However, it is anticipated that simple point‐of‐care (POC) molecular diagnostic qPCR systems that are currently available in human medicine will be incorporated into veterinary clinics in the near future.
Currently, qPCR assays typically cost around US $95 in most veterinary diagnostic laboratories, with results available in a few days. Microbial culture of fungal organisms and identification typically costs approximately $55, with results available in several days to weeks. One limitation of molecular assays is the inability to determine antimicrobial susceptibility patterns. Clinicians using qPCR to detect microbial organisms, such as fungal organisms, may use anecdotal experience to develop an appropriate treatment plan. As the mechanisms of resistance are better understood in fungal organisms, molecular techniques may be utilized to determine the potential for an organism to have inherent resistance to certain antifungal agents. We anticipate that in the future we should be able to detect, identify, and determine antifungal susceptibility patterns based on an organism’s DNA signature.
Uterine samples collected for qPCR should be collected and handled with minimal contamination. Samples that are “sterile” may not have microbial growth, but the DNA of microbial pathogens may still be present. DNA contamination is one cause of a false‐positive diagnosis when using a molecular‐based assay. Steam autoclaving will reduce the amount of microbial DNA present on equipment. Glass blood tubes without anticoagulant and new 15 or 50 ml centrifugation tubes appear to have minimal microbial DNA.
The equine uterus has historically been noted to be a “privileged site” that should be free of all microbial organisms and therefore no microbial DNA should be present in uterine samples from “normal mares.” However, recent studies have suggested that the equine uterus may contain a normal flora of non‐pathogenic microorganisms.
Deep‐seated infections and/or organisms in a biofilm may be present in an arrested state with a low replication rate, which may make detection difficult using traditional microbial culture. However, these quiescent organisms still contain DNA and may yield a positive qPCR result.
Detection of microbial DNA by qPCR and failure to detect organisms by traditional culture may be due to: (i) the ability of qPCR to amplify the DNA of non‐viable organisms and free fungal DNA; (ii) the failure to provide the appropriate growth conditions for fungal organisms (agar, temperature, etc.); or (iii) the organism having an extremely low replication rate in which growth may take weeks to months.
Clinicians should use caution in interpreting the results of qPCR assays as they are often extremely sensitive and may detect DNA from dead organisms and may not match the clinical picture of a case. Negative controls may exhibit microbial DNA detection (i.e., contamination with dead bacterial DNA) by 35 or more Ct cycles. A “positive” signal from a clinical sample beginning at 35 Ct cycles or more should be considered to be the result of contamination. A sample is considered to contain organisms that are biologically relevant if microbial DNA is detected in the first 30 Ct cycles. Adequate controls of known organisms at set CFUs should be utilized to confirm that the qPCR assay is functioning normally and to estimate the organism load in a clinical sample.
Further Reading
1 Byrne BA. 2007. Laboratory diagnosis of bacterial infections. In: Sellon DC, Long MT (eds). Equine Infectious Diseases. St Louis, MI: Saunders Elsevier, pp. 236–44.
2 Ferris RA, Dern K, Veir JK, Hawley JR, Lappin MR, McCue PM. 2013. Development of a broad‐range quantitative polymerase chain reaction assay to detect and identify fungal DNA in equine endometrial samples. Am J Vet Res 74: 161–5.
3 Ferris RA, Veir JK, Lappin MR, McCue PM. 2010. Development and clinical application of a broad range 16S quantitative PCR assay for detection of bacteria in the uterus of the mare. Anim Reprod Sci 121: S98–S100.
17 Uterine Cytology Collection: Swab/Brush
J. Dascanio1 and Ryan A. Ferris2
1 School of Veterinary Medicine, Texas Tech University, USA
2 Summit, Equine, USA
Introduction
A cytologic examination of the uterus should always be performed in conjunction with a uterine culture. Cytologic evaluation of the uterus involves the collection and interpretation of cells lining the uterus (endometrium) and within the uterine lumen. Endometrial cytology is a rapid, inexpensive technique to detect the presence of endometritis in the mare.
There are circumstances whereby a uterine cytology may be normal (no inflammatory cells) and an infection is still present. These include isolated infections, deep‐seated infections, or infections early in their course. It is also more common to have a negative cytology (i.e., no inflammatory cells) in the presence of an Escherichia coli infection than in the presence of a Streptococcus equi subspecies zooepidemicus infection.
Typically a guarded swab or a brush is used to obtain a uterine cytology sample; collection of a uterine sample with unguarded swabs may result in significant contamination from the vestibule, vagina, and cervix. A low volume uterine lavage may be more diagnostic than swab or brush samples in situations where isolated infections are present (see Chapter 18).
Equipment and Supplies
Tail wrap, tail rope, non‐irritant soap, roll cotton, stainless steel bucket, disposable liner for bucket, paper towels, sterile lubricant, sterile sleeve, guarded culture device (Kalayjian swab, single‐guarded; McCullough swab, double‐guarded or uterine brush), microscope slides, modified Wright’s stain (Diff‐Quik® or equivalent), microscope.
Technique
Remove feces from the rectum.
Place a tail wrap and tie the tail out of the way (see Chapter 4).
Clean and dry the perineum of the mare (see Chapter 3).
Place a sterile sleeve on the arm.
Place the guarded culture device into the palm of the hand.
Place sterile lubricant on the knuckles and down the length of the sleeve being careful not to get lubricant onto the palm. If the culture device becomes inundated with lubricant, it may be more difficult to obtain a diagnostic sample.
Rub lubricant from the knuckles onto the vulva, straighten the fingers and insert through the vulva, staying dorsal so as to not rub across the clitoris.
Using a slight rotating motion, pass the hand into the vagina so that the mid‐forearm is to about the level of the vulva. This should enable palpation of the external cervical os.
Gently insert the index finger into the external cervical os. Sometimes the os may be off‐center, located slightly downward, or to the left or right of center.
Pass the index finger through the cervix to the last knuckle (metacarpo‐phalangeal joint). Usually one can tell when the tip of the finger exits the internal
