Assessment of three commercial DNA extraction kits and a laboratory-developed method for detecting Cryptosporidium and Cyclospora in raspberry wash, basil wash and pesto.

Polymerase chain reaction (PCR) methods are often used to identify the parasitic protozoa Cryptosporidium parvum and Cyclospora cayetanensis in foods although little has been published regarding the efficacy of available DNA extraction methods. This study reviewed three commonly used commercial DNA extraction kits: FastDNA SPIN Kit for soil, QBiogene (FastDNA), UltraClean™ Soil DNA Isolation Kit, MO BIO Laboratories (MoBio), and QIAamp DNA Mini Stool Kit, Qiagen (QIAamp), as well as a 'homebrew' Universal Nucleic Acid Extraction (UNEX) method. Washes from raspberry and basil as well as commercial pesto samples were seeded with 5000, 500, or 50 C. parvum and C. cayetanensis oocysts. The protocols were assessed for: quantity and quality of the extracted DNA, time to completion, presence of PCR inhibitors and the percentage of samples correctly identified as positive for the two parasites. Real-time and conventional nested PCR assays were used to detect the seeded pathogens. Of the commercial kits, PCR results of samples extracted using FastDNA were statistically similar to QIAamp and both were superior to MoBio. Differences in PCR results among FastDNA, QIAamp and UNEX for detection of Cyclospora were not statistically significant although the UNEX method proved best with Cryptosporidium. Real-time PCR assays targeted the 18S rRNA and the hsp70 genes of C. cayetanensis; overall results were similar to those found using conventional nested PCR targeting the 18S rRNA gene.

Use of a common laboratory glassware detergent improves recovery of Cryptosporidium parvum and Cyclospora cayetanensis from lettuce, herbs and raspberries.

The success of any protocol designed to detect parasitic protozoa on produce must begin with an efficient initial wash step. Cryptosporidium parvum and Cyclospora cayetanensis oocysts were seeded onto herbs, lettuces and raspberries, eluted with one of four wash solutions and the recovered number of oocysts determined via fluorescent microscopy. Recovery rates for fluorescein thiosemicarbazide labeled C. parvum oocysts seeded onto spinach and raspberries and washed with de-ionized water were 38.4 ± 10.1% and 34.9 ± 6.2%, respectively. Two alternative wash solutions viz. 1M glycine, pH 5.5 and a detachment solution were tested also using labeled C. parvum seeded spinach and raspberries. No statistically significant difference was noted in the recovery rates. However, a wash solution containing 0.1% Alconox, a laboratory glassware detergent, resulted in a significant improvement in oocyst recovery. 72.6 ± 6.6% C. parvum oocysts were recovered from basil when washed with 0.1% Alconox compared to 47.9 ± 5.8% using detachment solution. Also, C. cayetanensis oocysts were seeded onto lettuces, herbs and raspberries and the recovery using de-ionized water were compared to 0.1% Alconox wash: basil 17.5 ± 5.0% to 76.1 ± 14.0%, lollo rosso lettuce 38.3 ± 5.5% to 72.5 ± 8.1%, Tango leaf lettuce 45.9 ± 5.4% to 71.1 ± 7.8% and spring mix (mesclun) 39.8 ± 0.7% to 80.2 ± 11.3%, respectively. These results suggest that the use of Alconox in a wash solution significantly improves recovery resulting in the detection of these parasitic protozoa on high risk foods.

The effect of cyanuric acid on the disinfection rate of Cryptosporidium parvum in 20-ppm free chlorine.

