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  Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 38  |  Issue : 3  |  Page : 430-439
 

Two novel genomic DNA sequences as common diagnostic targets to detect Cryptosporidium hominis and Cryptosporidium parvum: Development of quantitative polymerase chain reaction assays, and clinical evaluation


1 Infection Biology Laboratory, School of Biotechnology, KIIT Deemed to be University, Bhubaneswar, Odisha; Department of Microbiology, Virus Research and Diagnostic Laboratory, Atal Bihari Vajpayee Government Medical College, Vidisha, Madhya Pradesh, India
2 Infection Biology Laboratory, School of Biotechnology, KIIT Deemed to be University; Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, Odisha, India
3 Infection Biology Laboratory, School of Biotechnology, KIIT Deemed to be University, Bhubaneswar, Odisha, India
4 Infection Biology Laboratory, School of Biotechnology, KIIT Deemed to be University, Bhubaneswar, Odisha, India; Department of Microbiology and Immunology, Medical University of the Americas (R3 Education Inc), MA, USA

Date of Submission18-Mar-2020
Date of Decision05-Jul-2020
Date of Acceptance07-Sep-2020
Date of Web Publication4-Nov-2020

Correspondence Address:
Dr. Priyadarshi Soumyaranjan Sahu
Department of Microbiology and Immunology, Medical University of the Americas (R3 Education Inc.), 27 Jackson Road # 300, Devens, MA 01434

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_20_114

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 ~ Abstract 


Introduction: Cryptosporidium is an intestinal parasite responsible for gastroenteritis. Conventional diagnosis of Cryptosporidium is made by microscopy. The most frequent molecular detection method for this parasite is polymerase chain reaction (PCR). The objective of the present study was to identify the novel DNA targets and development of PCR-based assays for the specific detection of two major human infecting species Cryptosporidium parvum and Cryptosporidium hominis. Methodology: Sensitive and specific SYBR green quantitative PCR (qPCR) and TaqMan qPCR assays were developed and validated at both diagnostic and analytical level using the new identified targets TU502HP-1 and TU502HP-2. Results: Assay validation results showed that the newly developed real-time PCR assays are 100% specific with a reliable limit of detection. Overall repeatability and reproducibility of these assays showed good quality results over intra- and inter-laboratory analysis. Conclusion: Novel target-based qPCR assays can be rapid an efficient tool for simultaneous detection of a C. parvum and C. hominis. These genes could also be utilized for the development of innovative DNA-based Point-of-Care test development.


Keywords: Cryptosporidium hononis, Cryptosporidium parvum, quantitative polymerase chain reaction, TU502-1, TU502-2


How to cite this article:
Shrivastava AK, Panda S, Kumar S, Sahu PS. Two novel genomic DNA sequences as common diagnostic targets to detect Cryptosporidium hominis and Cryptosporidium parvum: Development of quantitative polymerase chain reaction assays, and clinical evaluation. Indian J Med Microbiol 2020;38:430-9

How to cite this URL:
Shrivastava AK, Panda S, Kumar S, Sahu PS. Two novel genomic DNA sequences as common diagnostic targets to detect Cryptosporidium hominis and Cryptosporidium parvum: Development of quantitative polymerase chain reaction assays, and clinical evaluation. Indian J Med Microbiol [serial online] 2020 [cited 2020 Nov 28];38:430-9. Available from: https://www.ijmm.org/text.asp?2020/38/3/430/299810





 ~ Introduction Top


Cryptosporidiosis caused by the protozoan parasite Cryptosporidium spp is responsible for gastrointestinal illness frequently in children and immunocompromised individuals.[1] Recent report suggests that Cryptosporidium infection is the fifth-leading diarrheal etiology in Africa and Asia; infection in children younger than 5 years caused more than 48,000 deaths and 4·2 million disability-adjusted life-years lost, and still, there is no routine molecular diagnostic test to screen Cryptosporidium infection.[2]

There is an increased estimate of the global burden of this disease being reported round the year from many different regions, despite this knowledge, cryptosporidiosis is considerably under-recognised.[3],[4] In developed nations, in spite of improved living conditions and healthcare infrastructures, cryptosporidiosis cases thought to be under-reported where modern diagnostics are expected to be available. In the United States of America, approximately there are 748,000 cases of cryptosporidiosis per year, and an estimated cost of $45.8 million due to hospitalisations from cryptosporidiosis as stated elsewhere.[5] Cryptosporidium hominis and Cryptosporidium parvum are the two most common species reported predominately in human cryptosporidiosis cases.[6] Therefore, these two species pose a major challenge on community population suggesting appropriate and prompt clinical diagnosis and treatment management.

