Improved Detection and Viability Assessment of Cryptospordium parvum using Real-Time NASBA and Light Cycler Detection

Lowery, CJ¹., Thompson, HP¹., Cruthers, L²., Finn, M¹., Millar, BC²., Moore, JE²., and Dooley, JSG¹.

¹School of Biological and Environmental Sciences, Faculty of Life and Health Sciences, University of Ulster, Cromore Road, Coleraine, County Derry, Northern Ireland BT52 1SA.²Northern Ireland Public Health Laboratory Service (PHLS), Belfast City Hospital, Lisburn Road. Belfast. BT9 7AB.

Corresponding Author: Dr Colm J. Lowery. School of Biological and Environmental Sciences, Faculty of Life and Health Sciences, University of Ulster, Cromore Road, Coleraine, County Derry, Northern Ireland. BT52 1SA. Tel: 028 70 323132

Email: cj.lowery@ulster.ac.uk

Abstract

A rapid real-time polymerase chain reaction (PCR) was developed using the Roche LightCycler technology. This method was optimised to enable the sensitive detection of Cryptosporidium oocysts isolated from both clinical and environmental water samples. The real-time detection system developed was shown to be consistently ten times more sensitive than nested PCR. A Nucleic Acid Sequence Based Amplification (NASBA) protocol was also designed to target the C. parvum replication protein A (RPA). The Cp-RPA1 gene, which encodes the large sub-unit of the C. parvum replication protein A (RPA), is expressed in both free sporozoites and parasite intracellular stages. NASBA of the Cp-RPA1 gene is proposed as a sensitive automated molecular detection system for Cryptosporidium oocysts with potential as a measure of viability.

Introduction

Cryptosporidium parvum is an enteric parasite belonging to the phylum Apicomplexa, subclass Coccidia, and family Cryptosporidiidae. All species of Cryptosporidium are obligate, intracellular, protozoan parasites that undergo endogenous development culminating in the production of an encysted stage discharged in the faeces of the host (Fayer et al., 2000). In farm animals, Cryptospoidium parasites cause disorders of the digestive and respiratory systems, which lead to poor health of infected animals and significant economic losses. In immunocompetent humans, Cryptosporidium parasites cause acute infections of the digestive system, but in immunocompromised patients they cause a chronic, life-threatening disease (Xiao et al., 1999). Failure to diagnose cryptosporidiosis in the immunocompetent patient with diarrhoea will rarely be of consequence because the disease is usually self-limiting. In contrast, the diagnosis of cryptosporidiosis is essential in the immunocompromised patient because it may influence therapeutical procedures, even though no effective therapy for cryptosporidiosis is currently known (Tzipori 1998; Bialek et al., 2002). There are currently 10 recognised species of Cryptosporidium infectingbirds, cats, mice, guinea pigs, cattle, fish, reptiles and skink. The species of concern from both a medical and veterinary perspective is C. parvum. C. parvum-like infections which have been reported in 152 species of mammals, including sea lions, polar bears and dugongs (a marine mammal similar to a manatee) (Fayer et al., 2000; Joan et al., 2002).

Transmission

Oocysts of Cryptosporidium, the infective stage, are spread via the faecal-oral route. The exact modes of infection of the parasite throughout the environment remain unclear although contaminated municipal water supplies and recreational waters, such as pools and lakes have previously been implicated in outbreaks of cryptosporidiosis (Kramer 1998). The exact importance of foreign travel, consumption of foods, beverages or water, and person-to-person transmission, as well as the role of infected animals in disease transmission remains to be ascertained (Casemore et al., 1997). Oocysts of C. parvum, from human faeces, can enter surface waters through wastewater, leaky septic tanks, or recreational activities, whilst oocysts from other mammals can enter surface waters either directly or through runoff (Fayer et al., 1999). Since the identification of C. parvum as a human pathogen in 1976 (Nimes 1976, Meisel 1976), several large outbreaks have occurred, most notably that which affected some 400,000 people in Milwaukee in the United States in 1993 (MacKenzie1994). Recently, there have been reports of at least three waterborne outbreaks of this organism in Northern Ireland in a 12 month period between May 2000 and May 2001 (Anon. 2000; Anon. 2000; Anon. 2001; Glaberman et al., 2001).