Cyanuric acid is used to stabilize free chlorine to reduce photodegradation in outdoor swimming pools. While there have been numerous studies examining its effect on the disinfection rates of bacteria and viruses, it is not known whether cyanuric acid can significantly impact the effectiveness of hyperchlorination for inactivating Cryptosporidium oocysts present in fecally-contaminated swimming pools. This study examined the effect of cyanuric acid on the disinfection rate of Cryptosporidium parvum under swimming pool hyperchlorination conditions (20 mg/ml free chlorine). When 50 mg/L cyanuric acid was present there was a 0.70-log10 reduction in oocyst viability after 10 hours as compared to a 3.7-log10 reduction without cyanuric acid. Aids to remediation, such as decreasing the pH to enhance the germicidal efficiency of the free chlorine and doubling the amount of free chlorine residual, were still unable to achieve a 3-log10 reduction. Current public health recommendations for hyperchlorination and pool remediation are insufficient for pools using cyanurate-stabilized chlorine to achieve a three log inactivation of the parasite.

Prevalence of Cryptosporidium spp. and Giardia intestinalis in swimming pools, Atlanta, Georgia.

Cryptosporidium spp. and Giardia intestinalis have been found in swimming pool filter backwash during outbreaks. To determine baseline prevalence, we sampled pools not associated with outbreaks and found that of 160 sampled pools, 13 (8.1%) were positive for 1 or both parasites; 10 (6.2%) for Giardia sp., 2 (1.2%) for Cryptosporidium spp., and 1 (0.6%) for both.

Inactivation of Cryptosporidium parvum under chlorinated recreational water conditions.

Cryptosporidium is a chlorine-resistant protozoan parasite and the etiological agent in many disinfected recreational water outbreaks. While previous studies have reported disinfection Ct values for Cryptosporidium parvum using sodium hypochlorite, these studies have employed conditions and procedures which are not ideal for establishing public health remediation recommendations for chlorinated recreational water venues. In the present study, free chlorine Ct values were measured at pH 7.5 using young oocysts (<1 month old) and tissue culture to determine oocyst viability. Two different oocyst isolates were used: one originating from Iowa and one from Maine (USA). This study determined that the Ct values for a 3-log reduction in oocyst viability were 10,400 (Iowa) and 15,300 (Maine) at pH 7.5. These Ct values are higher than the Centers for Disease Control and Prevention (USA) currently recommends (Ct = 9,600) for achieving a 3.0-log inactivation of Cryptosporidium oocysts during remediation of recreational water venues following fecal diarrhea accidents.

PCR-restriction fragment length polymorphism method for detection of Cyclospora cayetanensis in environmental waters without microscopic confirmation.

We developed an alternative nested-PCR-restriction fragment length polymorphism (RFLP) protocol for the detection of Cyclospora cayetanensis in environmental samples that obviates the need for microscopic examination. The RFLP method, with the restriction enzyme AluI, differentiates the amplified target sequence from C. cayetanensis from those that may cross-react. This new protocol was used to reexamine a subset (121 of 180) of surface water samples. Samples previously positive when the CYCF3E and CYCR4B primers (33) and RFLP with MnlI (20) were used were also PCR positive with the new primers; however, they were RFLP negative. We verified, by sequencing these amplicons, that while two were most likely other Cyclospora species, they were not C. cayetanensis. We can detect as few as one oocyst seeded into an autoclaved pellet flocculated from 10 liters of surface water. This new protocol should be of great use for environmental microbiologists and public health laboratories.

Cyclospora cayetanensis: a review of an emerging parasitic coccidian.

Cyclospora cayetanensis is a sporulating parasitic protozoan that infects the upper small intestinal tract. It has been identified as both a food and waterborne pathogen endemic in many developing countries. It is an important agent of Traveller's Diarrohea in developed countries and was responsible for numerous foodborne outbreaks in the United States and Canada in the late 1990s. Like Cryptosporidium, infection has been associated with a variety of sequelae such as Guillain-Barré syndrome, reactive arthritis syndrome (formally Reiter syndrome) and acalculous cholecystitis. There has been much debate as to where to place C. cayetanensis taxonomically due to its homology with Eimeria species. To date, the only genomic DNA sequences available are the ribosomal DNA of C. cayetanensis and three other species; within these a high degree of homology has been observed. This homology and the lack of sequence data from other Cyclospora species have hindered identification methods.