J-1503020-10_Samples_to_PrevMed.pdf

J‐1503020‐10

Mosquito Submission Guidelines for the Testing of

Zika, Dengue, and Chikungunya Viruses by the

Laboratory Sciences Division, Public Health Command – Atlantic (May 2016)

1. INTRODUCTION

a. Due to the emerging threat from Zika (ZIKV), dengue (DENV), and chikungunya (CHIKV) mosquito‐borne viruses, we, the staff of the Army Public Health Command‐Atlantic (PHC‐A) are broadening and enhancing our surveillance support to Army installations in our area of responsibility (AOR). As background, our Agency now supports all the Army installations east of the Mississippi as well as those in the Caribbean. You may have seen recent guidance provided by the Office of the Secretary of Defense, Assistant Secretary of the Army, and the Medical Command that identifies our Laboratory Sciences Division (LSD) as one of the military testing facilities for the detection of these pathogens in mosquitoes. To that end the PHC‐A LSD will test submitted mosquitoes as long as submissions are made in accordance with this laboratory protocol. This requires that mosquitoes be sorted, identified, pooled, and shipped using supplies that we provide. If you feel that you are prepared to enroll in our program please contact the LSD Laboratory Manager, Alexandra Spring (Alexandra.r.spring.civ@mail.mil) and copy the Chief, ESB (benedict.pagac.civ@mail.mil).

b. This protocol only addresses the procedures needed to prepare specimens for testing.

Guidance on mosquito collection methodology is provided in separate documents. ALL FIELD

COLLECTION ACTIVITIES SHOULD BE PERFORMED WHERE DUE REGARD IS GIVEN TO THE

SAFETY OF THE INDIVIDUALS PERFORMING SURVEILLANCE, INCLUDING THE USE OF

REPELLENTS AND THE WEARING OF PROTECTIVE, PERMETHRIN‐TREATED CLOTHING.

CONSULT WITH YOUR CHAIN‐OF‐COMMAND AND MEDICAL AUTHORITIES TO DETERMINE IF

THERE IS ANY REASON TO EXEMPT WORKERS THAT MIGHT BE AT INCREASED RISK FROM

ZIKV, DENV, OR CHIKV VIRUS ILLNESS.

2. SAMPLE SUBMISSION KIT

a. The PHC‐A LSD will supply the necessary materials for collaborators to submit mosquito samples for testing. The standard sample submission kit that PHC‐A supplies contains: pre‐ printed bar labels specific to your installation, desiccant packets, 2.0mL vials, THREE boxes with dividers to hold vials (“vial box”), , and THREE 11 X 7 X 2.5 inch foam‐lined shipping box (“shipping box”). Once a submission is received, the shipping box and a resupply of vials will be mailed back to the submitter for use.

Fig 1. The Standard Sample Submission Kit provided by the PHC‐A Laboratory includes: bar Code labels, vials, desiccant packets, vial box (3), and shipping box (3).

3. HANDLING MOSQUITO TRAP CATCHES

a. All efforts should be made to protect mosquitoes from damage so that they can be identified. Transport mosquitoes promptly from the field to the processing facility in a protected box. Place the specimens in a freezer (0 degrees F, ‐18 deg C) for at least 2 hours.

Dry ice in coolers can also be used and will normally kill mosquitoes in 15 minutes. If mosquitoes must be held for several days before processing (not recommended) they should be kept refrigerated to inhibit mold. Please submit your samples weekly.

b. If mosquitoes were trapped under especially high moisture conditions (e.g., rain), allow them to air‐dry on the bench‐top for about 1 hour, before processing, again to reduce mold.

Mold‐covered mosquitoes cannot be tested.

4. SORTING, IDENTIFYING, & POOLING

a. Empty the trap bag contents onto a light‐colored pan and sort and discard non‐ mosquitoes and male mosquitoes. It is good laboratory practice to wear disposable gloves when directly handling any biological sample. If there is an abundance of non‐target insects, gently tapping the pan at an angle will spread out the specimens and some convex non‐ mosquitoes will slide or roll to the edge, making the sort go faster. Gently push, using a small paint brush or the blunt end of forceps, all mosquitoes that look alike, into little “piles.” A sheet of paper can sometimes be used as a “scoop” to remove non‐mosquitoes or to gently move the same species. The “pushing” and “piling” method is less tedious and less damaging than grabbing each mosquito individually with forceps. Mosquitoes can be then identified as a group. Even though males are not to be submitted for testing, record their numbers on the datasheet (see para 4) using the ‘Notes/Comments’ section. Males do not need to be identified. Information on male mosquitoes is strictly for your records since the presence of males may be an indication of nearby breeding. Always handle mosquitoes over a pan. They are often not easy to find if dropped on the floor.