Cryptosporidium and the Water Supply

C. parvum oocysts are highly resistant to environmental factors, and can survive for several months in standing water (Unguen et al., 1997, Robertson et al., 1992). Surveys in the United Kingdom and the United States have shown that 50-80% of standing water is contaminated by C. parvum oocysts (Xiao et al., 2001, LeChevallier et al., 1991). The oocysts are resistant to chlorination at the concentrations used in water treatment (Payment 1999). These properties render it a problem for large-scale water suppliers and users of private water supplies. Statutory Instrument 1999 No. 1524 of the UK Water Supply (Water Quality) Regulations 2000 require water undertakers to carry out risk assessments to establish whether there is a significant risk from Cryptosporidium oocysts in water supplied from their treatment works for human consumption (regulation 2(1)). Where it is established that there is such a risk the relevant water undertakers must use a process for treating the water to ensure that the average number of Cryptosporidium oocysts per 10 litres of water is less than one. To verify compliance with this requirement water undertakers must ensure that the water leaving their treatment works is continuously sampled for Cryptosporidium oocysts. Breach of the standard is a criminal offence. In the event of breach of this standard this legislation also requires that the drinking water supplier must archive all slide material for a period of at least 3 months from the time of sampling.

Detection Methods

Prior to 1980, human cryptosporidiosis was diagnosed by histologic staining of gut or other biopsy specimens, and the subsequent identification of the life-stages of C. parvum (Tyzzer, 1910). The recognition of C. parvum as an emerging human and animal pathogen, and the global increase in immunocompromised populations have provided the impetus for research into sensitive and reliable detection and typing systems for both clinical and environmental samples. Currently, simple, rapid, and non-invasive acid fast staining techniques have found preference amongst many of todays clinical research laboratories. The high numbers of oocysts commonly found in clinical samples lends itself favourably to the application of simple acid fast methodology, and also enables the simultaneous identification of other parasites that would otherwise go unidentified if more specific stains were used. In the majority of modern clinical laboratories the successful diagnosisof cryptosporidiosis principally relies on the recognition ofcryptosporidial oocysts by light microscopy in stained faecal smears. Common diagnostic staining techniques include immunofluorescence (IF),modified Ziehl-Neelsen (MZN), and auramine phenol (AP) methods (Arrowood et al., 1998). Many of these stains require an experienced microscopist, however, and are labour-intensive laboratory procedures (Fayer et al., 2000). Most widely used in statutory body and commercial laboratories for the detection of Cryptosporidium sp. are immunofluorescent antibody (IFA) conjugates (Graczyk 1996, Sterling & Arrowood 1986, Stibbs 1986). Owing to the low numbers of oocysts commonly found in environmental samples it is often necessary to use an oocyst concentration technique such as IMS to maintain an acceptable level of assay detection sensitivity. IMS binds selectively to the oocyst wall proteins enabling them to be concentrated in a suspension free from inhibitory debris. As useful as these laboratory techniques are however, they are unable to determine if C. parvum oocysts are type I or II, nor do they directly allow for any further epidemiological study based on the data they generate.

Molecular Detection

PCR methods have beenshown to be more sensitive and specific than traditional microscopictechniques for detecting Cryptosporidium sp. in both clinical andenvironmental samples (Mayer et al., 1996; Morgan et al., 1998; Lowery et al., 2000). In a previous study 11 of the most common genotyping methods currently in use for Cryptosporidium sp. were evaluated for sensitivity and specificity (Sulaiman et al., 1999). A more recent study reviewing the use of PCR-based Cryptosporidium discriminating techniques recommends the direct sequencing of either the 18S rRNA or COWP gene as the most useful tool for accurate identification of Cryptosporidium species (Abe et al., 2002). More recently several real-time PCR procedures for the detection and genotyping of oocysts of Cryptosporidium parvum were developed and evaluated (Higgins et al., 2001; Limor et al., 2002; Tanriverdi et al., 2002; Fontaine, M and Guillot, E., 2002). In summary, the consensus of opinion from these previous real-time studies is that real-time PCR offers reliable, specific and rapid detection method alternative to nested PCR, with a baseline sensitivity of between one and ten oocysts.

