Replaces all aspects of the Cyclospora methodology in BAM Chapter 19a related to detection of C. cayetanensis in water.

Terminology

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Overview

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Chapter 19c

Cyclospora cayetanensis is a protozoan parasite that causes human diarrheal disease called cyclosporiasis (1). The symptoms of cyclosporiasis include explosive watery diarrhea, weight loss, cramping, bloating, increasing gas, nausea, fatigue and loss of appetite. Outbreaks and sporadic cases occur annually associated with the consumption of fresh produce such as basil, romaine, cilantro and berries and since 2013, there has been a surge in the number of reported cases of cyclosporiasis in the U.S. In 2018, 2,299 cases of domestically acquired cyclosporiasis were reported to the CDC with approximately one-third of the cases associated with two large multistate outbreaks linked to prepackaged vegetable trays and salads. In 2019, a total of 2,408 domestically acquired cases were reported to the Centers for Disease Control and Prevention by 37 states in the U.S. and approximately 10% of these cases were linked to an outbreak that implicated imported basil (2). It is not clear how produce becomes contaminated, but it is possible that agricultural water may play a major role in contaminating crops during the irrigation at the pre-harvest stage and/or during produce processing. Therefore, laboratory methods that can be used to detect the parasite in food and water are essential to identify potential sources of infection and provide critical support for outbreak investigations.

A method for recovering Cyclospora and Cryptosporidium oocysts from water was published in the FDA Bacteriological Analytical Manual (BAM) Chapter 19A (11) in 2004. The methodology described in Chapter 19A relies on the use of Envirochek™ sampling capsules to recover Cyclospora oocysts from large volume water samples by filtration and is based on a protocol published by the EPA, Method 1623 (3), for isolation of Cryptosporidium and Giardia from drinking water. In 2013, the U.S. FDA was involved in outbreak investigations which demanded the testing of turbid agricultural water collected from farms that harvested crops epidemiologically linked to cyclosporiasis outbreaks. The Envirochek® filters employed in this method clogged rapidly during the filtration and no effective analysis could be performed on the samples collected. In 2015, a study was initiated to define and compare performance characteristics of dead-end ultrafiltration (DEUF), continuous flow centrifugation and the Method 1623 for recovery of Cyclospora from agricultural water. It was demonstrated that the hollow fiber filters employed in the DEUF method were less prone to clogging in low quality water such as agricultural water having significantly high amounts of small debris and sediment. The DEUF method is also more practical for field applications and the filters are most cost effective. Based on these results, a new method that combined the DEUF with a robust DNA extraction and a sensitive qPCR detection was developed. A single laboratory validation (SLV) study of this newly developed method was conducted to demonstrate its effectiveness for recovery and detection of Cyclospora oocysts from agricultural water. This SLV study was followed by a multi-laboratory validation (MLV) study which was completed in July of 2019.

The MLV study demonstrated that the approach was specific and could detect approximately 6 C. cayetanensis oocysts in 10 L of agricultural water samples. The protocol described in the BAM Chapter 19C is divided in two parts: Part I, which describes the protocol for collection of agricultural water samples using ultrafilters in the field and Part II, which describes how the retentate in these ultrafilters is later recovered in the laboratory and tested for the presence of C. cayetanensis using molecular methods.

Dead-end Ultrafiltration

_Part I

Introduction

In July of 2019, a MLV study for a new method based on DEUF for recovery and detection of Cyclospora oocysts from agricultural water samples was completed and approved. In this validation study, it was demonstrated that the approach could detect approximately 6 C. cayetanensis oocysts in 10 L of agricultural water samples. Part I of BAM Chapter 19C describes assembly of the device used to collect samples of irrigation water and the procedure to collect the samples using ultrafilters in the field.

As instructed in the Part I protocol, investigators must charge the batteries in advance according to the manufacturer’s instructions. In the field, investigators must initially set aside a small sample of water and measure the turbidity using a field turbidimeter prior to executing the ultrafiltration procedure. If the turbidity is higher than 40 Nephelometric Turbidity Units (NTU), or a turbidimeter is not available in the field, the investigators must carefully monitor the effluent rate during the filtration step as instructed in the protocol below. If a turbidity meter is not available to measure the turbidity in the field, the sample set aside for turbidity measurements shall be shipped together with the ultrafilter to the laboratory which will perform the analysis described in Part II of BAM Chapter 19C. The filters can be stored at 4oC up to one week by the investigators before shipping to the laboratory. In the meantime, the laboratory should be informed of the collection and estimated shipping dates.

