Differences in staining intensities affect reported occurrences and concentrations ofGiardiaspp. in surface drinking water sources

Abstract

Aim USEPA Method 1623, or its equivalent, is currently used to monitor for protozoan contamination of surface drinking water sources worldwide. At least three approved staining kits used for detecting Cryptosporidium and Giardia are commercially available. This study focuses on understanding the differences among staining kits used for Method 1623. Methods and Results Merifluor and EasyStain labeling kits were used to monitor Cryptosporidium oocyst and Giardia cyst densities in New York City’s raw surface water sources. In the year following a change to the approved staining kits for use with Method 1623, an anomaly was noted in the occurrence of Giardia cysts in New York City’s raw surface water. Specifically, Merifluor-stained samples had higher Giardia cyst densities as compared with those stained with EasyStain. Side by side comparison revealed significantly lower fluorescence intensities of Giardia muris as compared with G. duodenalis cysts when labeled with EasyStain. Conclusions This study showed very poor fluorescence intensity signals by EasyStain on G. muris cysts results in lower cyst counts, while Merifluor, with its broader Giardia cyst staining specificity, results in higher cyst counts, when using Methods 1623. Significance and Impact of Study These results suggest that detected Giardia cyst concentrations are dependent on the staining kits used, which can result in a more or less conservative estimation of occurrences and densities of zoonotic Giardia cysts by detecting a broader range of Giardia species/Assemblages.

Ferric sulphate flocculation as a concentration method for Giardia and Cryptosporidium in filter backwash water

“…This suggests there are different efficiencies among microorganism's recovery from filter backwash water using ferric sulphate flocculation as a procedure for concentrating samples. Concerning this statistically significant difference found when pairing both target organisms, we assume that the lower G. muris RE may be explained by its poor fluorescence when observed by immunofluorescence using commercial kits for detection (Alderisio et al 2017). In other words, the low fluorescence intensity presented by this species may have affected RE, as some recovered cysts may not have been detected and accounted for.…”

Exploring challenges in Giardia cyst visualisation by common microscopy methods

“…Therefore, the monoclonal antibodies of the kit are likely to be specific to G. duodenalis antigens. This assumption could explain the difference in fluorescence intensity between the two species but could also be a limiting factor for the kit's use in environmental matrices, since there is no assurance which species might be present in the sample (Edelstein & Edelstein 1989;Alderisio et al 2017). From a public health perspective, this is particularly important, as differences in fluorescence staining may mislead reported cyst occurrences and concentrations in environmental samples (Alderisio et al 2017).…”

Métodos de recuperação e estimativa de viabilidade de cistos de >i<Giardia>/i< spp. e oocistos de >i<Cryptosporidium>/i< spp. em resíduos do tratamento de água de consumo

“…Pesquisas que trabalham com centrifugação buscam investigar os tempos requeridos, temperatura e gradiente, a exemplo dos testes realizados por Kourenti et al (2003) (ALDERISIO et al, 2017;JOHNSTON et al, 2003).…”

“…A análise dos custos permitiu constatar que a inclusão do teste confirmatório recomendado pelo Método 1623.1 (USEPA, 2012) utilizando o corante DAPI não altera a ordem de grandeza dos custos. Isto reforça sua utilização, principalmente, pois auxilia na indicação de organismos presentes na amostra que não tenham sido claramente visualizados sob fluorescência no filtro FITC, a exemplo do que ocorre com Giardia muris (ALDERISIO et al, 2017), o que, na ausência da análise com DAPI, subestimaria a ocorrência de Giardia spp., comprometendo os resultados de recuperação de cistos.…”

“…No caso da suspensão de Giardia muris, a visualização foi possível, mas dificultada em função da baixa fluorescência desta espécie sob o filtro FITC, conforme exibido na Figura 19 e reportado na literatura em comparação à Giardia duodenalis(ALDERISIO et al, 2017). A controversa fluorescência de G. muris foi um item a questionar ao tratar cistos com o Live/Dead Cell Assay, pois não era possível prever o comportamento das células supostamente "vivas" (a ficarem verdes sob FITC mediante ação do corante vital metabolizado por enzima intracelular) ou mesmo se o kit Merifluor® não iria interferir na análise de resultados.…”