Retrospective evaluation of an integrated molecular-epidemiological approach to cyclosporiasis outbreak investigations - United States, 2021.

Cyclosporiasis results from an infection of the small intestine by Cyclospora parasites after ingestion of contaminated food or water, often leading to gastrointestinal distress. Recent developments in temporally linking genetically related Cyclospora isolates demonstrated effectiveness in supporting epidemiological investigations. We used 'temporal-genetic clusters' (TGCs) to investigate reported cyclosporiasis cases in the United States during the 2021 peak-period (1 May - 31 August 2021). Our approach split 655 genotyped isolates into 55 genetic clusters and 31 TGCs. We linked two large multi-state epidemiological clusters (Epidemiologic Cluster 1 [n = 136 cases, 54 genotyped] and Epidemiologic Cluster 2 [n = 42 cases, 15 genotyped]) to consumption of lettuce varieties; however, product traceback did not identify a specific product for either cluster due to the lack of detailed product information. To evaluate the utility of TGCs, we performed a retrospective case study comparing investigation outcomes of outbreaks first detected using epidemiological methods with those of the same outbreaks had TGCs been used to first detect them. Our study results indicate that adjustments to routine epidemiological approaches could link additional cases to epidemiological clusters of cyclosporiasis. Overall, we show that CDC's integrated genotyping and epidemiological investigations provide valuable insights into cyclosporiasis outbreaks in the United States.

Investigation of US Cyclospora cayetanensis outbreaks in 2019 and evaluation of an improved Cyclospora genotyping system against 2019 cyclosporiasis outbreak clusters.

Cyclosporiasis is an illness characterised by watery diarrhoea caused by the food-borne parasite Cyclospora cayetanensis. The increase in annual US cyclosporiasis cases led public health agencies to develop genotyping tools that aid outbreak investigations. A team at the Centers for Disease Control and Prevention (CDC) developed a system based on deep amplicon sequencing and machine learning, for detecting genetically-related clusters of cyclosporiasis to aid epidemiologic investigations. An evaluation of this system during 2018 supported its robustness, indicating that it possessed sufficient utility to warrant further evaluation. However, the earliest version of CDC's system had some limitations from a bioinformatics standpoint. Namely, reliance on proprietary software, the inability to detect novel haplotypes and absence of a strategy to select an appropriate number of discrete genetic clusters would limit the system's future deployment potential. We recently introduced several improvements that address these limitations and the aim of this study was to reassess the system's performance to ensure that the changes introduced had no observable negative impacts. Comparison of epidemiologically-defined cyclosporiasis clusters from 2019 to analogous genetic clusters detected using CDC's improved system reaffirmed its excellent sensitivity (90%) and specificity (99%), and confirmed its high discriminatory power. This C. cayetanensis genotyping system is robust and with ongoing improvement will form the basis of a US-wide C. cayetanensis genotyping network for clinical specimens.

Single-strand conformation polymorphism-based genetic characterization of the Cyclospora cayetanensis strains collected from different provinces in Turkey.

INTRODUCTION AND OBJECTIVE: Cyclospora cayetanensis, a coccidian protozoan species, has been recently found to cause diarrhea in all age groups in immunocompetent and immunocompromised individuals in most regions of the world. This study aimed to conduct the molecular detection of C. cayetanensis and to determine the genetic diversity of the 18S ribosomal RNA (rRNA) gene sequence of C. cayetanensis isolated from individuals living in different provinces in Turkey by using PCR-single-strand conformation polymorphism (SSCP). MATERIAL AND METHODS: A total of 22 subjects were included in the study. Fourteen of the subjects were female and eight were male, with ages ranging between 7-65 years. Stool specimens were examined using wet mount and modified acid-fast staining methods, which revealed the presence of oocysts in the samples. The 18S rRNA ITS-1 Ccits37f-GCTTGCTATGTTTTAGCATGTGG and Ccits501r-GCACAATGAATGCACACACA gene regions were used as primers. The PCR products were analyzed by agarose gel electrophoresis and visualized on a UV transilluminator. For the SSCP, the PCR products were denatured with formamide, run for 16 h in 6% (49:1) polyacrylamide gel, and then imaged with silver staining. RESULTS: SSCP assay was performed given that the DNA strands demonstrated different folds; the DNA strands contain different nucleotides based on the PCR-SSCP results for the Cyclospora strains collected in 4 provinces. Moreover, 3 different band profiles were observed in the investigated samples. A slight mutation difference was observed among the strains collected. CONCLUSIONS: Further comprehensive studies involving more C. cayetanensis-positive specimens and utilizing different mutation screening methods are warranted to demonstrate mutation differences in Cyclopora strains in Turkey.