Conventional diagnosis of Cryptosporidium spp. is mainly made by the identification of oocysts by microscopic examination of fresh, unconcentrated stool smears staining.[7] In most of the reference laboratories from the developed countries, immunofluorescence microscopy is still used as a gold standard.[5],[8] While parasite specific antigen detection protocols, such as enzyme immune assay and immunochromatographic tests (lateral flow based), are also commercially available, which show diagnostic sensitivities ranging from 70% to 100%.[5],[9] Most of the Cryptosporidium detection and diagnostic assays allow reporting presence of Cryptosporidium oocysts; however, polymerase chain reaction (PCR) assays could identify the infecting species and also quantify the load of oocysts.

There might be possibility of one-time detection of C. parvum and C. hominis, however, those are not available for routine use. The current study highlights the validation of new PCR methods amplifying two novel DNA targets which are common to C. parvum and C. hominis for their specific detection as well as quantification.


 ~ Materials and Methods Top


Target selection/search for unique sequences

C. hominis and C. parvum are the two major causes of human cryptosporidiosis; therefore, we targeted those sequences, which were present in both C. hominis and C. parvum. Sequences common to both C. hominis and C. parvum were searched from 'CryptoDB'.[10] To find out unique genetic sequences for proposed new diagnostic development, BLAST analysis was done for C. parvum and C. hominis whole genome sequences considering expect threshold value 0.05. Further extensive literature-based confirmation was made to verify the novelty of the identified unique sequences as stated in the results section.

Source of Cryptosporidium oocysts and standard DNA

Viable whole C. parvum oocysts were commercially procured from Waterborne Inc (New Orleans, LA, USA). Extracted DNA of C. hominis, C. andersoni and C. felis parasites was obtained from the Christian Medical College, Vellore (India) upon request. All these DNA samples were preserved at −20°C till use for all PCR assays during standardisation and validation studies.

Stool specimens from patients for clinical validation

Stool specimens from patients with severe acute diarrhoea were obtained from our study repository from as these samples were collected for epidemiological and clinical studies. Samples were found to be positive for oocysts of Cryptosporidium by immunomagnetic separation (IMS) followed by conventional PCR detecting positive amplification for 18s SSU were considered as the positive controls where the diagnosis was confirmed at genus level only. Samples negative for IMS and 18s SSU PCR test was considered as the negative controls. These negative control participants were subsequently diagnosed to be infected with diarrhoeal pathogens other than Cryptosporidium spp. Genomic DNA extracted from the stool samples from these controls were used subsequently for the clinical validation of the newly developed assays.

Extraction of DNA

DNA was extracted from C. parvum oocysts employing commercially procured DNA extraction kit (Qiagen, Venlo, the Netherlands). Total 200 μl Cryptosporidium oocysts suspension was taken and 180 μl of ATL buffer was added followed by four freeze/thaw cycles (freezing in liquid N2 for 30 s and thawing at 95°C for 3 min). After freeze thaw cycles overnight protein kinase K digestion was carried out at 56°C and followed by incubated in lysis buffer for 10 min at 70°C. DNA was purified on silica gel columns employing a Wizard SV Gel/PCR Clean-Up Kit (Promega, Madison, WI).

Control plasmid DNA for assay standardization

Control DNA was prepared by cloning of the target Cryptosporidium sequences (TU502HP-1 and TU502HP-2 genes) in a plasmid employing a TOPO TA cloning kit according to manufacturer's protocol (Invitrogen, USA). These cloned plasmids were sequenced commercially from First BASE Laboratories, (Kuala Lumpur, Malaysia). BLAST analysis of these sequences was carried out to confirm the sequence similarity with the target sequence. In quantitative PCR (qPCR) standard curve generation, these cloned plasmids were used as control DNA.

Designing primers and probes

The primers and probes targeting TU502HP-1 and two genes were designed using IDT Sci Tools.[11] [Table 1] presents the list of primers and probes used in this study. Designed primers as well as TaqMan probes were synthesised commercially (Integrated DNA Technologies, Coralville, IA, U. S.).
Table 1: List of primers used for novel diagnostic development

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Real-time polymerase chain reaction conditions

qPCR development and optimization were attain using CFX Connect™ Real-Time PCR detection system (BIO-RAD). All SYBR Green PCR reactions were carried out in a final volume reaction mixture of 20 μl comprising QuantiTect SYBR Green PCR (Qiagen) and the appropriate concentrations of primers and DNA template. The best primer concentration and appropriate melting temperature was optimized using varying rage of primer concentrations and gradient PCR, respectively. For TU502HP-1 and TU502HP-2 simplex reactions, 200 nM of each primer was used, while in multiplex reaction, 150 nM primers concentration was used.

Reaction conditions for all qPCR assays comprises: Initial denaturation at 95°C for 2 min, then 40 consecutive cycles of first 10 s at 95°C and then 30 s at 57°C. Melting curve was analysed as per the default settings of the machine CFX Connect Real-Time PCR detection system (BIO-RAD).

