Comparison of real-time PCR with fungal culture for the diagnosis of Microsporum canis dermatophytosis in shelter cats: a field study

Abstract

Objectives

Fungal culture requires at least 14 days for a final result, compared with 1–3 days for PCR. The study compared a commercial real-time dermatophyte PCR panel with fungal culture in cats in a shelter setting for: (1) diagnosis of Microsporum canis infection; and (2) determination of mycological cure.

Methods

This was a cross-sectional, observational study of cats with suspicious skin lesions or suspected exposure to dermatophytosis. Hair samples were collected for fungal culture and PCR prior to treatment and at weekly intervals until two negative culture results were obtained.

Results

One hundred and thirty-two cats were included, of which 28 (21.2%) were culture positive and 104 (78.8%) culture-negative for M canis. PCR correctly identified all culture-positive cats and 92/104 culture negative cats; there were 12 false-positive PCR results. The sensitivity and specificity of PCR were 100% (95% confidence interval [CI] 87.7–100) and 88.5% (95% CI 80.7–93.9), respectively. Data from 17 cats were available for assessment of mycological cure. At the time of the first and second negative fungal cultures, 14/17 (82.4%) and 11/17 (64.7%) tested PCR positive, respectively.

Conclusions and relevance

PCR showed high sensitivity and specificity for diagnosis of M canis dermatophytosis compared with fungal culture, but was unreliable for identifying mycological cure. False-positive results were relatively common. There were no false-negative PCR results and a negative PCR test was a reliable finding in this study. The ability to rapidly diagnose or rule out dermatophytosis could be a valuable tool to increase life-saving capacity in animal shelters.

Introduction

Feline dermatophytosis is a superficial fungal infection of the skin caused primarily by Microsporum canis.^1–4 It is both contagious and zoonotic, and treatment is lengthy and expensive. The disease is most prevalent in kittens, the most vulnerable and adoptable population in a shelter. For these reasons, the disease presents major challenges for animal shelters, despite its benign clinical course.

Rapid identification of infectious and contagious diseases in a shelter is important for individual and population management. New diagnostic methodologies, such as SNAP ELISA and point-of-care antibody titer tests, have vastly improved diagnostic capacity and allow rapid identification of diseased animals. With respect to dermatophytosis, fungal culture is the gold standard diagnostic test to confirm the presence of fungal spores and/or hyphae in a lesion.^1,4–6 Arguably the main disadvantage of culture is the time required for confirmation of the results for cats with suspicious skin lesions or cats being treated, which can vary from 10–21 days.^1,7 A commercial PCR test for dermatophytosis offers rapid results within 1–3 business days and potentially a cost-effective alternative compared with the costs of establishing and outfitting a laboratory with personnel and equipment compliant with regulatory requirements.

The goals of this study were to assess a commercial real-time PCR test in a shelter setting for: (1) sensitivity and specificity compared with fungal culture for diagnosis of M canis dermatophytosis; and (2) agreement with fungal culture for determination of mycological cure.

Materials and methods

Institutional review and approval

The study was approved by the chief executive officer of the shelter. The cats were managed routinely, the only change being that the hair samples collected were larger than usual.

Shelter

The shelter was a private, limited admission, adoption-guarantee facility servicing 14,000 animals per year. Surrenders accounted for 4000 animals annually, of which 2600 were cats. Transfers from other shelters frequently included animals with surgical or medical conditions, including dermatophytosis, and accounted for 820 annual feline intakes.

Diagnostic sampling

Hair samples for fungal culture and PCR were collected prior to treatment. Culture-positive cases were sampled weekly during treatment, until mycological cure.^1,8

Samples were collected by plucking hairs at the periphery of lesions and brushing the coat with a sterile toothbrush, using recommended techniques.^1,2 A larger-than-normal hair sample was collected. Each sample was divided by one of the authors (LM, JM), with half retained for fungal culture and half submitted for PCR.

Fungal culture

Cultures were performed in-house by registered veterinary technicians (LM, JM). The samples were processed in a dedicated culture laboratory with a fume hood and incubator meeting the standards laid down by Health Canada’s Human Pathogens and Toxins Act.

Samples were inoculated onto Sabouraud dextrose agar with CCG antibiotic (Oxoid). Plates were stored at 2–8ºC, then warmed to 37ºC before plating. They were incubated at 37ºC for 3 days, then kept in the dark at room temperature for 21 days. Plates were checked at 3, 7, 10, 14 and 21 days. If fungal growth occurred, acetate tape was used to transfer the fungi to a microscope slide, where they were identified on a wet mount stained with lactophenol cotton blue, using classic microscopic morphology. The slides from all initial positive fungal cultures were submitted to a reference laboratory (IDEXX Laboratories Canada) for confirmation of the diagnosis.