Viability Assessment

Given the highly infective nature of Cryptosporidium oocysts, the development of an effective method of determining oocyst viability is of particular concern to researchers. IFA conjugates are usually used in conjunction with a 4',6-Diamidino-2-phenylindole (DAPI) stain for determining oocyst viability (Jenkins 1997, Campbell 1992). Molecular based techniques have previously been used to determine oocyst viability by reverse-transcriptase-PCR (RT-PCR) (Filkhorn et al 1994,Gobet et al., 2001,Rote et al., 2001). Wagner-Wiening and Kimmig (1995) used PCR to detect and specifically identify a 873-bp region of a 2359-bp DNA fragment encoding a repetitive oocyst protein of C. parvum using an excystation protocol before DNA extraction to allow the differentiation between live and dead oocysts. Survival and disinfection studies have shown that in vitro viability assays such as excystation and vital staining may or may not correlate with animal infectivity, depending on the disinfectant or animal model used (Belosevic et al., 1997, Bukhari et al., 1999, Campbell et al., 1992). Animal models have largely been discounted for use in infectivity studies owing to the long process time involved and specialist animal handling skills required. A recent study has also successfully demonstrated that tissue culture (HCT-8 cell line) can successfully be used to measure C. parvum infection and can be used for determining inactivation in disinfection studies (Slifko et al., 2002).

The presence of mRNA has been correlated with the viability of an organism, (Bej et al., 1991; Mahbubani et al., 1991). Heat shock proteins (hsps) are known to be synthesized with a high level of efficiency and the transcripts are present in large numbers in stressed organisms (Lindquist, 1986). Stinear et al., (1996) developed a (RT)-PCR coupled with IMS which can detect the presence of a single oocyst spiked into concentrated environmental water samples. The test is based on the detection of Hsp70 mRNA, produced only from viable oocysts, and then isolated by hybridization to oligo(dT)₂₅-coated beads. Zhu et al., 1999 described a 6kb Cp-RPA1 gene within the type 1 fatty acid synthase gene (Cp-FAS1) of C. parvum. This single-copy gene encodes a single-stranded-DNA binding protein involved in DNA replication and repair. Its product is a 473 amino acid, 53.9 kDa peptide which is considerably smaller than the replication protein A of C. parvum's mammalian hosts. It was determined that the Cp-RPA1 gene is expressed in both excysted free sporozoites and intracellular parasites 48 hours after infection. The role of RPA in DNA replication and repair means that its transcription is required by viable sporozoites, even in the apparently dormant oocyst. Therefore the mRNA transcript of this gene provides a potential target for viable C. parvum detection.

Nucleic Acid Sequence Based Amplification (NASBA)

Developed and patented by Kievits et al., 1991 NASBA is an isothermal, transcription-based amplification system. The activities of three enzymes - avian myeloblastosis virus reverse transcriptase (AMV-RT), T7 polymerase and RnaseH - are utilised in the amplification stages. Two modified primers, P1 and P2 are used to amplify the target sequence. Primer P1 has a T7 polymerase promoter sequence at its 5' end, whilst primer P2, which has a generic sequence tag at its 5'end, is later recognised by a detection probe. NASBA is a sensitive method for detection of RNA, which, unlike RT-PCR, does not require a DNAse step. It has been widely applied to the measurement of viral titre - examples include Epstein-Barr virus (EBV) (Brink et al., 1998), HIV-1 (Witt et al., 2000), human cytomegalovirus (CMV) (Degre et al., 2001). Several other previous studies also report on the use of NASBA to detect bacterial species, including Mycobacterium tuberculosis (Van der Vliet et al., 1993), Chlamydia trachomatis (Morre et al., 1998) and Neisseria gonorrhoeae (Mahony et al., 2001). NASBA has been previously been used to detect the hsp70 gene of Cryptosporidium parvum (Baeumner et al., 2001) and the ribosomal small subunit gene of Plasmodium falciparum (Schoone et al., 2000).

Materials and Methods

Cryptosporidium isolates

Two isolates were used in this study. A type I (Human) isolate was kindly provided by Dr L Xiao, Centres for Disease Control, Atlanta, Ga, USA. A type II (Animal) isolate was obtained from the Moredun Scientific Ltd, Penicuik, Scotland. Clinical samples were obtained from the Northern Ireland Public Health Laboratories at Belfast City Hospital.

Isolation of genomic DNA

Oocyst suspensions were then washed three times by centrifuging at 7000 x g for 10 minutes in double-distilled water (ddH₂O) and resuspending in 200µl lysis buffer (4M urea, 200mM Tris, 20mM NaCl, 200mM EDTA, pH 7.4),and 40µl protinease K (2.0 mg/ml) for 1 hour at 55^oC. The samples were then subjected to six cycles of freezing in liquid nitrogen for 2 minutes, followed by thawing at 95^oC for 5 minutes to release the target DNA which was then purified using a High Pure PCR Template Preparation Kit according to the manufacturers instructions (Boehringer Mannheim). Briefly, nucleic acids bind specifically to the surface of glass fibres in the presence of a chaotropic salt. Residual impurities such as salts, proteins and other cellular components are removed by a wash step and subsequently nucleic acids are eluted in an elution buffer.

