2 Laboratory tests
Unless the diagnosis is immediately obvious, many dermatology cases in veterinary medicine present something of a challenge to the clinician. In these more complex cases, consideration of the signalment, history and physical examination allows formulation of a differential diagnosis, and then various tests and therapeutic trials are employed to reach a definitive diagnosis. The tests available include those performed within the practice laboratory and those offered by external commercial labs.
The ideal diagnostic test is a procedure that gives a rapid, convenient and inexpensive indication of whether a patient has or has not a certain disease. Unfortunately, most diagnostic tests suffer from a problem of inherent unreliability and cannot always distinguish the normal from the abnormal, leading to false-positive and false-negative results. Biological variation, test methodology and the skill of the clinician all combine to account for test unreliability. It is important the clinician is aware of the limits of any diagnostic test, and test results should be interpreted in the light of the case history and clinical signs. This interpretation is of crucial clinical importance and is one of the most common sources of diagnostic error.
There are measures that the clinician can take to minimize the incidence of false-positive and false-negative test results. Firstly, take full histories and perform full physical and dermatological examinations and draw up a differential diagnosis (see Chapter 1). Whilst it is important to perform basic screening tests such as skin scrapes and cytology, diagnostic tests used should as far as possible be targeted to the diseases on the differential diagnosis list. The indiscriminate use of a wide variety of diagnostic tests will increase the likelihood of false-positive and false-negative test results. It is a common mistake to assume that because skin lesions look severe, autoimmune disease is involved. Statistically, the low prevalence of rare diseases will increase the chance of a false-positive test result, leading to an erroneous diagnosis. Much more frequently, severe skin lesions are just an unusual manifestation of a common disease.
Ectoparasitic diseases encountered in small animal practice are shown in Table 2.1. The tests available for the detection of external parasites are combings and coat brushings, the use of acetate strips, skin scrapes, hair plucks, a scabies IgG ELISA test and histopathological examination.
|Notoedres cati||Rare in cats|
|Pelodera, hookworms||Uncommon to rare|
Become familiar with the operation of your microscope. Apart from when using acetate strips, always apply a coverslip over any material to be examined microscopically. Having excessive material on the slide will make thorough examination difficult. Low power (×4 objective) is sufficient magnification for detection of ectoparasites, although a ×10 objective may be required for more detailed examination of specimens.
A flea comb may be used to collect material from the coat for gross examination. The test is useful for the detection of larger external parasites such as fleas and lice. If fleas are present in large numbers, they should be seen. However, fleas are only detected in around 60% of cases of canine flea allergy dermatitis and the figure is considerably lower in cats, who remove evidence of flea infestation by grooming. Thus, flea combing is an insensitive test for the diagnosis of flea infestation.
This is a useful and moderately sensitive test for the diagnosis of surface and superficial parasites such as fleas, lice, harvest mites and Cheyletiella spp. mites. Scale is dislodged from the dorsal trunk (Fig. 2.1) onto a piece of A4 paper by combing or vigorous brushing with the fingertips. The paper is folded and tapped so that the material collected falls into the crease. Hair is removed and the material can be examined grossly for the presence of flea faeces, as well as larger parasites such as lice. The material should be collected onto clear adhesive tape, mounted onto a glass slide and examined under the low-power light microscope. The complete area under the adhesive tape should be carefully examined.
Microscopic examination of hair plucks may be useful to detect Demodex spp. mites (Fig. 2.2) and Cheyletiella spp. or louse eggs. Hair plucks are very useful when taking samples from areas that are difficult to scrape, such as the feet in the case of pododemodicosis, when skin scraping would require sedation. Fifty to 100 hairs are plucked and mounted in liquid paraffin on a glass slide under a coverslip. The hair tips, shafts and bulbs should all be carefully examined. One study showed deep skin scraping to be more sensitive than hair plucks in cases of localized and squamous demodicosis, and therefore demodicosis should not be ruled out on the basis of not finding mites on hair plucks.
Skin scrapings are used to detect the presence of superficial and deep parasitic mites such as Cheyletiella, Sarcoptes and Demodex spp. Skin scrapings should be performed when there is evidence of erythema, scaling, crusting, alopecia, or a papular or pustular eruption. In cases of canine scabies, the hocks, elbows and pinnal margins are prime sites for finding mites. Avoid scraping areas that are excessively crusted or excoriated, as this may lead to false-negative results. Three to five different sites should be sampled (five in the case of suspected scabies or demodicosis).
Hair should be clipped with a No. 40 clipper blade. When scraping for Demodex spp., it helps to gently squeeze the skin between thumb and forefinger, as this extrudes mites from the hair follicles. A small amount of liquid paraffin to suspend collected material (or water if using potassium hydroxide) is applied to the area to be scraped. A blunted No. 10 scalpel blade is used to scrape material from the skin surface. Deep skin scrapes resulting in capillary oozing should be made when looking for Sarcoptes or Demodex spp. mites (Fig. 2.3). Collected material is mounted onto a glass slide in liquid paraffin or potassium hydroxide. A coverslip should be applied. Examine samples for ectoparasites under the low-power objective and scan the entire area under the coverslip.