Detection of Cyclospora cayetanensis on bagged pre-cut salad mixes within their shelf-life and after sell by date by the U.S. food and drug administration validated method.

Recently, outbreaks of Cyclospora cayetanensis in the U.S. were linked to the consumption of a variety of salads containing romaine and/or iceberg lettuce, carrots and/or red cabbage. The Bacteriological Analytical Manual (BAM) Chapter 19b method was validated for the detection of C. cayetanensis in carrots, cabbage and romaine lettuce, but has not been previously evaluated in ready-to-eat (RTE) salad mixes. In addition, the only samples available for traceback investigations are sometimes leftovers in bad conditions. This study evaluated the validated BAM method for detection of C. cayetanensis in two different RTE mixed salads (mix 1: romaine and iceberg lettuces, carrots, and red cabbage and mix 2: romaine and iceberg lettuces, carrots, red cabbage, radish, and pea pods) in good condition and after their sell by date. Individual samples (25 g) were seeded with five and 200 C. cayetanensis oocysts. Unseeded produce was used as negative control. The method included washing of the produce, concentration and extraction of C. cayetanensis DNA and molecular detection of C. cayetanensis 18 S rRNA gene. As few as five oocysts were detected in both fresh and after sell by date mix salads. All unseeded samples were negative, and all samples of both salad types seeded with 200 oocysts were positive. In samples seeded with 200 oocysts, average 18 S rRNA C. cayetanensis CT values were significantly higher in fresh salad mix 1 compared to fresh salad mix 2; CT values were significantly higher in the after sell by date salads compared to their respective fresh mixes (p < 0.05). In conclusion, the BAM method was able to detect as few as five oocysts even in after sell by date RTE mix salads. However, the differences in detection observed, highlight the importance of evaluating the performance of the validated C. cayetanensis detection method in different food matrices and conditions, in advance for future outbreak investigations.

Contamination of fresh produce sold on the Italian market with Cyclospora cayetanensis and Echinococcus multilocularis.

To investigate the presence of Cyclospora cayetanensis, Toxoplasma gondii and Echinococcus spp. in fresh produce sold in Italy, 324 locally produced 'ready-to-eat' (RTE) mixed-salad packages belonging to three brands and 324 berries packages (blueberries and blackberries imported from Peru and Mexico, respectively, and raspberries grown in Italy) were purchased at retail. Nine individual packages from each of the six types of fresh produce were collected monthly for one year, and with the same produce pooled, this resulted in a total of 72 pools for the whole year. Using microscopy (FLOTAC), a Cyclospora-like oocyst was detected in a blueberry sample and a taeniid egg was detected in a RTE-salad sample. Molecular tools confirmed these to be C. cayetanensis and Echinococcus multilocularis, respectively. Toxoplasma gondii was not detected in any of the samples. This study shows for the first time in Europe that imported berries on the Italian market may be contaminated with C. cayetanensis and RTE salads grown in Italy with E. multilocularis. The results indicate a new epidemiological scenario and highlight that current management of fresh produce, locally produced or imported, does not ensure products are free from parasite contamination.

Modifications of the U.S. food and drug administration validated method for detection of Cyclospora cayetanensis oocysts in prepared dishes: Mexican-style salsas and guacamole.

Although multiple outbreak clusters of Cyclospora cayetanensis have been traced back to consumption of dishes in Mexican-style restaurants, the FDA Bacteriological Analytical Manual (BAM) does not currently provide methods to detect C. cayetanensis in dishes that contain multiple produce ingredients, such as salsas and guacamole. These complex food matrices also may contain high levels of fats, which can interfere with the detection. Several modifications to the BAM Chapter 19b method (washing produce, DNA extraction, and a TaqMan real-time PCR assay targeting the 18S rRNA gene of C. cayetanensis) were assessed with the goal to detect as few as 5 oocysts of C. cayetanensis in 25 g samples of commercial salsa/pico de gallo, guacamole, and salsa verde. Both freshly prepared and frozen versions of these foods were seeded with 5, 10 and 200 oocysts. For salsa samples, using a gentler washing step than recommended by BAM, we achieved detection of 5 oocysts in the samples (81.8%, n = 11). Increasing the amount of Alconox® in the wash solution to 1%, rather than the 0.1% used in BAM, and adjusting the DNA extraction protocol to process large wash pellets, enabled detection of 5 oocysts in guacamole. To reach the desired level of detection in salsa verde, two types of modifications were necessary: gentler washing and DNA extraction modifications. The use of these same method modifications on previously frozen food samples, provided levels of detection similar to those achieved with fresh dishes. Our modifications enabled robust and reproducible detection of C. cayetanensis in multi-ingredient Mexican dishes, detecting as few as 5 oocysts in 25 g samples. Validating and deploying effective methods to detect C. cayetanensis in high risk fresh produce and prepared dishes are critically important for prevalence studies and outbreak investigations of this parasite.