Nested PCR-RFLP Analysis

Clinical faecal samples having previously tested positive for Cryptosporidium oocysts using a simple acid fast staining technique were genotyped as Type I or Type II using a previously described nested-PCR-RFLP technique based on amplification of the 18S rRNA gene and restriction digestion using Vsp I and Ssp I endonucleases (Promega Ltd. U.K). Briefly, 10µl of C. parvum nucleic acid extract was digested by each enzyme in a total volume of 50µl (Xiao L, et al., 1999).

NASBA reagents, generic ECL probe, ECL buffer and cleaning solutions were obtained from Organon Teknika (OT), Cambridge, England. The Nuclisens NASBA QR reader was kindly provided by OT. NASBA primers and probes P1 and P2 primers, and a sequence-specific capture probe were designed using Oligo 4.0 (MBInsights, Cascade, CO, USA and were obtained from Invitrogen BRL.

Oocyst nucleic acid isolation for NASBA 100µl of C. parvum isolate were centrifuged and washed three times in ddH20. After the third wash step, the pellet was re-suspended in 0.9ml of NASBA Lysis Buffer. The sample was subjected to six freeze-thaw cycles with nucleic acid extracted and purified using the Nuclisens NASBA Basic Kit protocol. A dilution series was created which gave a range of 1 to 1:10,000 of the original sample.

NASBA amplification reaction

NASBA amplification was carried out using the OT Nuclisens NASBA Amplification kit, following the manufacturer's protocol (Kievits et al., 1991). A magnesium chloride concentration of 70mM in the reaction mixture was used. All possible combinations of primer pairs were tested. Briefly, an amplification kit reagent sphere was re-constituted in 80µl of sphere diluent, and 14µl of potassium chloride and 16µl NASBA water added. Each sphere was sufficient for ten tests. For each test, 1µl of the chosen primer set and 10µl of the reconstituted sphere solution were added to a 0.5 ml RNase-free eppendorf. To this were added 5µl of the C.parvum nucleic acid extract or its serial dilutions. Hence all dilutions were tested with all primer sets. These were incubated at 65^°C for 5 min, followed by incubation at 41^°C for 5 min. 5µl of the NASBA enzyme mix were added to each sample, and incubation was continued at 41^°C for a further 90 mins. The amplified products were stored at -20^°C pending further analysis.

NASBA ECL detection

A 1:5 dilution was performed for each amplificate, and 5µl of each diluted sample were individually mixed with 10µl of the Cp-RPA1 capture probe-bead complex, and 10µl of the generic ECL probe. The mixture was incubated at 41^°C for 30 mins. Finally 300µl of ECL Assay Buffer were added to each test, and the samples analysed in the Nuclisens NASBA QR system. An instrument reference control and a negative control were also tested.

Roche LightCycler Real Time PCR

PCR was carried out in a LightCycler (Roche, Ireland) using 18S rRNA PCR primers as previously described by Xiao et al., 1999. Each PCR mixture was then subjected to 55 cycles of denaturation at 94^oC for 2 secs, annealing at 50^oC for 10 secs, and extension at 72 ^oC for 15 secs, with an initial denaturation at 95 ^oC for 3 mins. Serial dilutions were made from DNA extracted from a stock concentration of 10, 000 C. parvum type II oocysts (Moredun Strain).

Results

Speciation, Genotyping and Subgenotyping

A nested 18S rRNA endonuclease restriction protocol was optimised and used to speciate isolates of Cryptosporidium and further identify isolates of C. parvum as being either Type I or Type II (Figure 1). Following nested PCR, polymorphisms observed within the gp60 gene sequences of C. parvum isolates enabled them to be further grouped into distinct subgenotypes.

Real-Time PCR and Nested PCR

The real-time LightCycler system was found to be more sensitive than the nested 18S rRNA method and was found after multiple trials to be capable of detecting a single oocyst. (Table 1). This represents a 10-fold increase in sensitivity over the nested PCR protocol.

