8 research outputs found
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.
Household water purification system comprising cartridge filtration, UVC disinfection and chlorination to treat turbid raw water
Household slow sand filters in continuous and intermittent flows and their efficiency in microorganism’s removal from river water
Biological Layer in Household Slow Sand Filters: Characterization and Evaluation of the Impact on Systems Efficiency:Characterization and Evaluation of the Impact on Systems Efficiency
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.