A scoping review of the detection, epidemiology and control of Cyclospora cayetanensis with an emphasis on produce, water and soil.

Cyclospora cayetanensis is a parasite causing cyclosporiasis (an illness in humans). Produce (fruits, vegetables, herbs), water and soil contaminated with C. cayetanensis have been implicated in human infection. The objective was to conduct a scoping review of primary research in English on the detection, epidemiology and control of C. cayetanensis with an emphasis on produce, water and soil. MEDLINE® (Web of ScienceTM), Agricola (ProQuest), CABI Global Health, and Food Science and Technology Abstracts (EBSCOhost) were searched from 1979 to February 2020. Of the 349 relevant primary research studies identified, there were 75 detection-method studies, 40 molecular characterisation studies, 38 studies of Cyclospora in the environment (33 prevalence studies, 10 studies of factors associated with environmental contamination), 246 human infection studies (212 prevalence/incidence studies, 32 outbreak studies, 60 studies of environmental factors associated with non-outbreak human infection) and eight control studies. There appears to be sufficient literature for a systematic review of prevalence and factors associated with human infection with C. cayetanensis. There is a dearth of publicly available detection-method studies in soil (n = 0) and water (n = 2), prevalence studies on soil (n = 1) and studies of the control of Cyclospora (particularly on produce prior to retail (n = 0)).

Molecular characterization of the waterborne pathogens Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, Cyclospora cayetanensis and Eimeria spp. in wastewater and sewage in Guangzhou, China.

BACKGROUND: The waterborne pathogens Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Cyclospora cayetanensis can cause intestinal diseases in humans. An understanding of their occurrence and transport in the environment is essential for accurate quantitative microbial risk assessment. METHODS: A total of 238 influent samples were collected from four wastewater treatment plants (WWTPs) and 88 samples from eight sewer locations in Guangzhou, China. PCR-based tools were used to detect and genetically characterize Cryptosporidium spp., G. duodenalis and E. bieneusi. Eimeria spp. and Cyclospora spp. were also analyzed to assess the sources of Cryptosporidium spp., G. duodenalis and E. bieneusi in wastewater. RESULTS: The overall occurrence rates in the WWTP and sewer samples were 14.3% (34/238) and 13.6% (12/88) for Cryptosporidium spp., 55.5% (132/238) and 33.0% (29/88) for G. duodenalis, 56.3% (134/238) and 26.1% (23/88) for E. bieneusi and 45.4% (108/238) and 47.7% (42/88) for Eimeria spp., respectively. Altogether, 11 Cryptosporidium species and genotypes, six G. duodenalis genotypes, 11 E. bieneusi genotypes and four C. cayetanensis were found, together with the presence of nine Eimeria species. The common occurrence of Cryptosporidium rat genotype IV, C. muris and Eimeria papillata and E. nieschulzi suggested that rodents were significant sources of the enteric pathogens detected in the wastewater samples. CONCLUSIONS: While the dominant Cryptosporidium spp. detected in the raw wastewater sampled in this study are not pathogenic to humans, the widely detected G. duodenalis assemblage A and E. bieneusi genotypes D and Type IV are well-known zoonotic pathogens. Further studies are needed to monitor the occurrence of these waterborne pathogens in WWTPs to better understand their transmission and environmental transport in China.

Acute kidney injury associated with intestinal infection by Cyclospora cayetanensis in a kidney transplant patient. A case report.