TaqMan assay optimisation was done using CFX Connect Real-Time PCR detection system (BIO-RAD). Each TaqMan qPCR was done in a final volume of 20 μl of the reaction mixture containing SsoFast Probes Supermix (BIO-RAD), and 100nM primers, 50 nm probes along with the DNA template (appropriate concentration). The amplification reaction consisted of activation of the Taq DNA polymerase at 94°C (for 2 min), followed by 40 consecutive cycles of reactions at 95°C (10 s), 60.2°C (30 s) and 72°C (10 s), with thermal transitions between denaturation and primer annealing of 1.2°C/s, to enable probe hybridisation.

Evaluation of analytical sensitivity

The analytical sensitivity or limit of detection (LOD) being the lowest amount of the analyte detectable in a single reaction was calculated by performing the conventional PCR as well as the real-time PCR reactions employing a series of 10-fold dilutions of the template DNA. Well-characterised positive DNA control plasmids containing TU502HP-1 and TU502HP-2 were used with a dynamic log-diluted range 101–10-8.

A standard curve for each of the real-time qPCR assays was generated with decreasing dilution of DNA concentrations ranging from 101 to 10−5 ng/μl. PCR reaction efficiency (E) was calculated as per a 5-log dilution series initiating from 10.

Evaluation of analytical specificity

Initially, specificity of all PCR assays was evaluated in silico. The primers, probes and amplicon sequences were subjected to BLAST analysis to test for cross-reactions with non-targeted sequences. Specificity of each primer sets was further assessed by using target-containing and target-lacking strains for PCR amplification reaction. Subsequently, the estimation of assay specificity was also done with template DNAs from four Cryptosporidium species C. andersoni, C. felis. C. parvum and C. hominis.

Assay validation

Validation was performed to determine the sensitivity and specificity of the quantitative real-time PCR assays targeting TU502HP-1 and 2 genes. This was done testing against 15 confirmed C. parvum/C. hominis isolates and 15 isolates other than C. parvum/C. hominis.

Clinical validation

To validate the sensitivity and specificity (diagnostic) of the newly developed real-time PCR assays, DNA extracted from the stool samples of positive and negative controls were employed as the templates.

Evaluation of diagnostic reproducibility

Diagnostic reproducibility and repeatability of qPCR assays were assessed by inter-laboratory qPCR assessment. The primers, probes and template DNA controls were sent to the collaborating laboratories to perform TU502HP-1 and TU502HP-2 specific SYBR green and TaqMan assays. The participating laboratory performed all of the tests with no bias.

TU502HP-1 and TU502HP-2 specific diagnostic assays were checked with three different sets of primers targeting TU502HP-1 and TH502HP-2 genes. Three different PCR machines (BIO-RAD, Effendorf and Himedia) and various molecular reagents specifically PCR mater mix (BIORAD, Qiagen, Promega, NEB and TOYOBO) in different laboratory set ups were used for internal validation (School of Biotechnology in India) and external validation (International Medical University Kuala Lumpur Malaysia).


 ~ Results Top


Target identification

The CryptoDB database search reported a total of 105 genes that were unique to either C. parvum or C. hominis. BLAST analysis showed two unique sequences (CryptoDB Gene ID-Chro. 20272 and Chro. 20279) were restricted in common to C. parvum and C. hominis, which were therefore chosen for the further analysis and assay development. These two genes were named as TU502HP-1 and TU502HP-2.

In case of C. hominis, the query coverage and sequence identity for both TU502HP-1 and TU502HP-2 were 100% on BLAST analysis. Whereas, in case of C. parvum, the query coverage for TU502HP-1 and TU502HP-2 were 99% and 100%, respectively; similarly, the sequence identity for TU502HP-1 and TU502HP-2 was 95% and 97%, respectively, [Supplementary Figure 1].



Real-time polymerase chain reaction

Optimization results for SYBR green quantitative polymerase chain reaction

Optimal concentrations of the primers for SYBR Green real-time PCR assays were verified by using the varying range of primer concentration starting from 50 to 500 nM. The optimal concentrations of the primers for amplifying each target were confirmed based on obtaining an amplicon of expected band size when resolved on agarose gel. In assays for both targets a tendency of primer dimers formation was noticed, particularly when high primer concentrations were used. Therefore, most appropriate concentration of primers was selected to be 200 μM.

Setting up of gradient qPCR also optimised amplification conditions for each of the target under a range of melting temperatures with an appropriate positive control DNA as a template.

Optimization results for TaqMan quantitative polymerase chain reaction

All qPCR assays for both TU502HP-1 and TU502HP-2 were standardised at the varying concentrations of primers (ranging from 100nM to 500nM) and probes (ranging from 50nM to 250 nM). The optimal primer concentration for both SYBR green assays was 200nM while for TaqMan assay optimum primers concentration was 100nM. Optimal probe concentration for either target was standardised to be 50nM in final reaction mixture in all TaqMan qPCR reactions.

