brightfield (BF), fluorescence and darkfield (DF) microscopies for detecting Giardia duodenalis and Giardia muris cysts, by illustrating micrographs of such protocols applied to purified samples, as well as discussing advantages and constraints based on secondary information and collected data. BF analysis included Lugol's iodine staining. In fluorescence microscopy, samples were processed by immunofluorescence assay (IFA) with DAPI and by standalone DAPI dye. Cyst suspensions were also analysed by DF microscopy using a recently developed low-cost system. The three techniques enabled detecting Giardia spp. cysts, although they did not provide species identification by morphology. The overview of each method points out some relevant aspects to consider when selecting common optical microscopy techniques, and includes challenges and advantages regarding each of them. HIGHLIGHTS Comparisons of BF, DF, standalone DAPI and IFA-combined are provided.; BF and DF may be alternatives for low-cost detection of Giardia cysts.; Combinations of at least two diagnostic methods are recommended to minimise inherent errors.

Phylogenetic relationships of Toxocara spp. and Toxascaris sp. from different regions of the world

The nematodes of the genus Toxocara and Toxascaris are identified, traditionally, based on the morphologic aspects, however the genetic diversity among these parasites is recognized, being an accurate analysis of this variation necessary for the understanding of the biology of these organisms. This work aimed to establish the phylogenetic and phylogeographic relationships among specimens of Toxocara canis, Toxocara cati, Toxocara malaysiensis and Toxascaris leonina and to compare the results given by the morphologic and molecular techniques. In total, 436 helminths adults and four pools of eggs were collected from canids and felines from eight countries. The parasites were analyzed by stereoscopic microscopy, by Polymerase Chain Reaction (PCR) and by Restriction Fragment Length Polymorphism (PCR-RFLP). Primers TOXCOIF3 and TOXCOIR2 were designed in order to amplify 90pb of the mitochondrial gene Cox1, and the PCR-RFLP using the enzyme MseI could discriminate between these four species. The phylogenetic relationships among the isolates were observed in the phylogram and by the genetic variability (phylogenetic distance) rates. Of the 313 parasites adults, from dogs, 309 were identified, by microscopy, as T. canis and four as T. leonina. Of 123 originating from cats 118 were identified as T. cati, four as T. malaysiensis and one as T. leonina. Among the pools the three from lions were identified as T. leonina and that of dog as T. canis. PCR in all analyzed samples (n=440) failed in 3,6%. Of the amplified samples (n=424) 72 were selected to sequencing. With the exception of three samples, two from cats, were characterized as T. canis and one from dog as T. cati, all remaining were identified in agreement with their respective hosts. Of the 424 samples submitted to PCR-RFLP 351 were identified as T. canis/T.leonina, 70 as T. cati and three as T. malaysiensis. This technique presented similar results to sequencing. Homology and genotypic variation were observed within species, being the largest one 6.6%. Phylogeographic relationships within species could be observed by the phylogram. Among the techniques it was observed 9,7% of incongruence in the results between microscopy and sequencing, and 0,9% between microscopy and PCR-RFLP. Between the molecular techniques, it was considered only T. cati and T. malaysiensis which meant 100% of agreement. Based