Investigators may contact SMEs for questions related to environmental conditions and locations for water collections.

Procedures

The filtration protocol can be executed in the field.

Ultrafiltration Procedure for the Detection of Cyclospora cayetanensis

3.1

A. Ultrafiltration Setup

Figure 1 Diagram of the filtration system.

See Attached

B. Ultrafiltration Protocol

If the water to be collected contains free chlorine or chlorination is suspected, immediately after the filtration process, treat the filter with a 1% sodium thiosulfate solution following the procedure described in section iii.

Charge the pump batteries in advance according to the manufacturer’s instructions.

i. Check turbidity of water

1. Set aside a sample of approximately 500 ml for turbidity measurements in a 500 ml bottle.
2. If a turbidity meter is not available, ship the collected sample together with the filter to the laboratory that will perform the analysis.
3. If a turbidity meter is available, allow any debris to settle out of the water for approximately one minute and measure the turbidity according to the manufacturer’s instructions. (Do not transfer the settled debris into the turbidimeter).
4. If turbidity is higher than 40 Nephelometric Turbidity Units (NTU), or a turbidimeter is not available in the field, monitor the effluent flow rate during the filtration. If the rate decreases abruptly, stop the filtration and record the total volume of water that was filtered. If the turbidity is less than 40 NTU, the filter should not clog before filtering a 10 L sample.

ii. Ultrafiltration Procedure

1. Select the sampling locations and determine the number of filters to be used. This protocol was optimized for filtration of 10L water with each filter. The filters cannot be used under freezing conditions.
2. Assemble the filtration system with one filter as shown in the figure above, using the supplies and equipment listed above. Position the peristaltic pump on a flat surface or stand lift platform. Place the filter next to the pump.
3. Remove the end port cap from the blue end of the filter.
4. Screw in the DIN adapter to the blue end port.
5. Push the influent L/S 36 tubing onto the DIN adapter and secure with a SNP 8 tubing clamp. Use the pliers to secure the clamp in place if needed. Use the scissors to cut sufficient tubing to feed through the pump head and reach the water source.
6. Screw on the blood port (red end) cap or keep the original port cap in that port.
7. Feed the influent tubing through the pump head and close the pump head using the lever.
8. Remove the cap from the side port close to the red end. Push effluent L/S 36 tubing onto the open side port as shown above. Optionally, secure with a SNP 12 tubing clamp.
9. If a flow totalizer meter will be used to measure the volume of water filtered, screw a 3/4" GHT adapter onto each end, cut tubing attached to red side port and push tubing onto both sides of the meter (ensure directional flow of the meter is correct), no clamps are needed.
10. Place the influent tubing into the body of water and ensure the end of the tubing will stay below the surface of the water and away from plant material or other large debris which may be present. Be cautious not to disturb the water significantly near the filtration site.
11. Plug in the appropriate power cord into the outlet in the back of the pump and the other end of the power cord into the power source. The power source can be any external 12-18 V DC @ 70 watts or 90-260 V AC 47-65 Hz. Place the battery in a place where it will not get wet.
12. Determine the desired direction of flow and set the toggle switch for the flow direction. Ensure the speed dial is set to zero before starting the pump.
13. Turn the pump “ON” (the black filled circle), record the start time of filtration.
14. Once pumping has begun, the speed dial can be adjusted to gradually increase the flow to the maximum speed setting.
15. As soon a volume of 10 L is filtered, reduce the speed to the minimum and turn the pump “OFF”. Record the stop time of filtration.
16. If the water is chlorinated or chlorination is suspected follow the instructions in item iii below to treat the ultrafilter with 1% sodium thiosulfate. If the water is not chlorinated, skip this step.
17. Remove all tubing from the ultrafilter. Screw a blood port cap into the influent port and place a side storage port cap or the original port cap on the side port. Influent tubing can be discarded in the biohazard bags after collecting each sample. Effluent tubing can be re-used for subsequent samples. All tubing clamps and adapters that can be reused should be sterilized by autoclaving for future use.
18. Each filter must be labelled with the following information: Sample name, sample collection date and time, turbidity, and the amount of water that was filtered.
19. Place the filter in a cooler for transport to the laboratory. Use ice packs to keep the cooler cold if necessary for the target/suspected microbes collected.

iii. Procedure to treat ultrafilters with 1% sodium thiosulfate solution.