Limits of detection (LOD) of SYBR green and TaqMan quantitative polymerase chain reaction assays

LOD of the SYBR qPCR assay for both TU502HP-1 and TU502HP-2 were up to 4 × 10−5 ng/μl concentration of the template DNA. LOD of the SYBR qPCR assay in terms of gene copy numbers were calculated to be 1.68 × 104 for both TU502HP-1 and TU502HP-2.

Similarly, LOD of the TaqMan qPCR assay for both TU502HP-1 and TU502HP-2 were up to 2 × 10−5 ng/μl concentration of the template DNA. LOD of the TaqMan assay in terms of gene copy numbers were calculated to be 8.2 × 103 for both TU502HP-1 and TU502HP-2.

Evaluation of analytical sensitivity and specificity of quantitative polymerase chain reaction assays

Detailed in silico analysis of primers showed no sequences similarities with non-target DNA sequences. The in vitro tests confirmed that the SYBR and TaqMan assays for both TU502HP-1 and TU502HP-2 could recognise C. parvum and C. hominis only, and no other species of Cryptosporidium found positive which were tested in the present study [Table 2]. No false-negative or false-positive results were observed [Table 3]. In this study, the Tm values of the reactions amplifying respective targets were satisfactory, and in accordance with the values as obtained with reference plasmid and genomic DNAs. The assay was observed to be 100% sensitive and specific.
Table 2: Specificity of TU502HP-1 and TU502HP-2 assays using 4 different species of Cryptosporidium

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Table 3: Specificity of TU502HP-1 and TU502HP-2 assays using known positive and negative controls

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To evaluate the precision of the PCR amplicons in both SYBR green and TaqMan assays melting temperature (Tm) analysis was done. TU502HP-1 and TU502HP-2 specific SYBR green PCR results are shown in [Figure 1]a, [Figure 1]b, [Figure 1]c, and [Figure 2]a, [Figure 2]b, [Figure 2]c.
Figure 1: TU502HP-1 SYBR green assay: Amplification plot, melting peak, melting curve and standard curve using X-147 primer set. Fluorescence intensity as a function of the concentration of the template. For each assay, a series of 10-fold dilutions of cloned DNA was used as the template for polymerase chain reaction (1–7, sample dilutions; 8, no-template control). (a) Amplification plot for TU502HP-1 SYBR green assay. (b) Melting peak analysis (1–7, sample dilutions; 8, no-template control). The negative first derivative of the relative fluorescence units (–d[RFU]/dT) is plotted as a function of the temperature. The high peak indicates the amplified product; the low peak is for no-template control. (c) Melting curve analysis (1–7, sample dilutions; 8, no-template control). (c) Amplification plot (1–7, sample dilutions; 8, no-template control). (d) Standard curve derived from the amplification plot

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Figure 2: TU502HP-2 SYBR green assay: Amplification plot, melting peak, melting curve and standard curve using Y-119 primer set. Fluorescence intensity as a function of the concentration of the template. For each assay, a series of 10-fold dilutions of cloned DNA was used as the template for polymerase chain reaction (1–7, sample dilutions; 8, no-template control). (a) Amplification plot for TU502HP-2 SYBR green assay. (b) Melting peak analysis (1–7, sample dilutions; 8, no-template control). The negative first derivative of the relative fluorescence units (–d[RFU]/dT) is plotted as a function of the temperature. The high peak indicates the amplified product; the low peak is for no-template control. (c) Melting curve analysis (1–7, sample dilutions; 8, no-template control). (c) Amplification plot (1–7, sample dilutions; 8, no-template control). (d) Standard curve derived from the amplification plot

Click here to view


Single target-containing TA cloned plasmids were synthesised; these plasmid clones were used as reference DNA template for each of the targeted amplicon. Findings were compared with the results as obtained from genomic DNA from the Cryptosporidium spp. reference strains. Each amplification showed distinct peaks at a specific melting temperature. No significant difference was observed in the melting temperatures when positive control samples as well as unknown samples were tested. The amplified products of each assay were subjected to agarose gel electrophoresis and expected amplicon size on gel further confirmed the accuracy of tested qPCR assays [Figure 3]. Finally, sequencing of PCR amplified products verified the specificity of each of the qPCR assays.
Figure 3: SYBR quantitative polymerase chain reaction products gel image (a) TU502HP-1 specific product using X1-RT-147 primers (b) TU502HP-2 specific product using Y1-RT-119 primers. Lane 1-100 bp ladder. Lane 2- Template concentration 2 ng/μl. Lane 3 - Template concentration 2 × 10−1 ng/μl. Lane 4 - Template concentration 2 × 10−2 ng/μl. Lane 5- Template concentration 2 × 10−3 ng/μl. Lane 6 - Template concentration 2 × 10−4 ng/μl. Lane 7 - Negative Control