on the findings, of this study, it was concluded that some isolates of the same specie presented intragenotipic variability; Homologous sequences were identified in different countries, suggesting the occurrence of gene flow; The geographic origin was directed related to the genetic variation of the parasites; T. canis and T. cati presented variability rates similar to T. cati and T. leonina; And the three techniques used, herein, showed results that indicate a preference of T. canis, T. cati and T. malaysiensis for certain hosts.Tese (Doutorado)Os nematódeos dos gêneros Toxocara e Toxascaris são identificados, tradicionalmente, com base nos aspectos morfológicos, entretanto a diversidade genética entre esses parasitos é reconhecida, sendo a análise precisa dessa variação é necessária para o entendimento da biologia desses organismos. Esse trabalho teve como objetivos estabelecer relações filogenéticas e filogeográficas entre espécimes de Toxocara canis, Toxocara cati, Toxocara malaysiensis e Toxascaris leonina, e comparar os resultados das técnicas morfológica e moleculares. Foram coletados 436 helmintos adultos e quatro “pools” de ovos de canídeos e felídeos, procedentes de oito países. Os parasitos foram analisados por microscopia estereoscópica, pela Reação em Cadeia da Polimerase (PCR) e “Restriction Fragment Lenght Polymorfism” (PCR-RFLP). Os primers TOXCOIF3 e TOXCOIF2 foram desenhados para amplificar 490 pares de base do gene mitocrondrial Cox1 e a PCR-RFLP foi realizada utilizando-se a enzima MseI. As relações filogenéticas entre os isolados foram observados no filograma e pelo cálculo das taxas de variabilidade genotípicas (distância filogenética). Dos 313 parasitos adultos provenientes de cães, 309 foram identificados, por microscopia, como T.canis e quatro como T. leonina. Dos 123 oriundos de gatos, 118 foram identificados como T. cati, quatro T. malaysiensis e um T. leonina. Dentre os “pools” de ovos, os três de leoas foram identificados como T. leonina e o de cão como T. canis. A PCR em todas as amostras analisadas (n=440) apresentou falha de 3,6%. Das amostras amplificadas (n=424) 72 foram selecionadas para o sequenciamento. Com exceção de três amostras, duas provenientes de gato foram caracterizadas como T. canis, e uma proveniente de cão como T. cati, todas as restantes foram caracterizadas em concordância com os respectivos hospedeiros. Das 424 amostras submetidas a PCR-RFLP, 351 foram identificadas como T. canis/T.leonina, 70 como T. cati e três como T. malaysiensis. Essa técnica apresentou resultados semelhantes ao sequenciamento. Foram observadas homologia e variação genotípica nas espécies, sendo a maior 6,6%. Relações filogeográficas, dentro das espécies, puderam ser observadas pelo filograma. Entre as técnicas, observou-se 9,7% de incongruência de resultados entre microscopia e sequenciamento e 0,9% entre microscopia e PCR-RFLP. Entre as técnicas moleculares, considerou-se somente T. cati e T. malaysiensis, o que significou 100% de concordância entre resultados. A partir dos achados desse trabalho, conclui-se que alguns isolados, da mesma espécie, apresentaram variabilidade intragenotípica; Sequências homólogas foram identificadas em diferentes países, sugerindo a ocorrência de fluxo gênico; A procedência geográfica teve relação direta com a variação genética dos parasitos; T. canis e T. cati apresentaram taxa de variabilidade semelhante às de T. cati e T. leonina; E as três técnicas utilizadas apresentaram resultados que indicam a preferência de T. canis, T. cati e T. malaysiensis por determinados hospedeiros