1. Fill a 500-ml bottle with non-chlorinated water and add 5 g sodium thiosulfate, shake to dissolve.
2. Place the influent tubing into the 1% sodium thiosulfate solution immediately after the filtration process and pump the entire volume through the filter without pumping air into the ultrafilter.

Recovery and Detection of Cyclospora cayetanensis from Ultrafilters

_Part II

Introduction

Part II of BAM Chapter 19C describes the laboratory analysis protocol for backflush, recovery and detection of C. cayetanensis from the ultrafilter retentate. If an unfiltered water sample is provided for a turbidity measurement, analysts should use a turbidimeter to measure the turbidity according to the manufacturer’s instructions. Detailed information about how to measure the turbidity in the laboratory is provided in section 3.1 of this protocol. When ultrafilters containing sample are received in the laboratory, they should be stored at 4oC for no more than two weeks from date of collection before analysis. The analysis protocol comprises the following main steps: 1. Recovery of oocysts from filters by backflushing and concentration, 2. Disruption of oocysts and extraction of Cyclospora DNA, 3. Molecular detection by a real-time PCR assay targeting the C. cayetanensis 18S rRNA gene according to the BAM Chapter 19B with minor modifications.

Procedures

The backflush and concentration protocols to prepare samples for DNA extraction can be executed for a set of up to four filters in a single day using one centrifuge.

Turbidity Measurement

3.1

A. If an unfiltered water sample is provided for a turbidity measurement, use a turbidimeter to measure the turbidity according to the manufacturer’s instructions. The sample should be at room temperature before measuring turbidity. Measure the turbidity at any time before executing the backflush procedure. i. ii. Allow any debris to settle out of the provided unfiltered water sample for approximately one minute and do not transfer the settled debris into the turbidimeter. Record the turbidity of the water sample in Nephelometric Turbidity Units (NTU).

Backflush - Recovery of Cyclospora cayetanensis from Ultrafilters

3.2

A. Preparation of Backflush solution (0.5% Tween 80/0.01% NaPP/0.001% Antifoam Y-30). Note: the solution should be prepared freshly on the first day and stored at 4 °C for no more than 48 hours.

i.
ii.
Make 10 ml 10% NaPP/1% Antifoam Y-30 stock solution: a. Add 10 ml DI water to a 15 ml conical tube; b. c. Add 1 g NaPP and 100 µL Antifoam Y-30 to the water; Vortex to dissolve the NaPP.
For each ultrafilter to be processed, make 500 ml backflush solution: a. b. c. d. Add 500 ml DI water to a 500 ml or 1 L bottle;
Add 500 µL of the 10% NaPP/1% Antifoam Y-30 stock solution to the water; Add 2.5 ml of Tween 80 with a 10 ml serological pipette;
Stir with a sterile stir bar for at least 5 min to dissolve the Tween 80.

B. Backflush Procedure

i.
Remove the selected filters from the refrigerator at least one hour prior to executing the backflush procedure. ii.
Assemble the filtration system with one filter, as shown in Figure 1 or 2 depending on how the pump is supported, using the supplies and equipment listed above: a. Position the peristaltic pump on a level surface or on a lift platform at least 20” above the bench surface. b. c. d. Clamp the filter on a ring stand and make certain the red end of the filter is on the top. Remove the side port cap from the red end of the filter. Push L/S 36 tubing onto the side port and secure with a SNP 12 hose clamp. e. Feed the tubing through a peristaltic pump and place the end of the tubing into the 500 ml of backflush solution. Close the pump head lever. f. iii.
Hold the blue end port over a 1 L sterile bottle or beaker, and remove the port cap.
Set the pump dial setting to zero and turn on the pump. Slowly increase the flow rate by turning up the dial to a slow pumping rate. If the pump has a digital display, the flow rate can be set at ~ 650 ml/minute. iv.
v.
vi.
vii.
Continue pumping until no backflush solution remains in the container or the tubing and the out flow from the ultrafilter has slowed to a trickle. Do not pump air into the filter for more than approximately 10 seconds. Measure and record backflush volume or weight, if necessary.
Repeat the backflush procedure for the remaining filters of the set. Proceed to the concentration procedure.
Figure 2 Backflush assembly Figure 3 Backflush Assembly

Concentrating Cyclospora cayetanensis from Backflush Effluent

3.3.