NASBA requires optimisation using various combinations of 2 sets of primers which were designed to target the gene of interest. The best primer pair (i.e that which gave the most sensitive and consistent result) was determined after repeating the experiment 5 times. Primer set Piv enabled routine detection of as few as 50 oocysts of Cryptosporidium parvum per ml of water concentrate.

Lane 1: Molecular Weight 100 bp Ladder, Lane 2: Negative Control, Lane 3: Primary PCR product for Type I, Lane 4: Primary PCR product for Type II, Lane 5: Secondary PCR product for Type I, Lane 6: Secondary PCR product for Type II, Lane 7: Restriction of Type I with Vsp I, Lane 8: Restriction of Type II with Vsp I: Lane 9: Restriction of Type I with Ssp I, Lane 10: Restriction of Type II with Ssp I, Lane11: Blank, Lane 12: Molecular Weight 100 bp Ladder.

Table 1: Sensitivity of Detection of *Cryptosporidium* oocysts using Nested PCR and Real Time LightCycler Detection
No. of oocysts determined by IFA and Microscopic examination	Nested PCR	LightCycler Real-Time
1000, 1000	+ ,+	+ ,+
500, 500	+ ,+	+ ,+
100, 100	+ ,+	+, +
78, 74,	+ ,+	+, +
58, 53	+ ,+	+, +
13, 10	+ ,+	+, +
5, 3	-, -	+, +
1, 1	-, -	+, +

Discussion

The replication of DNA requires a range of protein co-factors, including helicases, topoisomerases, elongation factors and repair proteins, and expression of these proteins is an absolute requirement within viable cells. Furthermore, the need for a high level of conservancy in the DNA replication process means that efficient repair processes are required. These include nucleotide excision repair, which is highly conserved in all eukaryotes (Wold, 1997), and in which RPA is an absolute requirement. In the eukaryotic models Saccharomyces cerevisiae and Schizosaccharomyces pombe, RPA has been shown to be essential for DNA replication (Smith et al., 2000), with RPA^- mutants having no damage checkpoints (Longhese et al., 1996). This result compares well with results published by Schoone et al for detection of Plasmodium falciparum in blood samples and routinely detected as few as 50 oocysts of Cryptosporidium parvum per ml of water concentrate. NASBA of the single-copy Cp-RPA1 transcript therefore has potential as a sensitive detection system for C.parvum oocysts, with the advantage that, unlike RT-PCR, it does not require a DNase step. Since multiplex NASBA is possible, a system which would both type (eg by 18S) and detect viability should most certainly be considered.

UK PHLS scientists have long recognised the need for an increased use of real-time technology to aid in their patient management programmes (Moore and Millar, 2002). The results of this study highlight the potential of real-time PCR to be used in the rapid diagnosis of human Cryptosporidiosis, with the entire detection protocol taking less than 15 minutes to complete. Nested PCR is a robust and sensitive molecular technique capable of discriminating between all species of Cryptosporidium. Real-time PCR, however, allows for a quicker turn around time in diagnosis and was found to be consistently more sensitive, giving a ten-fold increase in sensitivity over the nested PCR technique. These results of this study also compare favourably with previously designed real-time systems were similar detection sensitivities were obtained (Higgins et al., 2001; Limor et al., 2002; Tanriverdi et al., 2002; Fontaine, M and Guillot, E., 2002). In future studies modification of the real-time system for use with specially designed hybridisation probes will enable not only quantification, speciation and subgenotyping of Cryptosporidium species, but will also allow for the analysis of multiple pathogens in a single PCR reaction. As well as being an ideal screening tool for the detection and genotyping of Cryptosporidium parasites in stool samples, real-time PCR will also enable the rapid, specific and sensitive detection of Cryptosporidium oocysts in drinking water. The ability to generate epidemiological data by molecular methods is of utmost importance in the event of any outbreak of cryptosporidiosis. Previously, several studies have linked acute waterborne outbreak situations to the source using molecular based detection methods (Glaberman et al., 2002, Jellison et al., 2002, Elwin et al., 2001, McLaughlin et al., 2000). Post-outbreak, archived environmental slides, and clinical slides produced from faecal smears, are potential sources of genetic material for further epidemiological studies (Amar et al., 2001., Amar et al., 2002). A preliminary study has recently shown that real-time PCR detection of genetic material from archive slides is also possible and highlights the major contribution that this technology can make to investigations of waterborne outbreaks of cryptosporidiosis.

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