This study shows a clinical case report of a kidney transplant patient who traveled from Mexico to The Netherlands and ate green vegetables in an international food restaurant. After 5 days, he started having diarrhea, nausea, colic, and a physical feeling of malaise. The patient only received symptomatic treatment after showing the characteristic symptoms of traveler's diarrhea. When he returned to Mexico, the clinical picture worsened, and he was hospitalized. Clinical analyses indicated dehydration and acute kidney injury stage II. Coproparasitoscopic study showed the presence of Cyclospora cayetanensis. Parenteral solutions, gastric mucosal protector, ciprofloxacin, and a soft diet were administrated as treatment. The patient was discharged 72 h later with an improvement of the kidney function.

Outbreak of Cyclosporiasis in Korean Travelers Returning from Nepal.

Cyclospora cayetanensis is an apicomplexan protozoan and is one of the most common pathogens causing chronic diarrhea worldwide. Eight stool samples with diarrheal symptom out of 18 Korean residents who traveled to Nepal were obtained, and examined for 25 enteropathogens including 16 bacterial species, 5 viral species, and 4 protozoans in stool samples as causative agents of water-borne and food-borne disease. Only C. cayetanensis was detected by nested PCR, and 3 PCR-positive samples were sequenced to confirm species identification. However, the oocysts of C. cayetanensis in fecal samples could not be detected by direct microscopy of the stained sample. As far as we know, this is the first report of a group infection with C. cayetanensis from a traveler visiting Nepal, and the second report of a traveler's diarrhea by C. cayetanensis imported in Korea.

Dead-End Ultrafiltration and DNA-Based Methods for Detection of Cyclospora cayetanensis in Agricultural Water.

Cyclospora cayetanensis is a protozoan parasite that causes foodborne and waterborne diarrheal illness outbreaks worldwide. Most of these outbreaks are associated with the consumption of fresh produce. Sensitive and specific methods to detect C. cayetanensis in agricultural water are needed to identify the parasite in agricultural water used to irrigate crops that have been implicated in outbreaks. In this study, a method to detect C. cayetanensis in water by combining dead-end ultrafiltration (DEUF) with sensitive and specific molecular detection was developed and evaluated. Triplicates of 10-liter agricultural water samples were seeded with 200, 100, 25, 12, and 6 C. cayetanensis oocysts. Surface water samples were also collected in the Mid-Atlantic region. All water samples were processed by DEUF and backflushed from the ultrafilters. DNA was extracted from concentrated samples and analyzed by quantitative PCR (qPCR) targeting the C. cayetanensis 18S rRNA gene. All water samples seeded with 12, 25, 100, and 200 oocysts were positive, and all unseeded samples were negative. Samples seeded with 6 oocysts had a detection rate of 66.6% (8/12). The method was also able to detect C. cayetanensis isolates in surface water samples from different locations of the Chesapeake and Ohio Canal (C&O Canal) in Maryland. This approach could consistently detect C. cayetanensis DNA in 10-liter agricultural water samples contaminated with low levels of oocysts, equivalent to the levels that may be found in naturally incurred environmental water sources. Our data demonstrate the robustness of the method as a useful tool to detect C. cayetanensis from environmental sources.IMPORTANCECyclospora cayetanensis is a protozoan parasite that causes foodborne and waterborne outbreaks of diarrheal illness worldwide. These foodborne outbreaks associated with the consumption of fresh produce and agricultural water could play a role in the contamination process. In this study, a method to detect C. cayetanensis in agricultural water by combining a robust filtration system with sensitive and specific molecular detection was developed and validated by the FDA. The results showed that this approach could consistently detect low levels of C. cayetanensis contamination in 10 liters of agricultural water, corresponding to the levels that may be found in naturally occurring environmental water sources. The method was also able to detect C. cayetanensis in surface water samples from a specific location in the Mid-Atlantic region. Our data demonstrate the robustness of the method to detect C. cayetanensis in agricultural water samples, which could be very useful to identify environmental sources of contamination.

Detection of human intestinal protozoan parasites in vegetables and fruits: a review.

Diarrheal diseases caused by intestinal protozoan parasites are a major food-borne public health problem across the world. Vegetables and fruits provide important nutrients and minerals, but are also common sources of some food-borne human pathogenic microorganisms. The contamination of raw vegetables and fruits with human pathogenic parasites are now a global public health threat, despite the health benefits of these foods in non-pharmacological prophylaxes against diseases. A large number of reports have documented the contamination of vegetables or fruits with human pathogenic microorganisms. In this paper, we reviewed the contamination and detection methods of human pathogenic intestinal protozoans that are frequently recovered from raw vegetables and fruits. The protozoan parasites include Cryptosporidium spp., Giardia duodenalis, Cyclospora cayetanensis, Entamoeba spp., Toxoplasma gondii, Balantioides coli, Blastocystis sp., Cystoisospora belli and Enterocytozoon bieneusi. The risk factors involved in the contamination of vegetables and fruits with parasites are also assessed.