A. Each filter yields approximately 600 ml of effluent. Divide the effluent collected from each filter into four 225 ml conical centrifuge bottles. You will have a total of four 225 ml conical bottles for each filter.

B. Balance the four conical bottles with PBS and centrifuge at 4,000 x g for 45 min with maximal acceleration and a brake setting of 6 (on a scale of 0 9) for deceleration.

C. Carefully remove and discard all but ~25-30 ml of the supernatant from each centrifuge bottle using a glass Pasteur pipette connected to a vacuum aspirator or a 50 ml serological pipette. Using a vacuum aspiration system will be faster and more efficient than using a serological pipette.

D. Re-suspend each pellet in the remaining supernatant by pipetting up/down and vortexing. Transfer approximately 13-15 ml aliquots of the re suspended material from the four 225 ml centrifuge bottles to multiple 15 ml centrifuge tubes. Use a single 10 ml aliquot of PBS to rinse all four empty bottles one by one and add the rinse material to the 15 ml tubes. Between eight to ten 15 ml centrifuge tubes will be required to accommodate the re-suspended material and the rinse from the four 225 ml centrifuge bottles for each filter. Keep all centrifuge tubes for the same filter in a single Styrofoam stand or rack. At this point, if needed, samples can be stored at 4oC overnight before continuing the procedure.

E. Centrifuge the 15 ml centrifuge tubes at 4,000 x g for 45 min.

F. After the centrifugation, aspirate all but ~1.5 ml of the supernatant from each of the 15 ml tubes to waste without disturbing the pellets.

G. Re-suspend the pellets in the remaining supernatant and pool the pellets for each filter by transferring the re-suspended material into a single 15 ml tube. Use a single 2 ml aliquot of PBS to rinse all of the eight to ten tubes one by one and add the rinse to the single 15 ml tube. Centrifuge at 4,000 x g for 45 min.

H. Carefully aspirate all but approximately 300 μl of the supernatant above the pellet in each of the 15 ml tubes to waste without disturbing the pellets. At this point, the content of each 15 ml tube represents the sample from each ultrafilter.

I. Store the pellets at 4°C for no more than 24 hours.

J. Autoclave the supernatant waste.

DNA Isolation and Purification

3.4

A. Modified DNA Isolation Protocol Using the FastDNA® SPIN Kit for Soil

i. Material preparation: a. Add 100 ml of 100% ethanol to the 12 ml concentrated SEWS-M wash solution when a new kit is opened. b. Fill up an ice bucket for cooling the samples after the FastPrep-24 bead beater homogenizing steps (Step vi and vii below). c. Set a heat block for 2.0 ml tubes to 55 °C. d. Label the DNase-free 2.0 ml tubes appropriately. These are the tubes to be used in step ix and are NOT provided in the kit. e. Program the FastPrep-24 bead beater to a setting of 6.5 m/s (corresponds to approximately 4000 rpm) for 60 sec.

ii. Assemble the sample pellets in the 15 ml tubes for DNA isolation on a workbench cleaned with DNA AWAY or a cleaned Bio-Safety Cabinet (BSC). Include a Lysing Matrix E (LME) tube (supplied with the FastDNA Spin Kit) for the negative DNA extraction control.

iii. Add 122 µl MT buffer to each of the pellets in the 15 ml conical tubes (and to the DNA extraction control LME tube). (FastDNA kit protocol step 3).

iv. Add 830 µl Sodium Phosphate Buffer (SPB) to the mixture in the 15 ml conical tubes (and to the DNA extraction control LME tube). Mix each of the pellets in the 15 ml conical tubes with the buffers using a sterile wooden stick or a sterile transfer pipette. Try to minimize generating bubbles.

v. Carefully transfer the mixture (pellet, MT and SPB buffers) from each 15 ml conical tube into an LME tube using a sterile transfer pipette. Screw the caps on securely. The LME tubes have limited capacity. Make sure that there is at least 1.0 cm of air space at the top of the tube to allow for efficient bead-beating; do not fill beyond the maximum fill height as shown in the Figure 3. If the total volume is too large, split the mixture in two LME tubes. The sample can be recombined later as described in step xv.

