Clinical and prognostic relevance of Mycoplasma felis PCR detection in feline lower respiratory tract disease

Abstract

Objectives

The aim of this retrospective cohort study was to compare two groups of cats with lower respiratory tract disease, one with Mycoplasma felis detected by PCR in the bronchoalveolar lavage fluid (BALF) (M+) and the other without (M–), with regard to signalment, clinical signs, diagnostic results, treatment response and survival.

Methods

All cats for which M felis was investigated by PCR in BALF between 2016 and 2023 were included. Cats with evidence of oropharyngeal contamination, or for which PCR results were under the quantification level, or without follow-up information were excluded. Cats that had received antibiotics effective against M felis before BALF collection were excluded if PCR results were negative. Follow-up information was retrieved from the medical records and by contacting referring veterinarians and owners.

Results

A total of 55 cats were included (19 in the M+ group and 36 in the M– group). Significant differences were detected between the two groups in the prevalence of systemic signs (M+: 0%, M–: 28%; P = 0.01), bronchial collapse on bronchoscopy (M+: 28%, M–: 6%; P = 0.03), radiographic alveolar lesions (M+: 57%, M–: 24%; P = 0.04), and percentage of neutrophils (M+: 65%, M–: 35%; P = 0.002) and eosinophils (M+: 9%, M–: 25%; P = 0.03) in the BALF. Antibiotics were used more frequently in M+ cats (M+: 90%, M–: 42%; P = 0.001) than in M– cats. No significant difference was found in treatment response (short term: P = 0.94, long term: P = 0.28) and risk of death (P = 0.42) between the two groups.

Conclusions and relevance

The presence of radiographic alveolar lesions and neutrophilia in BALF was significantly associated with the detection of M felis in BALF. This association might be causal, consequential or contextual (ie, sharing the same cause). The detection of M felis in BALF did not negatively impact prognosis but the necessity to treat M felis using targeted antibiotics remains to be determined.

Keywords

respiratory prognosis bronchoalveolar lavage radiography

Introduction

In humans, Mycoplasma pneumoniae is a worldwide distributed pathogen causing lower respiratory tract (LRT) infections. It accounts for 4–39% of the pathogens detected by PCR or serology in children with community-acquired pneumonia.¹ However, diagnostic PCR and serology cannot discriminate between M pneumoniae infection and carriage, and this pathogen also has been detected in up to 19.9% of healthy children.^1,2 Unnecessary macrolide usage likely leads to the emergence of macrolide-resistant M pneumoniae; however, M pneumoniae must be treated when it contributes to respiratory diseases, such as in childhood asthma, with its acute presentation and necessity of hospitalisation.^2,3

In veterinary medicine, Mycoplasma felis has been commonly detected by PCR through oropharyngeal or nasal swabs in cats with upper respiratory tract (URT) disease, although its prevalence varies widely (46.5–78%).^4–6 Like M pneumoniae in humans, M felis has also been detected frequently in the upper airways of cats without clinical signs (20–31%).^4,7,8 One study failed to demonstrate a significant association between the detection of M felis and the development of URT disease in shelter cats.⁷ Coinfection with M felis might represent a risk factor for feline calicivirus infection, but M felis was highly prevalent in both URT disease and healthy cats in that study.⁸ Therefore, M felis is frequently considered a commensal of feline URT, although it may participate to URT disease.⁹

Conversely, M felis is commonly assumed to be pathogenic in the LRT of cats; however, it could be detected by PCR from the bronchoalveolar lavage fluid (BALF) in up to 50% of sick cats without respiratory signs.¹⁰ Furthermore, a meta-analysis failed to show a significant association between the detection of M felis from BALF and the presence of LRT disease in cats, suggesting that detecting M felis from the LRT could also be incidental.¹¹

The aim of this retrospective cohort study was to compare two groups of cats with LRT diseases (one with M felis detected in the BALF and the other without), in terms of signalment, clinical signs, diagnostic results, treatment response and survival.

Materials and methods

Study design, case selection and data collection

This retrospective cohort study included all cats presented for LRT signs or bronchopulmonary lesions on thoracic radiographs at our referral hospital between 2016 and 2023 for which M felis was searched by PCR in BALF. Cats with evidence of oropharyngeal contamination on BALF cytology (ie, presence of squamous epithelial cells or Simonsiella species) or for which PCR results were under the quantification level were excluded. Cats that had received antibiotics effective against M felis within the month before BALF collection were excluded only if the PCR results were negative. Follow-up information was retrieved from medical records and by contacting referring veterinarians and owners. Cats were excluded if follow-up information could not be retrieved. Two groups of cats were formed: one with M felis detected in the BALF by PCR (M+) and the other without (M–).

