FREQUENTLY ASKED QUESTIONS FOR DIAGNOSTICS

 

GENERAL

1. How do you choose the preferred sample types.  Can I submit a sample that is not on the list?
2. There are multiple sample types per pathogen/panel listed on your submission forms.  Is it necessary to send all of the sample types?
3. What do you mean by “pool” samples? How does pooling affect the cost?
4. What is the specificity/sensitivity/efficiency of your assays?
5. Why is serum not an acceptable sample type?
6. How long can I hold a sample before shipping?
7. Do you have an IDEXX account?
8. How do I ship a sample to you?
9. Is it worthwhile to send a sample collected after the animal has started treatment?
10. What is your turn-around time?
11. Why is whole blood not recommended for the testing of Borrelia burgdorferi (Lyme Disease)?
12. Will your assays detect vaccine?

QUALITY CONTROL (QC)

1. What does it mean when a sample fails QC?
2. What are the possible reasons for failure to pass QC?
3. What procedures are in place to ensure the accuracy and quality of your laboratory’s work/tests?

BILLING

1. What types of payment are acceptable?
2. What is a FEIN# and why do you require it for billing?
3. I submitted a sample a couple weeks ago and still have not received a bill.  How long does it typically take to receive an invoice?

LARGE ANIMAL

1. Do you test for the neurotropic strain of Equine Herpesvirus 1 (EHV-1)?
2. Why do you recommend submitting dual samples (whole blood and nasal swab) for EHV-1 testing?
3. Do you test for EPM (Equine Protozoal Myeloencephalitis)?
4. What is the most accurate method for diagnosis of Streptococcus equi subspecies equi (S.equi equi )? How do I interpret the bacterial load?
5. I think my patient has an internal Streptococcus equi subspecies equi abscess. Can I submit a whole blood sample for diagnosis?

SMALL ANIMAL

1. Do you test for Feline Immunodeficiency Virus (FIV)?
2. Why isn’t Mycoplasma felis offered as part of the feline respiratory panel?
3. Does the Chlamydia felis/psittaci assay detect the isolate that infects guinea pigs/hamsters?

 

ANSWERS

GENERAL

How do you choose the preferred sample types.  Can I submit a sample that is not on the list?

The preferred sample types are those suggested by our veterinarians to be the most likely to host the specific pathogen(s) of interest. You may submit other sample types that are not on the list.  It is important to note, however, that the likelihood of detecting the pathogen in other sample types may not be as great. If you do decide to use a different sample type, it is a good idea to call the lab before collecting the specimen to make sure we have a means of processing the sample. 

There are multiple sample types per pathogen/panel listed on your submission forms.  Is it necessary to send all of the sample types? 

No, it is not necessary to send each listed sample type.  It is up to you to send the sample type that you think is most suitable for diagnosis based on your patient’s clinical signs/symptoms.  If you do send multiple types, please indicate on the form if you would like them pooled (see question 3) or tested individually.  If you wish to test them individually we will charge you per sample type as outlined below.

What do you mean by “pool” samples?

We pool multiple samples by mixing an aliquot of DNA from each sample type and testing the combined mixture for the specific pathogen(s) of interest.  This is a great option if you are not sure where the pathogen might be present as you can combine as many sample types as you like. It is important to note, however that pooling a large number of samples may diminish pathogen detection. We recommend pooling no more than three sample types.  For billing, if you pool samples you will only be charged for one test, instead of one test per sample type.  The example below outlines these two options; pooling multiple sample types, or testing each sample type individually. 

 

Option A: Pool samples
Patient Name:	Sample type(s):	Test requested:	Price:
“Harley”	POOLED whole blood, nasal swab	Canine Herpes Virus	$42.00
		Total:	$42.00
Option B: Samples tested individually
Patient Name:	Sample type(s):	Test requested:	Price:
“Harley”	whole blood	Canine Herpes Virus	$42.00
“Harley”	nasal swab	Canine Herpes Virus	$21.00*
		Total:	$63.00
			*half price for the same test on a different sample type for the same patient.

    We do not recommend pooling samples for EHV-1 (nasal swab and whole blood) and PHF (feces and whole blood). At different disease stages one or both of the samples may be positive.  Based on the results you can get an indication of the severity or disease stage of the infection.

What is the specificity/sensitivity/efficiency of your assays?