Figure 4 Maximum fill height for LME tubes See Attached

vi. Transfer the samples to the FastPrep-24 bead beater, making sure to balance and secure the tubes in the sample holder according to the manufacturer’s instructions. Homogenize at a setting of 6.5 m/s for 60 sec. Immediately remove the sample holder containing the tubes from the instrument and place on ice for 3 minutes.

vii. Return the sample holder to the bead beater and repeat the homogenization and the incubation on ice as above for 3 minutes.

viii. Remove the tubes from the sample holder and centrifuge at 14,000 x g for 15 min (FastDNA kit protocol step 5).

ix. Transfer the supernatant to a clean 2 ml tube (use the 2 ml tubes indicated in step 3.4.A.i.d which are NOT included in the kit). Add 250 µl PPS solution and mix by inverting by hand 10 times (FastDNA kit protocol step 6).

x. Centrifuge at 14,000 x g for 5 min (FastDNA kit protocol step 7).

xi. During the above centrifugation, re-suspend the Binding Matrix provided in the kit by shaking well before use. Transfer 1.0 ml of re-suspended Binding Matrix to a clean 15 ml conical tube for each sample. (For easy pipetting, 5 ml Eppendorf tubes may be used for this step instead of 15 ml tubes).

xii. Transfer the supernatant from each sample to a tube containing 1.0 ml of re-suspended Binding Matrix.

xiii. Place the tubes in a rack and mix by inverting the rack for 2 min and then allow the silica matrix to settle for 3 min (FastDNA kit step 9). Centrifuge the tubes briefly at 1000 x g for 1 min in a centrifuge (using a swinging bucket rotor).

xiv. Remove 1.4 ml (700 µl x 2) of the supernatant from each tube by pipetting.

xv. Re-suspend the matrix in the remaining supernatant (with a pipette) and transfer all of the volume to a SPIN Filter in a catch tube provided in the kit. Centrifuge at 14,000 x g for 1 min (FastDNA kit protocol step 11). Empty the catch tube. If a sample was split into two tubes at step v above, transfer the matrix from the first tube to one spin filter and perform the centrifugation.
Then, discard the flow through and transfer the matrix from the second tube to the same spin filter and perform another centrifugation.

xvi. Add 500 µl prepared SEWS-M to each filter. Gently re-suspend each by stirring (with a P-200 tip) for ~ 10 sec. Do not poke the membrane. (FastDNA kit protocol step 12).

xvii. Centrifuge at 14,000 x g for 1 min. Empty the catch tube and replace the filter back into the same tube. (FastDNA kit protocol step 13).
Note: if the sample is too thick, some liquid won’t go through and you will need to centrifuge the sample for another 1 or 2 min.

xviii. Centrifuge at 14,000 x g for 2 min to dry the matrix. Discard the catch tube and replace with a new catch tube provided in the kit (FastDNA kit protocol step 14).

xix. Air dry the filter for 5 min at room temperature (kit protocol step 15).

xx. Add 75 µl DES to the matrix in the spin filter. Re-suspend the Binding Matrix by gently stirring with a P-10 pipette tip. Do not puncture the membrane. Incubate the tubes at 55 °C in a heat block for 5 min. (FastDNA kit protocol step 16).

xxi. Centrifuge at 14,000 x g for 1 min to recover the eluted DNA and discard the SPIN Filter (FastDNA kit protocol step 17).

xxii. Proceed to DNA purification.