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Melt peak and melt curve for TU502HP-1 and TU502HP-2 specific TaqMan assays are shown in [Figure 4]a, [Figure 4]b, [Figure 4]c, and [Figure 5]a, [Figure 5]b, [Figure 5]c. Similar to SYBR green assay, the results were analysed and compared with those obtained from reference strain genomic DNAs. There were discrete peaks with a specific Tm value for each target when compared in case of each of the different protocols. Our experiments using all of these platforms revealed no significantly different Tm values when control and unknown samples were analysed. The amplified products of each assay were subjected to agarose gel electrophoresis expected amplicon size on gel further confirmed the accuracy of tested qPCR assays [Figure 6].
Figure 4: TU502HP-1 TaqMan assay: Amplification plot, melting peak, melting curve and standard curve using TqM-502-1 primers and probe. Fluorescence intensity as a function of the concentration of the template. For each assay, a series of 10-fold dilutions of cloned DNA was used as the template for polymerase chain reaction (1–7, sample dilutions; 8, no-template control). (a) Amplification plot for TU502HP-1TaqMan assay. (b) Melting peak analysis (1–6, sample dilutions; 7, no-template control). The negative first derivative of the relative fluorescence units (–d[RFU]/dT) is plotted as a function of the temperature. The high peak indicates the amplified product; the low peak is for no- template control. (c) Melting curve analysis (1–7, sample dilutions; 8, no-template control). (c) Amplification plot (1–7, sample dilutions; 8, no-template control). (d) Standard curve derived from the amplification plot

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Figure 5: TU502HP-2 TaqMan assay: Amplification plot, melting peak, melting curve and standard curve using TqM-502-2 primers and probe. Fluorescence intensity as a function of the concentration of the template. For each assay, a series of 10-fold dilutions of cloned DNA was used as the template for polymerase chain reaction (1–7, sample dilutions; 8, no-template control). (a) Amplification plot for TU502HP-2TaqMan assay. (b) Melting peak analysis (1–6, sample dilutions; 7, no-template control). The negative first derivative of the relative fluorescence units (–d[RFU]/dT) is plotted as a function of the temperature. The high peak indicates the amplified product; the low peak is for no- template control. (c) Melting curve analysis (1–7, sample dilutions; 8, no-template control). (c) Amplification plot (1–7, sample dilutions; 8, no-template control). (d) Standard curve derived from the amplification plot

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Figure 6: TaqMan quantitative polymerase chain reaction products gel image (a) TU502HP-1 specific product (b) TU502HP-2 specific product. Lane 1-100 bp ladder. Lane 2 - Template concentration 2 ng/μl. Lane 2 - Template concentration 2 × 10−1 ng/μl. Lane 3 - Template concentration 2 × 10-2ng/μl. Lane 4 - Template concentration 2 × 10−3 ng/μl. Lane 5 - Template concentration 2 × 10−4 ng/μl. Lane 6 - Template concentration 2 × 10−5 ng/μl. Lane 6 - Negative Control

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For TU502HP-1 and TU502HP-2 SYBR green assay, standard curves were generated according to the serial dilutions of control DNA template, as illustrated in [Figure 1]d and [Figure 2]d. When Cryptosporidium DNA was not added as a template into the PCR mixture, then no amplification was detected. For each assay, a linear relationship was observed over the dynamic range (1 ng/μl DNA to 10−4–10−5 ng/μl DNA per reaction) using control DNA. As indicated in [Figure 1]d, TU502HP-1-specific SYBR green assay showed slope values 3.552, square correlation coefficients (R2) 0.998 and efficiencies was 91.2% and TU502HP-2 specific SYBR green assay showed slope values 3.377, square correlation coefficients 0.998 and efficiencies was 97.7%, as shown in [Figure 2]d.

TU502HP-1 specific TaqMan assay showed slope values 3.456, square correlation coefficients (R2) 0.999 and efficiencies was 94.7% and TU502HP-2 specific TaqMan assay showed slope values 3.392 square correlation coefficients 0.998 and efficiencies was 97.2%, as shown in [Figure 4]d and [Figure 5]d.