Each assay is 99-100% specific to the available sequence information (National Center for Biotechnology, NCBI), sensitive enough to detect as few as five copies of the gene, and 95-100% efficient.  Our primer/probe sequences are checked annually to make sure we use the most up to date sequence information. All our diagnostic assays undergo a strict validation process including testing on positive/negative controls to confirm proper assay design.

Why is serum not an acceptable sample type?

Serum separates the blood cells from the plasma.  Because some pathogens invade the blood cells sending a serum sample would eliminate detection by PCR. To ensure that our tests yield accurate results, the DNA contained within the blood cells must be tested. This is why whole blood (with EDTA additive) is preferable over serum.

How long can I hold a sample before shipping?

We recommend freezing the sample if you need to hold it over the weekend or for longer periods of time (up to a week).  If freezing is not an option, refrigeration can keep a sample fresh for up to five days.  EXCEPTION:  Refrigerate, DO NOT freeze, all fecal/environmental samples that will be submitted for Salmonellla spp PCR.  Our lab tests the sample directly for Salmonella and once again after a 20-hour selenite enrichment culture.  The bacteria will be killed during freezing and will not grow in culture. 

Do you have an IDEXX account?

While IDEXX delivers to our facility every morning, we do not have an IDEXX account number.  We recommend calling the IDEXX office in West Sacramento (916-373-9792) if you have questions in regards to shipping. 

How do I ship a sample to you?

Complete shipping instructions can be found in our sample submission packet (available through FAX or on our website: http://www.vetmed.ucdavis.edu/vme/taqmanservice/forms.html).  All samples need to be shipped on ice in a Styrofoam container via overnight delivery.  This preserves the integrity of the DNA/RNA during outside temperature fluctuations.  If possible, please put the sample submission form in a separate plastic bag to protect it from condensation from the ice pack or leaky samples.  All tubes should be wrapped in something padded (i.e. bubble wrap, vet wrap, paper) to prevent them from breaking during shipping.  We receive deliveries from FedEx, IDEXX, DHL, and UPS.  We DO NOT recommend shipping samples through USPS.  Many times the packages are retained at the main post office on central campus for several days before being distributed. 

Is it worthwhile to send a sample collected after the animal has started treatment?

It is up to the discretion of the veterinarian. While antibiotics or other various treatments will not inhibit the PCR process, there is a chance treatment will diminish the presence of the pathogen(s) beyond detection.  If possible, a pre-treated sample that has been kept cold (either in the freezer or refrigerator) would be preferable unless of course you are testing for confirmation of post treatment recovery.

What is your turn-around time?

In most cases, if we receive the sample by 11am we are able to FAX results by 5pm the same day.  If there is a large sample load that day, results will be faxed the next morning.  There are also a few exceptions.  If your sample does not pass quality control (see below) then extra time is needed for retesting.  In addition, paraffin (FFPE) samples take 24-48 hours due to intricate processing techniques.  Please keep in mind any time zone differences as well.  If you are in dire need of results and will be out of the office, we will be more than happy to call or email the veterinarian with results if you let us know ahead of time.

Why is whole blood not recommended for the testing of Borrelia burgdorferi (Lyme Disease)?

Whole blood is not a preferable sample type for PCR-based detection for the presence of Borrelia burgdorferi DNA because spirochetes may be present for a limited time in blood. Therefore, a negative PCR result on blood does not rule-out Lyme disease. Tissues most likely to harbor B. burgdorferi are lymph nodes in the region of the tick bite, muscle, fascia, and synovial (joint) membrane. Less frequently, the spirochetes end up in meninges.

Will your assay detect vaccine?

The general rule is that any vaccine using DNA of the pathogen to illicit an immune response has the possibility of being detected by PCR. This includes modified live vaccines which use an attenuated form of the virus/bacteria.  The trace amount of DNA used in these vaccines will eventually be metabolized by the patient’s immune system and will no longer be detectable by PCR.  The amount of time this takes varies depending on the vaccine and how it is administered (ex. intranasal versus intravenous).  Unfortunately, not a lot of literature is available establishing concrete timelines and our lab does not have the funds to perform our own research projects.  In general, however, most residual DNA will be shed/metabolized within one month of vaccination.  If vaccination history is unknown, this should be taken into consideration.  It is also important to remember to use PCR results in context with clinical symptoms/signs.  Please inquire about specific vaccines.