Sporadic cyclosporiasis in symptomatic Cuban patients: Confirmation of positive results from conventional diagnostic methods by molecular assay.

In Cuba, there are few studies on cyclosporiasis. Here, we report results from 1247 stool samples from symptomatic patients that were examined by microscopy methods and positive cases confirmed by nested PCR targeting the 18S rRNA gene, followed by sequencing. Seven positive samples, all diagnosed during May-June, were confirmed by the molecular method, indicating an occurrence in this patient cohort of 0.56%.

Comparative evaluation of UNEX-based DNA extraction for molecular detection of Cyclospora cayetanensis, Toxoplasma gondii, and Cryptosporidium parvum as contaminants of berries.

The potential public health impact of foodborne parasites (FBP) transmitted via contaminated fresh produces indicates the necessity for robust and reliable laboratory methods for their detection and identification on this infection vehicle. Standardization of methods for detection of common FBP in fresh produce is to be expected and ensuring that the DNA extraction approach is most appropriate for the FBP of interest and for the matrix being analyzed is also important. Therefore, the aim of the present study was to compare the efficacy of two commercially available DNA extraction procedures, the UNEX-based method and DNeasy PowerSoil kit in the detection of three protozoan parasites, C. cayetanensis, C. parvum, and T. gondii, on contaminated berries. Oocysts of each parasite were spiked into the pellets of raspberry and blueberry washes. The spiked pellets were then randomly assigned to DNA extraction using either the PowerSoil or UNEX method, with DNA extraction with both methods performed by two independent analysts. The detection rate when berry washes were spiked with 20 oocysts of C. cayetanensis, T. gondii, and C. parvum was 95%, 85%, and 40%, respectively, when using the PowerSoil kit; whereas the equivalent results using the UNEX method were 55%, 60%, and 5%, respectively. In addition, significantly lower Cq values were achieved for each parasite in the samples spiked with 500 oocysts when the PowerSoil kit was used. Possible reasons for these results are discussed, and include the composition of both the beads and the buffers in each method.

Molecular typing of Cyclospora cayetanensis in produce and clinical samples using targeted enrichment of complete mitochondrial genomes and next-generation sequencing.

BACKGROUND: Outbreaks of cyclosporiasis, a diarrheal illness caused by Cyclospora cayetanensis, have been a public health issue in the USA since the mid 1990's. In 2018, 2299 domestically acquired cases of cyclosporiasis were reported in the USA as a result of multiple large outbreaks linked to different fresh produce commodities. Outbreak investigations are hindered by the absence of standardized molecular epidemiological tools for C. cayetanensis. For other apicomplexan coccidian parasites, multicopy organellar DNA such as mitochondrial genomes have been used for detection and molecular typing. METHODS: We developed a workflow to obtain complete mitochondrial genome sequences from cilantro samples and clinical samples for typing of C. cayetanensis isolates. The 6.3 kb long C. cayetanensis mitochondrial genome was amplified by PCR in four overlapping amplicons from genomic DNA extracted from cilantro, seeded with oocysts, and from stool samples positive for C. cayetanensis by diagnostic methods. DNA sequence libraries of pooled amplicons were prepared and sequenced via next-generation sequencing (NGS). Sequence reads were assembled using a custom bioinformatics pipeline. RESULTS: This approach allowed us to sequence complete mitochondrial genomes from the samples studied. Sequence alterations, such as single nucleotide polymorphism (SNP) profiles and insertion and deletions (InDels), in mitochondrial genomes of 24 stool samples from patients with cyclosporiasis diagnosed in 2014, exhibited discriminatory power. The cluster dendrogram that was created based on distance matrices of the complete mitochondrial genome sequences, indicated distinct strain-level diversity among the 2014 C. cayetanensis outbreak isolates analyzed in this study. CONCLUSIONS: Our results suggest that genomic analyses of mitochondrial genome sequences may help to link outbreak cases to the source.

Evaluation of the U.S. Food and Drug Administration validated molecular method for detection of Cyclospora cayetanensis oocysts on fresh and frozen berries.