Medical records were searched to retrieve information on signalment (age, sex, breed), clinical signs (duration of clinical signs before presentation and presence of cough, abnormal breathing pattern, abnormal auscultation, systemic signs [ie, non-specific extrarespiratory signs such as dysrexia or anorexia, weight loss, lethargy and hyperthermia], URT signs and vomiting), bloodwork results (alanine aminotransferase [ALT] and alkaline phosphatase [ALP] activities), radiographic findings (signs of hyperinflation, bronchial pattern and its severity, bronchial collapse, bronchiectasis, mineralised lesions, pulmonary nodules, alveolar pattern and/or atelectasis, diffuse interstitial pattern, cardiovascular abnormalities), bronchoscopic lesions (presence of erythema, mucus/pus, obvious pus, bronchiectasis, airway collapse and nodules) and BALF analysis results (total nucleated cell count [TNCC], percentage of neutrophils, eosinophils, lymphocytes, macrophages and results of bacterial culture). M felis PCR was performed on pooled BALF samples. Treatments were retrieved from medical records and were categorised as antibiotics, steroids, bronchodilators, dewormers and anti-gastroesophageal reflux treatments. Finally, each cat was assigned a respiratory disease diagnosis based on predefined definitions (see Table S1A in the supplementary material), and the presence of extrarespiratory comorbidity was noted.

Thoracic radiograph procedure

Thoracic radiographs were taken in non-anaesthetised cats. Two planes were typically taken (right lateral recumbency and dorsal recumbency). More planes could have been obtained in some cats at the clinician’s discretion. Thoracic radiographs were blindly reviewed by a European board-certified specialist in diagnostic imaging (CT) to assess the presence of the radiographic lesions previously listed.

Bronchoscopic procedure

Bronchoscopy was performed using a 5.0 mm diameter flexible paediatric videobronchoscope (Olympus). Before each use, the bronchoscope was sterilised by immersion in an activated solution of peracetic acid (Anioxyde 1000; Lille-Hellemmes) for 30 mins, and then thoroughly rinsed with 0.9% sterile saline solution. For each procedure, anaesthesia was induced with propofol (2–5 mg/kg IV) after premedication (butorphanol 0.3 mg/kg IV, midazolam 0.25 mg/kg IV, dexmedetomidine 1–3 μg/kg IV, or some combination of these), and was maintained with propofol boluses. No animals were intubated and 100% oxygen was delivered via the biopsy channel.

Bronchoscopy was performed with the cat in sternal recumbency. The right and left principal and all lobar bronchi were examined and 1–2 bronchoalveolar lavage (BAL) sites were selected on the basis of imaging and gross bronchoscopic findings. Where gross lesions had not been identified, the right and left caudal lung lobes (RB4 and LB2) were chosen. A 0.9% sterile saline solution was used for BAL. Although bronchoscopy was performed by different clinicians, a standardised institutional protocol was followed, and the BAL volume used per site was 5 ml per cat. After gently wedging the tip of the bronchoscope in a distal bronchus, the sterile saline was rapidly instilled through the biopsy channel using a syringe, followed by 4 ml of air to clear the channel. The sample was then immediately collected into the same syringe via gentle pulsatile aspiration. Where necessary, the same syringe was emptied of air to continue aspiration. If the quality of the BALF samples was judged inadequate, a third BAL was performed.

Aliquots of BALF from the selected lobes were combined for bacterial culture analysis and M felis PCR analysis. Aliquots were analysed separately for cytological examination. Upon completion of the procedure, all animals were intubated to receive supplemental oxygen during anaesthesia recovery. Endoscopic images were blindly reviewed by a European and American board-certified specialist in small animal internal medicine (KLB) to assess the presence of the bronchoscopic lesions cited herein.

BALF cytology

Samples of BALF were delivered to the clinical pathology laboratory (Vebio Laboratory, Arcueil, France) within 10 mins of collection. Total nucleated cell count was determined using an automated cell counter (ADVIA 2120; Siemens) and was then verified manually. Direct smear slides were prepared by cytocentrifugation (Cytospin 4; Thermo Shandon) and stained with May–Grünwald Giemsa stain. The slides were examined and reported by a clinical pathologist at the time of diagnosis and were not re-examined for the purpose of this study. Differential cell counts were performed by counting 100–300 nucleated cells with an oil-immersion objective lens at × 500 magnification.

Bacterial culture

The BALF samples were quantitatively cultured for bacteria by inoculation of 100 μl onto a medium for the growth of fastidious organisms with clearly visible haemolytic reactions (agar plate with 5% sheep blood PLUS; Thermo Scientific). Subsequently, samples were serially diluted in sterile water to 10⁻¹, 10⁻³ and 10⁻⁵. Then, 100 μl of each dilution was inoculated onto a Columbia agar plate with 5% sheep blood PLUS (Thermo Scientific). Plates were incubated at 37°C for at least 24 h. Plates were examined for growth after both 24 and 48 h of incubation. When growth was observed, the number of colony-forming units was calculated by multiplying the number of colonies by 10 and applying the dilution factor. Bacterial identification and antibiotic susceptibility testing were also performed (VITEK-2 analyser; Biomerieux). Specific mycoplasma cultures were not performed.