PCR

Real-time PCR was performed by a commercial reference laboratory (IDEXX Laboratories Canada), using proprietary methodology. The PCR result was reported as positive or negative for Trichophyton or Microsporum species.

Study design

This was a cross-sectional observational study conducted from January 2014 to May 2015. Cats were assessed for skin lesions by a veterinarian or registered veterinary technician. They were enrolled in the study if they had alopecia with or without crusting or scaling, or were considered to be at high risk for dermatophytosis, owing to exposure to positive cats.

Cats were classified into three groups (see Table 1): those with a high risk of dermatophyte infection (compatible lesions ± exposure to infected cats); those with only exposure but no lesions; and those considered low risk, having lesions not compatible with dermatophytosis and no history of exposure. Low-risk cats were usually rinsed once in lime sulfur diluted 1:16 (LimePlus Dip; Aventix). Dermatophytosis suspects and those with a high risk of exposure were isolated and treated with itraconazole (5 mg/kg q24h, Sporanox; Janssen Pharmaceuticals) and twice-weekly lime sulfur rinses until release from isolation. ⁸ Itraconazole was administered for a total of 21 days to culture-positive cats. ⁸

Table 1.

Summary data

Risk group	Criteria	Culture positive	Culture negative	Total (n)
High risk	Suspicious skin lesions ± history of exposure	24 (39.3)	37 (60.7)	61
Exposed	Non-lesional, high risk of exposure	2 (6.7)	28 (93.3)	30
Low risk	Skin lesions, not typical for dermatophytosis	2 (4.9)	39 (95.1)	41
Total		28 (21.2)	104 (78.8)	132

Data are n (%) unless otherwise indicated

In this study, cats were classified as dermatophytosis-positive if M canis was grown on the initial fungal culture, regardless of the presence or absence of skin lesions. Mycological cure was defined as two consecutive negative cultures.^1,8

Statistical analysis

Sensitivity and specificity were calculated using a 2 × 2 contingency table, and 95% confidence intervals (CIs) were determined, using GraphPad Prism Version 7.

Results

Study population

One hundred and fifty cats were entered into the study. Eighteen were excluded from data analysis for the following reasons: insufficient quantity of sample for PCR (n = 8), culture plate overgrown by contaminant (n = 8), fungal culture positive for Microsporum gypseum (n = 1) and Trichophyton mentagrophytes (n = 1). Of the 132 cats included in the analysis, 28 (21.2%) cultured M canis-positive and 104 (78.8%) were culture-negative. Eleven of the 28 positive cats were excluded from assessment of mycological cure because of missing data points during treatment.

Based on the history and the nature of their skin lesions, 61 cats were considered to be at high risk for dermatophytosis, 30 exposed and 41 low risk (Table 1). Of those in the high-risk group, 39.3% were fungal culture-positive vs 6.7% of cats in the exposed group and 4.9% of cats in the low-risk group.

Performance of PCR for diagnosis of M canis dermatophytosis

The sensitivity of PCR was 100% (95% CI 87.7–100) and specificity was 88.5% (95% CI 80.7–93.9) (Table 2).

Table 2.

Performance of dermatophyte real-time PCR panel for diagnosis of Microsporum canis in cats in a shelter

	Culture positive	Culture negative	Total
PCR positive	28	12	40
PCR negative	0	92	92
Total	28	104	132
Sensitivity	100 (87.7–100)
Specificity	88.5 (80.7–93.9)

Values are shown as % (95% confidence interval)

Using fungal culture as the ‘gold standard’, there were 12 instances where PCR was positive and the initial fungal culture was negative. Repeat fungal cultures were performed in 9/12 cases. In 2/9, repeat fungal cultures were positive for M canis, indicating that the original negative fungal culture was inaccurate but the positive PCR results were accurate. In seven cases, repeat fungal cultures were again negative and in these cases PCR results were, in fact, false-positive findings. Five of these seven cats had a history of exposure to dermatophytosis.

Performance of PCR for confirmation of mycological cure

Seventeen cats had PCR and culture results for all time points until the second negative culture (NC2). Fourteen (82.4%) remained PCR positive at the time of the first negative culture (NC1) and 11 (64.7%) were PCR positive at the second negative culture (Table 3). Eight cats were PCR positive at both NC1 and NC2. The three cats that were PCR negative at NC1 were positive at NC2, and six different cats were PCR negative at NC1.

Table 3.

Agreement between fungal culture and PCR in 17 cats at mycological cure

	NC1	NC2	Total (n)
PCR positive	14 (82.4)	11 (64.7)	25
PCR negative	3 (17.6)	6 (35.3)	9
Total	17	17

Data are n (%) unless otherwise indicated

NC1 = first negative culture; NC2 = second negative culture

Of the 11 cats that were PCR positive at NC2, four tested negative the following week, three remained positive and four were not recorded. Of the six cats that were PCR negative at NC2, five remained negative a week later and one was not recorded.

