Development and evaluation of a molecular based protocol for detection and quantification of Cryptosporidium spp. in wastewater

Mthethwa, Nonsikelelo Precios; Amoah, Isaac Dennis; Reddy, Poovendhree; Bux, Faizal; Kumari, Sheena

doi:10.1016/j.exppara.2022.108216

Cited by 8 publications

(11 citation statements)

References 68 publications

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“…The lower DNA yields from freeze-thaw pretreatments could be due to DNA degradation, as has been reported in other studies using freeze-thaw pretreatment for DNA extraction from Cryptosporidium oocysts and other protozoa and parasites (Babaei et al, 2011;Mthethwa et al, 2022). Additionally, a reduction in detection sensitivity has been noted as a drawback associated with freeze-thaw cycles.…”

Section: Comparison Of Dna Extraction Protocolsmentioning

confidence: 74%

“…To determine the most effective DNA extraction protocol for enhancing Cryptosporidium DNA recovery in wastewater, our study evaluated the DNA yields of eight different protocols which used combinations of two pretreatments (freeze-thaw and bead beating) and two commercial extraction kits (QIAamp DNA mini kit and DNeasy Powersoil Pro kit). Both pretreatments and commercial extraction kits have been previously utilizing in Cryptosporidium quantification from wastewater (Elwin et al, 2012; Lindergard et al, 2003; Mthethwa et al, 2022; Valeix et al, 2020). However, the specific effectiveness of these approaches in extracting Cryptosporidium DNA, both individually and in combination, had not been thoroughly explored.…”

Section: Resultsmentioning

confidence: 99%

“…Similarly, DNA extraction protocols can differ widely, especially in terms of different pretreatments to effectively disrupt the thick and robust Cryptosporidium oocyst wall along with commercial DNA extraction protocols. Pretreatments can either be mechanical such as beadbeating (Elwin et al, 2012;Valeix et al, 2020), freeze-thawing (Nichols et al, 2004;Lindergard et al, 2012), andsonication (Anceno et al, 2007;Adamska et al, 2011) (Amar et al, 2004;Kitajima et al, 2014;Mthethwa et al, 2022;Pomari et al, 2019;Wiedenmann et al, 1998;Yang et al, 2014). Finally, the target gene for the molecular detection methods can vary from 18S small subunit ribosomal RNA (18S rRNA), actin gene, 60-kDa glycoprotein gene (GP60), 70-kDa heat shock protein (HSP70) to Cryptosporidium outer wall protein (COWP) gene (Gobet & Toze, 2001;Mthethwa et al, 2022;Shin et al, 2018;Yang et al, 2014;Zahedi et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

“…Pretreatments can either be mechanical such as bead-beating ( Elwin et al, 2012; Valeix et al, 2020 ), freeze-thawing (Nichols et al, 2004; Lindergard et al, 2012), and sonication (Anceno et al, 2007; Adamska et al, 2011), or chemical using different lysis buffers (Paulos et al, 2016). Thus far, there have been limited comparison studies on how these different DNA extraction techniques affect Cryptosporidium gDNA recovery from wastewater samples (Mthethwa et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

“…Molecular detection methods can also vary widely from qualitative methods of endpoint polymerase chain reaction (PCR), restriction fragment length polymorphism, and nested PCR, to quantitative methods of quantitative PCR (qPCR), droplet digital PCR (ddPCR), and digital PCR (dPCR) (Amar et al, 2004; Kitajima et al, 2014; Mthethwa et al, 2022; Pomari et al, 2019; Wiedenmann et al, 1998; Yang et al, 2014). Finally, the target gene for the molecular detection methods can vary from 18S small subunit ribosomal RNA (18S rRNA), actin gene, 60-kDa glycoprotein gene (GP60), 70-kDa heat shock protein (HSP70) to Cryptosporidium outer wall protein (COWP) gene (Gobet & Toze, 2001; Mthethwa et al, 2022; Shin et al, 2018; Yang et al, 2014; Zahedi et al, 2018). These differences in sample concentration, DNA extraction, and molecular detection methods for the detection of Cryptosporidium in wastewater result in highly variable detection limits, specificity, and recovery, making it difficult to compare between studies (King et al, 2015; Kitajima et al, 2014; Mthethwa et al, 2022; Ramo et al, 2017; Ward et al, 2002; Yamashiro et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of Molecular-based Methods for the Detection and Quantification ofCryptosporidiumspp. in Wastewater

Hachimi,

Falender,

Davis

et al. 2024

Preprint

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Cryptosporidium, a eukaryotic protozoan parasite, poses a significant public health risk as a cause of waterborne disease worldwide. Clinical surveillance of cryptosporidiosis is estimated to be largely underreported due to the asymptomatic and mildly symptomatic infections, clinical misdiagnoses, and barriers to access testing. Unlike clinical surveillance, wastewater surveillance overcomes these limitations and could serve as an effective tool for identifying cryptosporidiosis at the population level. Despite its potential, the lack of standardized methods forCryptosporidiumspp. detection in wastewater challenges the comparability of studies. Additionally, the use of standard methods for the detection and quantification ofCryptosporidiumspp. in surface waters may not be appropriate for wastewater samples due to the more complex composition of the wastewater matrix. Thus, in this study, we evaluated methods for concentratingCryptosporidiumoocysts from wastewater, extracting DNA, and detecting its genetic markers, using wastewater seeded withC. parvumoocysts. The evaluated concentration methods included electronegative membrane filtration, Envirocheck HV capsule filtration, centrifugation, and Nanotrap Microbiome particles. All methods except the Nanotrap Microbiome particles were conducted with or without additional purification via immunomagnetic separation. For DNA extractions, we tested the DNeasy Powersoil Pro kit and the QIAamp DNA Mini kit, while assessing the impact of bead beating and freeze-thaw cycles on DNA yield. Genetic detection was performed using qPCR targeting theCryptosporidium18S rRNA gene. Our results indicated that centrifugation yielded the highest oocyst recoveries (39-77%), followed by the Nanotrap Microbiome particles (24%), electronegative filtration with a phosphate buffered saline with 20% Tween 80 elution (22%), and Envirocheck HV capsule filtration (13%). IMS was found to be unsuitable due to interference from the wastewater matrix, significantly reducing recovery rates to 0.03 to 4%. DNA yields were highest with bead-beating pretreatment with either the DNeasy Powersoil Pro kit (314 gc/μL DNA) or the QIAamp DNA Mini kit (238 gc/μL DNA). In contrast, freeze-thaw pretreatment reduced DNA yields to under 92 gc/μL DNA, likely through DNA degradation. This study is amongst the first to compare different concentration methods, including Nanotrap Microbiome particles, and DNA extraction methods that can be utilized forCryptosporidiumspp. wastewater surveillance, highlighting the importance of method selection for accurate detection and quantification.

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Section: Comparison Of Dna Extraction Protocolsmentioning

confidence: 74%