A flow cytometric protocol for detection of Cryptosporidium spp.

Abstract

Cryptosporidium parvum is transmitted through water and can cause severe diarrhea. The diagnosis is usually based upon observer-dependent microscopic detection of oocysts, with rather low sensitivity and specificity. Our objective was to optimize a flow cytometric (FC) protocol for the detection of C. parvum. A specific monoclonal antibody conjugated with R-phycoerythrin was incubated with dead oocysts to determine the optimal antibody concentration. Serial concentrations of oocysts were stained with the optimized concentration and analyzed by FC. The lower detection limit was determined, and the possibility of cross-reaction was investigated using prokaryotic and eukaryotic microorganisms. A FC protocol was optimized to detect oocysts in spiked human stools. The optimal antibody concentration was found to be 3.0 mg/ml. The lowest number detectable was 2 × 10 3 oocysts/ml. Staining procedure was specific, as no cross-reactions were observed. This reliable and easy FC protocol allow the specific detection of Cryptosporidium oocysts, even at very low concentrations, which is important for public health and further studies of treatment efficacy. ' 2007 International Society for Analytical Cytology

An improved quantitative method to assess adhesive properties of Trichomonas vaginalis to host vaginal ectocervical cells using flow cytometry

“…Flow cytometry has commonly been used to quantify and characterise eukaryotic cells. In addition, some microbiologic applications have been described, for instance, for the detection and determination of viability of microbes (Barbosa et al, 2008;Pina-Vaz et al, 2004Pina-Vaz and Rodrigues, 2010). Although flow cytometry has proved to be advantageous when compared to conventional methods, its potential use for more complex applications in microbiology has been underestimated.…”

Applications of Flow Cytometry to Clinical Microbiology

“…The diagnosis is usually based upon observer-dependent microscopic detection of oocysts, with rather low sensitivity and specificity. Barbosa et al (2008) recently proposed a study with an objective to optimize a FCM protocol to detect Criptosporidium parvum oocysts in spiked human stools, using specific monoclonal antibodies. In particular, a specific monoclonal antibody conjugated with R-phycoerythrin was incubated with dead oocysts to determine the optimal antibody concentration, who was calculated in 3.0 mg/ml.…”

“…Comparison of fluorescence signal intensities of Cryptosporidium parvum oocysts analyzed at FL2 showing autofluorescence of 2x10 5 oocysts/ml and different concentrations of labeled oocysts with specific antibody (R-Phycoerythrin vs Counts) (Barbosa, 2008). Dixon et al (2005) involves an evaluation of the effectiveness of flow cytometry for the detection and enumeration of Cyclospora cayetanensis oocysts in human fecal specimens.…”

Detection of viable hookworm ova from wastewater and sludge

“…As a result, the method may significantly over-estimate the numbers of specific helminths ova in treated wastewater and sludge (Table 4.3). In addition, treated wastewater or sludge can have a high level of background debris which may provide a similar profile to helminths ova during detection/quantification and compromise the sensitivity of the method (Barbosa et al, 2008). (Bowman et al, 2003;Nocker et al, 2007b;McCarthy et al, 2012) Vital stain  Cheap and easy process  Assessment can be done in few hours  Less chemical and equipment  Can be done in small scale laboratory  Difficulty on viability assessment  Sensitivity depends on detection threshold of a microscope  Stain might not effective on recently inactivate ova  Lack specificity (Weber et al, 1991;Nelson and Darby, 2001;Cabaret et al, 2002;Victorica and Galvan, 2003;Verweij et al, 2007;McCarthy et al, 2012;Gyawali et al, 2016b) Flow cytometry  Quick and easy process  Higher sensitivity than microscopy  Automated process  Quantification can be done  Lack of specificity  Difficulty on viability assessment of ova  Difficulty distinguishing background debris from ova (Hammes et al, 2008;Barbosa et al, 2008;Wang et al, 2010) Molecular (PCR/qPCR)  Quick and easy process  Higher sensitivity and specificity  Viability could be possible  Automated process  a Multiple species can be identified from single sample  b Quantification is possible  Require advance laboratory and equipment  Genomic information is essential  Require right genomic target for viability  Possibility of providing false positive result by extracting DNA from inactivated ova  Possibility of false negative result via inhibitors present in the samples  a Multiple sets of primers require which can reduce the sensitivity  b Triplicate sample required (Pecson et al, 2006;Verweij et al, 2007;Traub et al, 2007;Traub et al, 2008;Janwan et al, 2011;Jonker et al, 2012, Ngui et al, 2012b a = Multiplex PCR, b =Quantitative PCR…”

“…In addition, hookworm ova recovered from raw wastewater and sludge can have a high level of foreign particles that are similar to the ova which makes quantification very difficult for trained staff ( Weber et al, 1991;Barbosa et al, 2008). The detection limit of both methods depends on detection sensitivity of a microscope which may not be satisfactory particularly of low numbers of hookworm ova are present in a sample (Weber et al, 1991).…”