Discussion

The dermatophyte real-time PCR panel showed high sensitivity and specificity for the diagnosis of M canis dermatophytosis in cats in this study. Comparison of fungal culture (± microscopy) and PCR in human studies has shown excellent agreement, often with superior detection rates by PCR.^6,9–14 A veterinary study reported accurate results for nested PCR, with an area under the curve of 93.6% for feline samples. ¹⁵ Nested PCR had specificity of 94.4% and sensitivity of 94.9% for feline samples, and there was no cross-reaction with non-dermatophytic fungi on the culture plates.

Recent improvements in fungal PCR technology have overcome challenges posed by the homogenous genetics of fungi, rigid fungal cell walls and inhibitors in patient samples.^6,13 Real-time PCR is faster and less susceptible to contamination than previous PCR techniques. ⁵ Current techniques also provide significant advantages for species identification.^10,15,16

Caution should be used when extrapolating results from one laboratory to another, because different DNA extraction methods may be used. ⁵ Caution is also warranted when comparing results between shelters, given differences in animal numbers, staffing, isolation facilities, disease prevalence and fungal loads. In southwestern Ontario, where the study took place, dermatophytosis is uncommon, with no positive fungal cultures among 400 shelter cats screened. ¹⁷ Fungal loads may be low and clinical infection mild in a low-prevalence area; however, because this is a contagious and zoonotic disease, any evidence of infection warrants investigation and appropriate intervention.

Two of the false-positive PCR results in this study revealed dermatophytosis-positive cases that were confirmed by a subsequent positive fungal culture. PCR has the potential to detect disease in cases where fungal culture sampling is not properly performed, as well as early clinical disease prior to shedding of large numbers of infective spores. In the five culture-negative, dermatophytosis-exposed cats, the positive PCR result may have revealed the presence of fungal elements that were not detectable by culture. The high sensitivity of PCR, reported in many studies,^{5,6,9,13–15,18} means that a positive test result could reflect true disease, fomite contamination or non-viable spores. In regions such as the study setting, where dermatophytosis is rare, ¹⁷ and colony counts often low (personal observation), the high sensitivity of PCR could be an advantage, but this might not be the case in an endemic area. Neither PCR nor culture can differentiate between transient colonization and true infection, ⁶ and careful clinical and Wood’s lamp examination, trichograms and contextual clinical judgment should be used to distinguish clinical dermatophytosis from fomite carriage.^1,4 For shelters that euthanize cats with confirmed dermatophytosis, fungal culture has the advantage of a lower potential for false-positive results and established methods for identifying fomite carriers.^1,4

The high proportion of false-positive results at the time of mycological cure could result from the ability of the PCR to identify very small fungal loads that might be too low to cause clinical infection, coat contamination, sample contamination in a busy shelter environment and the presence of dead organisms during treatment.^6,13,19 The finding suggests that while a negative result during treatment is likely to be reliable, routine use of PCR to determine mycological cure could result in a longer duration of isolation for many animals. This might, to some extent, be balanced by the 14 day wait time for culture results. However, until more data are available, existing recommendations to consider an animal mycologically cured after two negative cultures should continue to be followed.^1,2

A number of cats initially included in the study had to be dropped from data analysis. This reflects the realities of a shelter setting, where resources are limited and managing contagious diseases can stretch those resources to their utmost. This could be considered a strength of the study, rather than a weakness, in that the results reflect the realities of a field setting, as intended. Eight cats were excluded from analysis because the PCR samples were returned as ‘NSQ’, indicating that insufficient follicular material was present. Greater attention must be paid to hair plucks for PCR compared with fungal culture, and a certain amount of trial and error is to be expected when first using this test. In eight cases a contaminant species overgrew the plates. This was a temporary problem and did not represent an ongoing issue in the culture laboratory.

In the current study, 78.8% of all cats and 60.7% of the high-risk group were culture-negative. Ninety-two of the 104 culture negative cats were also PCR negative. At Can$56 per test, a conservative cost estimate of Can$20 per cat per day, and assuming a 14 day period to release these cats from isolation using culture, vs a 3 day waiting period using PCR, this would translate to savings of Can$15,088 and 1012 cat care days. Therefore, despite the higher direct costs of the PCR test compared with in-house culture, the ability to determine negative status within 1–3 days could result in significant savings for animal shelters, particularly if the financial and mission-related costs of isolation and treatment, increased length of stay and reduced life-saving capacity are also taken into account.

Conclusions

Real-time PCR showed high sensitivity and specificity for diagnosing dermatophytosis in shelter cats. The lack of false-negative results in this study indicates that PCR could be particularly useful for rapidly establishing that a suspect animal is not infected. The PCR was unreliable for identifying mycological cure, and fungal culture remains the test of choice for this purpose.