Outbreaks and sporadic cases of Cyclospora cayetanensis have been linked to consumption of berries. The efficacy of the U.S. Food and Drug Administration (FDA) method for detection of C. cayetanensis was evaluated in fresh berries (blackberries, strawberries, blueberries and mixed berries) and in frozen mixed berries. The protocol included seeding with C. cayetanensis oocysts, produce washing, DNA extraction and a dual TaqMan assay. As few as five oocysts were detected in every type of fresh berry analyzed. All berry samples seeded with 200 oocysts were positive and all unseeded berry samples were negative. No significant differences were observed among any of the berry types analyzed in detection rates, CT values and estimated oocyst recovery percentages. Mixed berries were seeded and frozen for up to seven weeks. As few as five oocysts were also detected. No significant differences were observed in C. cayetanensis CT values between fresh and frozen mixed berries at any seeding level. In conclusion, the FDA BAM Chapter 19B method for the detection of Cyclospora was robust, consistent, and showed high sensitivity in all types of berries analyzed. Evaluation of the FDA detection method in berries will provide reliable laboratory support for surveillance programs and for outbreak investigations.

Silver nanoparticles as a therapeutic agent in experimental cyclosporiasis.

Cyclosporiasis is an emerging worldwide infection caused by an obligate intracellular protozoan parasite, Cyclospora cayetanensis. In immunocompetent patients, it is mainly manifested by self-limited diarrhea, which is persistent and may be fatal in immunocompromised patients. The standard treatment for cyclosporiasis is a combination of two antibiotics, trimethoprim and sulfamethoxazole. Gastrointestinal, haematologic and renal side effects were reported with this combination. Moreover, sulfa allergy, foetal anomalies and recurrence were recorded with no alternative drug treatment option. In this study, silver nanoparticles were chemically synthesized to be evaluated for the first time for their anti-cyclospora effects in both immunocompetent and immunosuppressed experimental mice in comparison to the standard treatment. The effect of silver nanoparticles was assessed through studying stool oocyst load, oocyst viability, ultrastructural changes in oocysts, and estimation of serum gamma interferon. Toxic effect of the therapeutic agents was evaluated by measuring liver enzymes, urea and creatinine in mouse sera. Results showed that silver nanoparticles had promising anti-cyclospora potentials. The animals that received these nanoparticles showed a statistically significant decrease in the oocyst burden and number of viable oocysts in stool and a statistically significant increase in serum gamma interferon in comparison to the corresponding group receiving the standard treatment and to the infected non-treated control group. Scanning electron microscopic examination revealed mutilated oocysts with irregularities, poring and perforations. Biochemical results showed no evidence of toxicity of silver nanoparticles, as the sera of the mice showed a statistically non-significant decrease in liver enzymes in immunocompetent subgroups, and a statistically significant decrease in immunosuppressed subgroups. Furthermore, a statistically non-significant decrease in urea and creatinine was recorded in all subgroups. Thus, silver nanoparticles proved their effectiveness against Cyclospora infection, and this will draw the attention to its use as an alternative to the standard therapy.

A novel multiplex real-time PCR for the detection of Echinococcus multilocularis, Toxoplasma gondii, and Cyclospora cayetanensis on berries.

Foodborne parasites (FBP) are of major public health importance and warrant appropriate detection and control strategies. Most of the FBP considered for risk-ranking by a panel of experts are potentially transmitted via consumption of contaminated fresh produce, including berries. In this study we focused on the potential of three FBP, namely Echinococcus multilocularis, Toxoplamsa gondii, and Cyclospora cayetanensis, as contaminants of berries. Surveys to assess these parasites as contaminants of fresh produce in general, and berries in particular, are scanty or non-existent mainly due to the lack of optimized laboratory methods for detection. The aim of the present study was to develop and evaluate a novel multiplex qPCR for the simultaneous detection of E. multilocularis, T. gondii, and C. cayetanensis from berry fruits. The efficiency and linearity of each channel in the multiplex qPCR were within the acceptable limits for the range of concentrations tested. Furthermore, the method was shown to have good repeatability (standard deviation ≤0.2 Cq) and intermediate precision (pooled standard deviation of 0.3-0.6 Cq). The limit of detection was estimated to 10 oocysts for Toxoplasma and Cyclospora, and 5 eggs for Echinococcus per 30 g of raspberries or blueberries. In conclusion, evaluation of the present method showed that the newly developed multiplex qPCR is highly specific, precise, and robust method that has potential for application in food-testing laboratories.