Commercial laboratories offer a Sarcoptes IgG ELISA test. This is a highly sensitive (∼90%) test, although false-negative reactions may occur in early cases because seroconversion may take up to 4 weeks following mite exposure. False-positive reactions may be seen in cases of canine atopic dermatitis due to house dust mite hypersensitivity because of cross-reaction between Dermatophagoides spp. house dust mites and Sarcoptes scabiei.
Histopathological examination is a very sensitive test for the diagnosis of demodicosis and if skin scrapes have been unrewarding, but demodicosis is still suspected, then histopathology would be the definitive rule out. Histopathologically, canine demodicosis results in interface mural folliculitis, perifolliculitis, folliculitis and furunculosis, and nodular dermatitis. Mites should be evident on histopathological sections. Histopathology is a highly insensitive test for other ectoparasitic diseases.
With the exception of demodicosis, tests for ectoparasites are of low sensitivity but 100% specificity. Sarcoptes spp. mites are only found on skin scrape examination in around 50% of cases of canine scabies and Cheyletiella spp. mites may also be difficult to detect in some cases. Indeed, in multi-animal households, it can be helpful to check in contact (and frequently unaffected) animals for the presence of the parasite. Fleas are notoriously difficult to find, particularly in cats with any of the feline manifestations of pruritus.
The situation is different in the case of demodicosis. Provided at least five carefully taken and thoroughly examined deep skin scrapes are negative, then the clinician can be confident in ruling out demodicosis as a cause of the skin disease, although there are rare exceptions. Possibly due to the thickness of their skin, it can be difficult to detect mites in the Shar Pei and occasionally mites can be difficult to find in cases of pododemodicosis. With such cases, histopathological examination is indicated to rule the disease in or out. Trial therapy is inappropriate for suspected demodicosis.
Wood’s lamp: Wood’s lamp is an ultraviolet light with a wavelength of 360 nm. Only lamps with two bulbs and a magnifier should be used. It is important to switch the lamp on and allow it to warm up for 5 minutes prior to examination. Examination of the animal should be conducted in a darkened room. Hair shafts infected with certain strains of Microsporum canis fluoresce an apple green colour under Wood’s lamp examination due to tryptophan metabolites. Wood’s lamp examination is a test with high specificity (100% in the right hands) but low sensitivity, as only 50% of strains of Microsporum canis fluoresce. Rare infections with M. audounii, M. distortum and Trichophyton schoenlenii may also result in fluorescence.
Direct microscopy: Most dermatophytosis cases in domestic animals involve ectothrix invasion of hair shafts by fungal spores which can be visualized under ×40 magnification using the light microscope. Fluorescing hairs or hairs from lesions may be plucked for direct microscopic examination. Samples should be mounted on the slide in liquid paraffin or potassium hydroxide. Hair shafts with distorted or damaged cuticles should be examined under higher power for the presence of fungal spores (Fig. 2.4). Although a test with high specificity in the right hands, this is not a sensitive technique for the diagnosis of dermatophytosis in the hands of the inexperienced clinician.
Fungal culture: Fungal culture is arguably the most sensitive test for dermatophytosis and should be performed whenever this disease is suspected. The simplest method of collection of material for culture is the MacKenzie brush technique. This is most commonly used for routine screening of cats for dermatophytosis. A new toothbrush is used and hair and scale are collected on the bristles by brushing the hair coat for 30–60 seconds, paying particular attention to lesional skin. The shaft of the brush can be cut off and the entire head of the brush submitted to the laboratory. In addition to using the brush method, it is advisable to culture scale scraped from lesions and also hair plucks from lesion margins.
Dermatophyte test medium (DTM) is used as an in-practice growth medium for the diagnosis of dermatophytosis. DTM is Sabouraud’s dextrose agar with various antimicrobials that suppress bacterial and some saprophytic fungal growth, along with phenol red as an indicator. Dermatophytes metabolize protein in the medium first, giving off alkaline metabolites which turn the pH indicator red (Fig. 2.5). This should happen within 10 days and should occur as the fungal colony grows. Saprophytic species of fungi metabolize carbohydrates first and the red colour change should only appear after 10 or more days and subsequent to colony growth. There are potential problems associated with the use of DTM. The agar should be inspected daily for evidence of fungal growth and colour change; some saprophytic species of fungi can induce a colour change within 10 days and Microsporum persicolor may produce a colour change after 10 days. Thus, the identity of any fungal mycelium should be confirmed by microscopic examination, which requires specialist knowledge. Furthermore, DTM may not be a suitable growth medium for the identification of some fungi that will only sporulate on Sabouraud’s dextrose agar. Nevertheless, DTM culture remains a useful in-practice tool for screening for dermatophytosis, but the clinician should be aware of the limitations.
Figure 2.5 Dermatophyte test medium showing red colour change and white colony growth on the surface at 10 days. Note the darkly pigmented growth of saprophytic fungal organism on the edge of the plate.