Multiplex TaqMan qPCR assay by using TqM 502 1 and TqM-502-2 primers and probe demonstrated simultaneous detection of TU502HP-1 and TU502HP-2 targets in a single run [Figure 7]. The optimal primer and probe concentration for final reaction mixture was found 100nM and 50 nM respectively. Multiplex TaqMan qPCR assay showed sensitive and specific amplification with slope values of -3.86 for TU502HP-1 and -3.55 for TU502HP-2; efficiencies of 81.4% for TU502HP-1 and 91% for TU502HP-1; square correlation coefficients (R2) of 0.99 for both TU502HP-1and TU502HP-2.
Figure 7: Multiplex TaqMan assay: (a) Amplification plot, (b) Melting peak, (C) Melting curve and (d) Standard curve using TqM 502 1 and TqM-502-2 primers and probe. Fluorescence intensity as a function of the concentration of the template. A series of 10 fold dilutions of cloned DNA was used as the template for polymerase chain reaction (1–4, sample dilutions; 5, no template control)

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Repeatability and reproducibility

The repeatability and reproducibility of novel diagnostic assays are shown in [Table 4]. All these assays performed well in different laboratory set ups using different PCR reagents and machines. The comparisons of the limits of detection for conventional and real-time PCR assays for both genetic targets are also depicted in [Table 4].
Table 4: Repeatability and Reproducibility of newly developed diagnostic assays

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 ~ Discussion Top


Cryptosporidiosis is such an important disease, which can be present concurrently with other diarrheal pathogens.[12] Sensitive and specific diagnostic development is always on a priority to diagnose the underlying etiology of each disease for which a rapid diagnosis is essential for prompt and appropriate treatment management. Diagnostic tests for Cryptosporidium infection are suboptimum, novel stool diagnostics, serodiagnostics and biomarkers are need to be developed for more accurate identification of active cryptosporidiosis.[13]

Previously, PCR-based methods have been found to have higher sensitivity, to have greater specificity, and to be faster than conventional culture-based or any other method used to identify infectious causes of gastroenteritis including Cryptosporidiosis.[9],[14],[15],[16] PCR-based tests provide highly reliable results compared to stool-smear microscopy because environmental factors can interfere with the results of direct stool examination.[17] Thus, PCR-based tests for identifying the species or strains of any pathogen might be very helpful when considering all the difference possible pathogens, their epidemiology and their differing clinical manifestations. Information on the distribution pattern of different species in different regions, seasons, populations and environmental conditions would also be useful.[18] The use of real-time PCR, together with microscopy and immunochromatography techniques, would result in a more precise diagnosis of cryptosporidiosis.[19] Therefore, this study used PCR-based techniques to develop reliable new diagnostic tools for detecting C. parvum and C. hominis.

Real-time qPCR is a simple, fast and automatised amplification system with a decreased risk of cross-contamination compared to conventional PCR.[20],[21] Currently, qPCR allows original target quantification by using fluorescently labelled agents (e.g. SYBR Green, TaqMan probes, Förster resonance energy transfer and Scorpion primers.[22] The present study showed successful development of SYBR Green and TaqMan assays targeting TU502HP-1 and TU502HP-2 genes.

Our study is the first among all in the recent time where previously unexplored two genes (TU502HP-1 and TU502HP-2) are targeted for the detection of C. parvum and C. hominis together; new methods are developed and subsequently validated clinically employing latest techniques with high sensitivity, specificity and reproducibility of the tests that is discussed in the following section.

Real-time PCR has been known to be advantageous over conventional PCR assays. A number of samples can be analysed simultaneously by the real-time PCR assay even with low concentration roughly about 6 logs units of the template DNA, and also there is no post handling process needed for reading results.[19] These methods are easy to operate and less turnaround time from receipt of samples to results. In the present study, initially, qPCR assay was done using SYBR green for both the target genes, and subsequently, TaqMan qPCR was employed. The TU502HP-1 and TU502HP-2 gene-based TaqMan real-time PCR assays developed in the present study were found to be much faster than conventional PCR and microscopy-based methods for the identification of Cryptosporidium spp. The TaqMan assay results could be obtained within 2 h with much greater sensitivity and higher specificity in detecting C. parvum and C. hominis even in the clinical specimens.

We found TaqMan assays were found to be more sensitive in comparison to SYBR Green assays and conventional PCR assays with lowest detection limit up to 2 × 10-5 ng/ul of template DNA. Upon optimisation, TaqMan qPCR assays showed a LOD 8.2 × 103 copies of target per reaction, which is comparatively lower than the LOD from previously reported C. parvum specific qPCR assays.[23]

The ability of primers to hybridise to one another (especially at the 3'-end) might lead to primer extension during PCR and the formation of target-independent products; the primer dimer formation reduces the reaction efficiency and sensitivity. Validation of primer design is important, each of the primer and probes were designed considering all the necessary parameters, which are required.[24] We have used IDT primer quest software for sensitive and specific designing of primer. All the targeted sequences of TU502HP-1 gene and TU502HP-2 gene were also subjected for BLAST analysis to their presence only in C. hominis and C. parvum.