 

QUALITY CONTROL (QC)

What does it mean when a sample fails QC?

The DNA, cDNA (synthesized from the RNA) of every diagnostic sample is run with a standard housekeeping gene(s) to ensure successful nucleic acid extraction.  If the housekeeping gene is negative (Cycle Threshold value of 40 - after 40 thermocycle repetitions no amplification detected), or very weak (CT value greater than 30), we re-extract the nucleic acids from the back up sample, and run it through PCR a second time.  If the housekeeping gene for the backup sample is also negative or weak, we consider the sample to “fail quality control”; that is, there are not enough cells or the cells that were present have degraded beyond detection. 

What are the possible reasons for failure to pass QC?

Samples fail quality control for a variety of reasons, most of which depend on the sample type, or how the sample is handled.  The longer a sample sits without being processed, the more likely the DNA/RNA will degrade.  The sample should be shipped overnight on an ice pack to preserve the integrity of the nucleic acids. Also, some sample types are more susceptible to fail QC than others.  Samples with typically low cell concentrations (mainly urine, CSF, and joint fluid) fail quality control simply because there is not enough genetic material.  In these cases we spike the sample before the gDNA extraction with an unrelated gene from a potato.  After extraction, we test for the presence of this gene.  The PCR should yield a positive result if the DNA/RNA extraction was successful. 

What procedures are in place to ensure the accuracy and quality of your laboratory’s work/tests?

Below is a condensed version of our quality assurance procedures.  These control measures include, but are not limited to the following:

• All researchers are extensively trained in the areas of laboratory safety, sample preparation, reagent preparation, and data analysis.  Training records are held onsite. 
• The Real-time PCR Core Facility has physically separate rooms for sample receiving/preparation, assay preparation, amplification, and analysis.  Separation of workflow greatly decreases the risk of cross contamination between samples and reagents.
• All samples are tested with a standard housekeeping gene.  This ensures proper nucleic acid extraction and absence of inhibitors. 
• Positive and negative controls are run for the diagnostic assays daily.  This guarantees the assays are made correctly and no contaminants are present.
• Various areas within the lab are swabbed on a biweekly basis and tested for any pathogens that have tested positive within the previous two week interval. This ensures work areas are free from contamination.

*This is a small portion of the Quality Assurance program in place at the Real-time PCR Core Facility.  A complete guide can be found on our website.

 

BILLING/PAYMENT

What types of payment are acceptable?

We do not accept cash or credit card payments.  Personal or business checks are accepted and need to be made out to “The Regents of UC Davis”.  Please include the Invoice # on the check.  You will see two different numbers on your bill.  One starts with VME ____D, the other 01-_______. Please include the 01-_______ invoice number on the check.  If sending a check with your sample, please call prior to submission to find out exact charges.

What is a FEIN# and why do you require it for billing?

A Federal Tax Identification Number (FEIN# or FTID#) is a nine digit number; two digits followed by a dash and seven more digits (ex: 12-3456789). Every business is required to have one by law as it is used to identify your company by the Internal Revenue Service (IRS).  We require an FEIN# for all first time clients in order to set up an account in our University accounting database.  You may either call us with the FEIN# or fill in the field marked “Your Federal Tax ID (FTID) #” on the sample submission form. Please keep in mind that FEIN#’s are public domain, and should not be regarded as the equivalent to your company’s social security number.  Once an account is created for your company, an invoice will be mailed with the amount due and the address of the Cashier’s Office where the check should be sent.

I submitted a sample a couple weeks ago and still have not received a bill.  How long does it typically take to receive an invoice?

Because the University has so many checks and balances in regards to billing, receiving an invoice can take up to three weeks.  If you need to know the exact cost of our services so you can bill your client, please feel free to call us!

LARGE ANIMAL

Do you test for the neurotropic strain of Equine Herpesvirus 1 (EHV-1)?

Yes, we use two separate assays designed to detect a mutation on the ORF 30 gene of EHV-1. These assays differentiate the neurological (neurotropic) strain from the wild type (non-neurotropic) stain of EHV-1.  All samples submitted for EHV-1 testing will be run with neurotropic and non-neurotropic assays.  In positive cases, the report will include the strain type (neurotropic/non-neurotropic) and a viral load concentration. Guidelines for interpreting the viral load (see next question) will be included at the bottom of the report.