Fig 2. Sorting of trap catches is made easier by using a white pan and by pushing like‐appearing mosquitoes into groups. Identifications can then be made of groups (rather than individuals) by microscopic examination.

b. Identify female mosquitoes using locally available regional keys. Keys and identification guidance are available from PHC‐A by request. Below is a basic identification flow chart for the primary and secondary vectors of ZIKV, DENV, and CHKV in our AOR. We would like all Aedes albopictus and Aedes aegypti identified and pooled separately. Other Aedes spp can be minimally pooled as “Aedes.” The “Other Mosquitoes” can be minimally pooled at the genus level (e.g., Culex, Anopheles, Psorophora, Coquilletidia, Culiseta). Specimens will be spot‐ checked by the lab. Vector species will be tested for appropriate pathogens (e.g., Culex spp.

will be tested for West Nile virus). Other, non‐vector genera and species will be entered into the database, even if not tested.

Fig 3. Basic flow chart for primary and secondary vectors of ZIKV, DENV, and CHKV in the PHC‐A AOR.

c. Pool up to 25 female mosquitoes, of the same genus, from the same trap, collected on the same day, and place them in the vial provided in the PHC‐A supply kit. These vials are of specific dimensions for the PHC‐A analytical instruments, so please do not substitute other vials. Pool sizes can range anywhere from 1 to 25 female mosquitoes, but not more than 25.

Please wipe your forceps with a clean tissue or kimwipe after closing each pooling vial. This reduces the possibility of cross‐contamination between pools.

Fig 4. Pooling one to twenty five mosquitoes that are the same Genus (and species), from the same trap, collected on the same night. Wipe the forceps clean between each pool.

d. Fold one small desiccant packet in half and insert it in the vial opening, PRESSING IT TO

JUST THE POINT WHERE IT IS FLUSH WITH THE LIP. There should be ample room in the lower half of the vial for mosquitoes. Gently close the vial cap making sure all mosquitoes are in the lower portion of the vial. The desiccant prevents mold.

Fig 5. Gently placing the folded desiccant packet in the top portion of the vial.

e. Apply the pre‐printed bar code label lengthwise down the side of the sample tube. Write, with an indelible pen, the four character code and the last three digits of the corresponding log number (e.g., WRFG‐001) on the cap. Writing on the cap may not be easy (especially if you are a coffee drinker), but this helps you to place them, in order, in the vial box, and helps us to view them without handling them.

Fig 6. Bar code label applied lengthwise.

Fig 7. Sample number and location code written on cap with indelible pen.

5. DATA RECORDING

a. The PHC‐A data form required for sample submission is entitled “Mosquito Pooling Data Sheet.” On the back of that sheet are “Codes and Explanations.” These detailed instructions and codes must be used when filling out the form.

Figs 7 & 8. Front and back of the Mosquito Pooling Data Sheet showing Codes and Explanations.

b. It is asked that entries be made as neatly as possible. This prevents errors and speeds up database entry. Please leave the mosquito ID column block empty if no mosquitoes are collected in that particular catch. The form can be supplied electronically (by request) so that entries may be made by computer, but we still ask that a hard copy always be included in the box containing samples.

Fig 9. Example of properly filled‐out Mosquito Pooling Data Sheet.

6. SHIPMENT

a. Place vials, in order, in the vial box and place the vial box inside the padded shipping box.

Be sure to include a copy of the completed “Mosquito Pooling Data Sheet” while retaining a copy of the datasheet for your records. The foam insert should be sufficient to hold the box in place. If there is any chance of it moving in transit (i.e., the foam insert is missing, please add packing material to stabilize it. Arrange for timely delivery of the samples. Ship samples using a commercial carrier that provides tracking (e.g., FedEX, UPS, DHL)

Fig 10. Sequence of procedure for packing samples for shipment.

b. The testing laboratory is not open on weekends so please time shipping to be received no later than close of business Friday. Ship samples to:

Public Health Command –Atlantic

Laboratory Sciences Division, ATTN: Alexandra Spring

4411 Llewellyn Avenue

Ft George G .Meade, MD 20755

Phone: 301 677‐2607

7. ASSISTANCE. For lab support or questions regarding shipping supplies, vials, bar codes, database issues or sample status, contact: Alexandra Spring (301) 677‐2607. For operational support including surveillance, control, and response planning contact: Ben Pagac (301) 677‐ 3932.