Source of the standard template DNA during assay validation of a newly developed PCR tool might play a role in the standardization outcome in general. Usually, the standard curve is generated by amplifying a range of serially diluted concentrations of the standard DNA that could be either a plasmid carrying the DNA target, a genomic DNA, a PCR amplicon, a cDNA, or even a synthesised oligonucleotide. Out of the above different types, especially plasmid DNA, (the uncut circular one) is the most common choice because of its high reproducibility and stability. In the case of plasmid, one can calculate the exact number of molecules in each dilution, hence could be useful for an accurate quantification of the amount of DNA molecules in terms of gene copy numbers that one need to measure during the assay development. In the present study, plasmid carrying the target DNA (TA cloned TU502HP-1 and TU502HP-2) was the choice for all the PCR standardisation protocol.

The TU502HP-1 and 2 specific SYBR green and TaqMan assays did not amplify DNA from Cryptosporidium species other than C. parvum and C. hominis. We did not aim to discriminate between C. parvum and C. hominis because other quantitative real-time PCR protocols have been developed for the same purpose.[25],[26] These newly identified target based diagnostic assays were further validated by using different laboratory settings (Infection Biology Lab and Immunology Lab, from School of Biotechnology, Bhubaneswar, India; Molecular Biology Lab from International Medical University, Kuala Lumpur, Malaysia) with different sets of machines (BIO-RAD, Eppendorf and Himedia) and reagents (BIORAD, Promega, Qiagene, Toyobo). All the conventional and real-time PCR assays worked well with different settings of laboratory using different machines and reagents. These targets and newly developed assays can be useful for tracking anthroponotic transmission in close group populations and outbreak disease surveillance of cryptosporidiosis.

The present study findings could also provide an opportunity for gene cloning and protein expression to investigate their serodiagnostic value for the development of low-cost antibody-based diagnostic tests for cryptosporidiosis; which was beyond the scope of the present study.


 ~ Conclusion Top


In the present study, two novel gene targets (TU502HP-1 and TU502HP-2) common to C. homins and C. parvum were identified. Based on these novel target sequences, SYBR green and TaqMan qPCR assays were developed and validated for the detection of both species, which are often associated with human cryptosporidiosis. The assay quality parameters and their clinical validation results sounds promising to be employed in large scale clinical as well as epidemiological studies on C. hominis and C. parvum in future. More interestingly, developing new assay like ICT-TU502HP-1 could be considered as a preliminary approach to innovate highly sensitive and rapid point of care diagnostics for the specific detection of these tested targets.

Acknowledgement

This study is a part of student's PhD works institute provided partial support for this study. The authors duly acknowledge Prof. Mrutyunjay Suar, (Director, School of Biotechnology, KIIT Deemed to be University), for the institutional support. Authors are also thankful to Dr. Gopal Krishna Purohit for providing guidance on qPCR methods.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

1.
Cavalier-Smith T. Gregarine site-heterogeneous 18S rDNA trees, revision of gregarine higher classification, and the evolutionary diversification of Sporozoa. Eur J Protistol 2014;50:472-95.  Back to cited text no. 1
    
2.
Khalil IA, Troeger C, Rao PC, Blacker BF, Brown A, Brewer TG, et al. Morbidity, mortality, and long-term consequences associated with diarrhoea from Cryptosporidium infection in children younger than 5 years: A meta-analyses study. Lancet Glob Health 2018;6:e758-e768.  Back to cited text no. 2
    
3.
Mahmoudi MR, Ongerth JE, Karanis P. Cryptosporidium and cryptosporidiosis: The Asian perspective. Int J Hyg Environ Health 2017;220:1098-109.  Back to cited text no. 3
    
4.
Pisarski K. The global burden of disease of zoonotic parasitic diseases: Top 5 contenders for priority consideration. Trop Med Infect Dis 2019;4:44.  Back to cited text no. 4
    
5.
Chalmers RM, Campbell BM, Crouch N, Charlett A, Davies AP. Comparison of diagnostic sensitivity and specificity of seven Cryptosporidium assays used in the UK. J Med Microbiol 2011;60:1598-604.  Back to cited text no. 5
    
6.
Khan A, Shaik JS, Grigg ME. Genomics and molecular epidemiology of Cryptosporidium species. Acta Trop 2018;184:1-4.  Back to cited text no. 6
    
7.
Vohra P, Sharma M, Chaudary UA. Comprehensive review of diagnostic techniques for detection of Cryptosporidium parvum in stool samples. J Pharm 2012;2:15-26.  Back to cited text no. 7
    
8.
Van den Bossche D, Cnops L, Verschueren J, van Esbroeck M. Comparison of four rapid diagnostic tests, ELISA, microscopy and PCR for the detection of Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in feces. J Microbiol Methods 2015;110:78-84.  Back to cited text no. 8
    