Why do you recommend submitting dual samples (whole blood and nasal swab) for EHV-1 testing?

The disease stage can be determined using the individual results for each sample type. At different times of the infection the nasal swab and the whole blood can be positive or negative.  The following is what we include in the report if the nasal swab and/or the whole blood samples are positive:

PCR results for EHV-1 are expressed qualitatively (positive or negative) and quantitatively in the case of a positive result. The viral load of a sample is calculated by the absolute number of EHV-1 (glycoprotein B gene) per million cells (either blood cells or nasopharyngeal cells). We are in the process of validating thresholds in order to better understand the viral kinetics of diseased and subclinical horses. Diseased horses (neurological or febrile) commonly have high (greater than 1x10^4) viral loads in blood and nasal secretions. Subclinical horses commonly have low (less than 1x10^3) to moderate (1x10^3 – 1x10^4) viral loads only in nasal secretions. The absolute number should be used judiciously and allow comparisons between samples taken at different time points. Viral load ranges are only suggestions and are not currently well-defined.

Do you test for EPM (Equine Protozoal Myeloencephalitis)?

We do have assays designed to detect the causing agents of EPM, Sarcocystis neurona and Neospora hughesi.  It is important to note, however, that these pathogens are found in the blood or cerebral spinal fluid (CSF) of infected horses for a very short period of time during infection.  Therefore, PCR testing of neurologic horses using either blood or CSF is not recommended as it has the possibility of generating false negative results.  Concrete diagnosis of EPM relies on the detection of antibodies against the two Apicomplexan parasites in serum and/or CSF using the quantitative serological IFAT.  The Immunology Lab at the Veterinary Medical Teaching Hospital at UC Davis offers such antibody testing (530-752-7373).

What is the most accurate method for diagnosis of Streptococcus equi subspecies equi (S.equi equi)?  How do I interpret the bacterial load?

You have one of two options for diagnosing S.equi equi:

1. Submit a nasal swab or guttural pouch wash for PCR testing.  If negative, no infection. If positive, retesting at a later time to determine carrier state is recommended.
2. Perform a single culture.

We include a calculated bacterial load with the results if the horse is positive for S.equi equi.  The bacterial load represents the number of S.equi equi cells per million nasopharyngeal cells.  Even though we do not have specific guidelines established for interpretation, the bacterial load is meant to give you an idea the bacterial presence.  We recommend testing a positive S.equi equi patient at a later date to see if the bacterial load has decreased or increased over time/treatment. 

I think my patient has an internal Streptococcus equi subspecies equi abscess. Can I submit a whole blood sample for diagnosis?

It is highly unlikely that an internal Streptococcus equi subspecies equi abscess will be detect by PCR in a whole blood sample.  Once the bacteria is harbored in the abscess site, it does not circulate in the blood stream frequently. The odds of collecting a blood sample containing the bacteria is very small. 

SMALL ANIMAL

Do you test for Feline Immunodeficiency Virus (FIV)?

As you may or may not be aware of, the Real-time PCR Research and Diagnostics Core Facility previously tested for FIV free of charge.  While we could not charge for the test, we still ran it because we firmly believed it was an important diagnostic tool for differentiating between active infection and exposure/vaccine. 

With that said, thanks to the legalities of patenting we are no longer allowed to offer a PCR test for FIV.  At this time, we are aware of only one facility in the country that does offer this test:  Auburn University, Alabama.  Because we have not used their facility, nor have we confirmed the accuracy of their tests, we cannot endorse them in any way.  We recommend calling their facility and inquiring about their test: 

http://www.vetmed.auburn.edu/molecular-diagnostics

Why isn’t Mycoplasma felis offered as part of the feline respiratory panel?

Instances of false positives are extremely high (~50%) in clinical cases when veterinarians submit corneal or nasal swabs.  That is, subsets of felines are carriers for M.felis and will test PCR positive even though they show no signs of infection. To prevent misdiagnosis, and therefore unnecessary treatment, only paraffin or fresh tissue should be submitted for M.felis.

Does the Chlamydia felis/psittaci assay detect the isolate that infects guinea pigs/hamsters?

Yes, the assay will pick up the caviae strain of Chlamydia that commonly infects guinea pigs/hamsters.