B. DNA Purification Using the QIAquick® PCR Purification Kit

i. Upon opening a new kit:
a. Add 96-100% ethanol to Buffer PE wash solution (see bottle label for ethanol volume)
b. Add the pH indicator to Buffer PB.

ii. Add 375 µl of Buffer PB to the DNA sample and mix. If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.2, and mix.

iii. To bind DNA, apply the DNA sample to the QIAquick column and centrifuge at 14,000 x g for 30–60 s. Discard the flow-through and place the QIAquick column back in the same tube.

iv. To wash, add 750 μl Buffer PE to the QIAquick column, centrifuge at 14,000 x g for 30–60 s, discard the flow-through, and place the QIAquick column back in the same tube.

v. Centrifuge the QIAquick column once more for 1 min to remove residual wash buffer.

vi. Place each QIAquick column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 30 μl Buffer EB (10 mM Tris·Cl, pH 8.5)
to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge at 14,000 x g for 30-60 seconds to elute the purified DNA. Discard the columns and cap the tubes.

vii. Proceed to real-time PCR detection, or you may store the DNA samples at 4 °C for up to 2 days or at -20 or -80 °C for longer term storage.

Detection of Cyclospora cayetanensis by Real-time PCR Targeting the 18S rRNA Gene

3.5

A. Real-time PCR

For real-time PCR detection of C. cayetanensis in DNA extracts, follow the protocol described in BAM Chapter 19B, “Cyclospora cayetanensis Real-Time PCR Detection Method”, sections 1-5 for reagent ordering and preparation, reaction setup, and execution with three modifications to the method described in Chapter 19B:

• The real-time PCR reactions are run for 40 cycles instead of 45.
• Each DNA sample is analyzed in triplicate both undiluted and at a 1/10 dilution instead of a ¼ dilution.
  • 1/10 Sample Dilution Protocol: Transfer 2.0 µL of sample to a clean microfuge tube containing 18.0 µL of TE. Mix well and centrifuge briefly.
• The threshold for the C. cayetanensis target is manually set at 0.03 instead of 0.02.

B. Interpretation of Results

Positive DNA Samples i.

DNA samples are only considered Positive for the presence of C. cayetanensis if one (1) or more real-time PCR replicates produces a smooth exponential amplification signal crossing the threshold for the Ccay18S target reaction and the IAC target reaction is either Negative or Positive.

ONLY ONE REAL-TIME PCR REPLICATE OF AN UNKNOWN DNA SAMPLE OR 1/10 DILUTION OF THAT DNA SAMPLE NEEDS BE POSITIVE FOR THE Ccay18S TARGET IN ORDER TO CONSIDER A DNA SAMPLE POSITIVE.

Negative DNA Samples ii.

If a DNA sample Ccay18S target reaction produces all replicates with undetermined Ct and all replicates of the undiluted IAC target reactions produce a positive result crossing the threshold: Sample is NEGATIVE, no further action.

Invalid Results iii.

If one (1) or more replicates of the NTC sample or the DNA extraction control sample Ccay18S target reactions produces a positive result crossing the threshold, the experimental run is invalid and must be repeated.

If after repeat of an invalid experimental run, the DNA extraction control repeatedly produces a positive result and the NTC sample is negative, the DNA extraction procedure was likely contaminated, and the results must be considered invalid for the entire set of samples.

If one (1) or more replicates of the positive control sample Ccay18S target reaction is undetermined, the experimental run is invalid and must be repeated.

Inconclusive Results iv.

If a DNA sample Ccay18S target produces all replicates with undetermined Ct and one or more of the undiluted DNA sample IAC target reactions is undetermined: DNA sample is INCONCLUSIVE → consult CFSAN SME.

Appendix

Sanitizing Polypropylene (PP) Carboys and 225 ml Bottles

• Spray a liberal amount of a 0.65% bleach solution into the carboy/bottle. Add water to create approximately a 1:1 by volume bleach/water mixture at the bottom of the carboy/bottle.
• Screw the cap of the carboy/bottle back on and gently shake the carboy/bottle, ensuring that the bleach/water mixture rinses the entire inside of the carboy/bottle and cap.
• If you are cleaning a 225ml bottle, brush the bottle with a tube brush to remove any residual pellet material.
• Dispose of the bleach/water mixture into a waste container carefully. Leave the carboy/bottle to air dry for approximately two hours. The carboy/bottle does not need to be completely dry for the next step.
• Rinse the carboy/bottle with DI water once to remove residual bleach. Discard the liquid into the waste container.
• Autoclave the carboy/bottle at 121oC, 15 psi for approximately 20 minutes.