9.
Youn S, Kabir M, Haque R, Petri WA Jr. Evaluation of a screening test for detection of giardia and Cryptosporidium parasites. J Clin Microbiol 2009;47:451-2.  Back to cited text no. 9
    
10.
Heiges M, Wang H, Robinson E, Aurrecoechea C, Gao X, Kaluskar N, et al. CryptoDB: A Cryptosporidium bioinformatics resource update. Nucleic Acids Res 2006;34:D419-22.  Back to cited text no. 10
    
11.
Owczarzy R, Tataurov AV, Wu Y, Manthey JA, McQuisten KA, Almabrazi HG, et al. IDT SciTools: A suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 2008;36:W163-9.  Back to cited text no. 11
    
12.
Shrivastava AK, Kumar S, Mohakud NK, Suar M, Sahu PS. Multiple etiologies of infectious diarrhea and concurrent infections in a pediatric outpatient-based screening study in Odisha, India. Gut Pathog 2017;9:16.  Back to cited text no. 12
    
13.
Checkley W, White AC Jr, Jaganath D, Arrowood MJ, Chalmers RM, Chen XM, et al. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for Cryptosporidium. Lancet Infect Dis 2015;15:85-94.  Back to cited text no. 13
    
14.
Bruijnesteijn van Coppenraet LE, Wallinga JA, Ruijs GJ, Bruins MJ, Verweij JJ. Parasitological diagnosis combining an internally controlled real-time PCR assay for the detection of four protozoa in stool samples with a testing algorithm for microscopy. Clin Microbiol Infect 2009;15:869-74.  Back to cited text no. 14
    
15.
Stark D, Al-Qassab SE, Barratt JL, Stanley K, Roberts T, Marriott D, et al. Evaluation of multiplex tandem real-time PCR for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical stool samples. J Clin Microbiol 2011;49:257-62.  Back to cited text no. 15
    
16.
Johnson AM, Giovanni GD, Rochelle PA. Comparison of assays for sensitive and reproducible detection of cell culture-infectious Cryptosporidium parvum and Cryptosporidium hominis in drinking water. Appl Environ Microbiol 2012;78:156-62.  Back to cited text no. 16
    
17.
Schuurman T, Lankamp P, van Belkum A, Kooistra-Smid M, Van Zwet A. Comparison of microscopy, real-time PCR and a rapid immunoassay for the detection of Giardia lamblia in human stool specimens. Clin Microbiol Infec 2007;13:1186-91.  Back to cited text no. 17
    
18.
Xiao L. Molecular epidemiology of cryptosporidiosis: An update. Exp Parasitol 2010;124:80-9.  Back to cited text no. 18
    
19.
Cunha FS, Peralta RHS, Peralta JM. New insights into the detection and molecular characterization of Cryptosporidium with emphasis in Brazilian studies: A review. Rev Inst Med Trop Sao Paulo 2019;61:e28.  Back to cited text no. 19
    
20.
Farcas GA, Soeller R, Zhong K, Zahirieh A, Kain KC. Real-time polymerase chain reaction assay for the rapid detection and characterization of chloroquine-resistant Plasmodium falciparum malaria in returned travelers. Clin Infect Dis 2006;42:622-7.  Back to cited text no. 20
    
21.
Shokoples SE, Ndao M, Kowalewska-Grochowska K, Yanow SK. Multiplexed real-time PCR assay for discrimination of Plasmodium species with improved sensitivity for mixed infections. J Clin Microbiol 2009;47:975-80.  Back to cited text no. 21
    
22.
Ndao M. Diagnosis of parasitic diseases: Old and new approaches. Interdiscip Perspect Infect Dis 2009;2009:278246.  Back to cited text no. 22
    
23.
Fontaine M, Guillot E. Development of a TaqMan quantitative PCR assay specific for Cryptosporidium parvum. FEMS Microbiol Lett 2002;214:13-7.  Back to cited text no. 23
    
24.
Thornton B, Basu C. Real-time PCR (qPCR) primer design using free online software. Biochem Mol Biol Educ 2011;39:145-54.  Back to cited text no. 24
    
25.
Burnet JB, Ogorzaly L, Tissier A, Penny C, Cauchie HM. Novel quantitative TaqMan real-time PCR assays for detection of Cryptosporidium at the genus level and genotyping of major human and cattle-infecting species. J Appl Microbiol 2013;114:1211-22.  Back to cited text no. 25
    
26.
Mary C, Chapey E, Dutoit E, Guyot K, Hasseine L, Jeddi F, et al. Multicentric evaluation of a new real-time PCR assay for quantification of Cryptosporidium spp. and identification of Cryptosporidium parvum and Cryptosporidium hominis. J Clin Microbiol 2013;51:2556-63.  Back to cited text no. 26
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

Online since April 2001, new site since 1st August '04