Variabilidade genotípica dos isolados de Giardia duodenalis em diferentes espécies de animais

Giardia duodenalis is parasite of small intestine of several mammalian species, including humans, and it is found worldwide. Among domestics and wild animals this specie is prevalent regardless of race, sex, age and fitness, with significant clinical and economic importance. For a long time the parasite was considered specific for the host where it was found, but recently it was observed the existence of a relationship between assemblage of isolate and its host. Currently, G. duodenalis have been considered a complex, and molecular characterization of parasite isolates is critical for the knowledge about the transmission of giardiasis. Different results have been identified when various loci are sequenced, therefore, multilocus genotyping becomes the main ally in the trusted attribution of assemblages/subassemblages to the isolates. The aim of this study was to genetically characterize G.duodenalis cysts from dogs, cattle, pigs and lambs from kennels, pet shops, and farms from microregion of Uberlândia, using four markers. To determine the positivity of G. duodenalis cysts a flotation technique was employed. For genotyping, the genes glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), β-giardin (bg) and SSU-rRNA was used. Cysts of G.duodenalis were found in dogs, cattle, pigs and lambs and the infection rates were 21,66%, 18,75%, 5.5% e 24,8%, respectively. In association between positivity and sex, age, race and fitness, only age was significant regarding the infection. Fragments of gdh, tpi and bg was amplified in 33,3%, 36,8 and 7,2% of samples, respectively. PCR detection of SSU- rRNA failed to amplify and to sequence DNA samples. A total of 70 sequences were obtained, 34 (48,6%) from gdh, 25 (35,7%) from tpi and 11 (15,7%) from bg. The predominace of host-adapted assemblages was observed in all genes, and tpi was the only gene that classified isolates as zoonotic assemblage. Heterogeneous samples were found in gdh (pigs) and tpi (cattle and lambs). On multilocus genotyping (MLG) only three samples demonstrated concordance using the three genes, thus it was not possible to establish any relationship between assemblage and the variables sex, age, race fitness and fecal score.Mestre em Imunologia e Parasitologia AplicadasGiardia duodenalis é parasito do intestino delgado de várias espécies de mamíferos, incluindo o homem, tendo distribuição mundial. Entre os animais, domésticos e silvestres é prevalente, independente da raça, sexo, idade e aptidão, com importância clínica e econômica. Durante muito tempo, foi considerado específico para a espécie de hospedeiro onde era encontrado, porém recentemente descobriu-se a existência de relação de especificidade entre o grupo genotípico ou assemblage do parasito e seu hospedeiro. Baseado nisso, atualmente, a Giardia duodenalis é considerada um complexo, sendo a caracterização molecular, de isolados do parasito, de fundamental importância para o entendimento da transmissão da giardíase. Resultados inconsistentes têm sido identificados quando diferentes loci são sequenciados. Por isso a genotipagem multilocus torna-se a principal aliada na atribuição fidedigna das assemblages e subassemblages aos isolados. O objetivo desse trabalho foi caracterizar molecularmente os cistos de Giardia duodenalis, procedentes de cães, bovinos, ovinos e suínos provenientes de propriedades rurais, canis, pet shops e granjas da microrregião de Uberlândia, utilizando-se quatro marcadores. Para a determinação de positividade para cistos de G. duodenalis foi utilizada a técnica de Centrífugo-Flutuação em Sulfato de Zinco a 33%. Para a genotipagem dos isolados utilizou-se os genes glutamato dehidrogenase (gdh), triose fosfato isomerase (tpi), β-giardin (bg) e SSU-rRNA. Foram observados cistos de G. duodenalis em cães, bovinos, suínos e ovinos e as taxas de infecção foram 21,66%, 18,75%, 5.5% e 24, 8%, respectivamente. Na associação entre positividade e sexo, faixa etária, raça e aptidão a faixa etária foi a única variável que apresentou significância estatística em relação à infecção. Fragmentos do gene gdh foram amplificados em 33,3% das amostras, do tpi em 36,8% e do bg em 7,2%. A PCR falhou em amplificar e sequenciar amostras do gene alvo SSU-rRNA. Foram obtidas ao todo 70 sequencias, sendo 34 (48,6%) do gdh, 25 (35,7%) do tpi e 11 (15,7%) do bg. Observou-se predominância de assemblages espécie-específicas no sequenciamento dos três genes, sendo o tpi o único gene que classificou isolados como assemblage zoonótica; Foram observadas amostras heterogêneas, de suínos, no sequenciamento do gene gdh, e de bovinos e ovinos, no sequenciamento do tpi. Na análise da genotipagem multilocus (MLG), somente três isolados apresentaram concordância utilizando-se os três genes, não sendo possível estabelecer relação entre a assemblage e as variáveis sexo, faixa etária, raça, aptidão e escore fecal

Exploring challenges in Giardia cyst visualisation by common microscopy methods

Giardia spp. is an intestinal parasite responsible for worldwide disease outbreaks. Guiding researchers and practitioners to choose among current methods for microscopy detection of the infectious forms may be directly beneficial to public health and the environment. This study provides an overall comparison of brightfield (BF), fluorescence and darkfield (DF) microscopies for detecting Giardia duodenalis and Giardia muris cysts, by illustrating micrographs of such protocols applied to purified samples, as well as discussing advantages and constraints based on secondary information and collected data. BF analysis included Lugol's iodine staining. In fluorescence microscopy, samples were processed by immunofluorescence assay (IFA) with DAPI and by standalone DAPI dye. Cyst suspensions were also analysed by DF microscopy using a recently developed low-cost system. The three techniques enabled detecting Giardia spp. cysts, although they did not provide species identification by morphology. The overview of each method points out some relevant aspects to consider when selecting common optical microscopy techniques, and includes challenges and advantages regarding each of them. HIGHLIGHTS Comparisons of BF, DF, standalone DAPI and IFA-combined are provided.; BF and DF may be alternatives for low-cost detection of Giardia cysts.; Combinations of at least two diagnostic methods are recommended to minimise inherent errors.