M felis PCR

Real-time quantitative PCR (qPCR) was performed using the GENESIG Advanced Kit (Primerdesign) specific for M felis. Unless otherwise stated, reagents were sourced from Primerdesign. The molecular diagnostics were performed with quality controls, including a PCR-positive control of known quantity, a PCR-negative control and an internal extraction control. Suspension protocol, complete DNA extraction (using the GENESIG Easy DNA/RNA extraction kit) and qPCR detection protocol were performed according to the manufacturer’s instructions. Amplification was performed for 50 cycles with an annealing temperature of 60°C.

Outcome

The day of bronchoscopy was defined as T0. Two follow-up times were used: the first follow-up after the bronchoscopy visit (T1) and the last follow-up available (T2). Time elapsed between T0 and T1 or T2 was recorded. Clinical evolution was categorised as complete response (CR), when all respiratory signs resolved according to the owner or to the medical record; partial response (PR), when an improvement was noted according to the owner or to the medical record but complete resolution was not achieved; and no response (NR), when respiratory signs did not improve according to the owner or to the medical record.

The date of death was obtained from the medical record or by contacting the owner if the medical record did not mention that the cat had died. If the cat was still alive at the date the owner was contacted, this date was used as the last follow-up date before censoring in the survival analysis.

Statistical analysis

Data were coded as continuous (age, duration of clinical signs, PCR quantification, TNCC and percentage of inflammatory cells in the BALF), binary (group, sex, pure breed, indoor only, presence of clinical signs, abnormal bloodwork values, presence of radiographic and bronchoscopic lesions, other infection, treatment type and presence of comorbidity), ordinal (severity of radiographic lesions and evolution at T1 and T2) or categorical (respiratory disease diagnosis). The normality of continuous data was assessed using the Shapiro–Wilk test and inspection of distribution histograms.¹² As none of the continuous variables were normally distributed, median (range) was used to describe the data, and a univariate analysis was performed using the Mann–Whitney test. χ² or Fisher’s exact test, as appropriate, was performed for univariate analysis of binary and categorical data. Ordered logistic regression was used for ordinal data. As BALF was usually collected from 2–3 sites, mixed effects regression was performed to take into account the repeated measures per each cat. Averaged values per group and their 95% confidence intervals (CIs) were estimated using the Margins command. Survival was analysed using Cox proportional hazards regression models with the Breslow method for ties. A bivariate analysis was performed to adjust on the confounding effect of increasing age on survival. Finally, multivariable analyses were performed to assess the independent impact of detecting M felis in the BALF on response to treatment and survival. All variables with P <0.2 on univariate analysis were selected for inclusion in the multivariable analysis. Backward elimination was then applied to all variables except for group to filter out features not significant enough for the model and optimise the accuracy of the coefficient estimates.

When ordered logistic regression and Cox proportional hazards regression models were used, the approximate likelihood-ratio test of proportionality of odds and proportional hazards assumption test were performed to ensure that model assumptions were not violated. When mixed regression was used, Gaussian distribution and homoscedasticity of the residuals were checked by graphical assessment of frequency distribution histograms and residual plots, respectively.

Statistical analyses were performed using Stata 17.0 and the significance level was set at P <0.05 for all statistical analyses, unless otherwise specified.

Results

Population characteristics

A total of 55 cats were included: 36 in the M– group and 19 in the M+ group. Demographic data are presented in Table 1. No significant difference in signalment was found between the two groups.

Table 1

Demographical and clinical data of cats with M felis (M+) and without M felis (M–) detected in BALF

	M+ (n = 19)	M– (n = 36)	P value
Age (years)	6.6 (0.4–14.1)	5.8 (0.8–15.6)	0.81
Sex			0.88
Male	12 (63.2)	22 (61.1)
Female	7 (36.8)	14 (38.9)
Purebred	6 (31.6)	5 (13.9)	0.16
Indoor only			0.62
Yes	6 (31.6)	15 (41.7)
No	5 (26.3)	12 (33.3)
Unknown	8 (42.1)	9 (25)
Duration of clinical signs (days)	183 (0–2190)	256 (1–4380)	0.20
Cough	16 (84.2)	29 (80.6)	1.00
Abnormal breathing	10 (52.6)	23 (63.9)	0.42
Upper respiratory signs	3 (15.8)	10 (27.8)	0.51
Abnormal auscultation	9 (47.4)	16 (44.4)	0.84
Systemic signs	0 (0)	10 (27.8)	0.01*
Vomiting	4 (21.1)	13 (36.1)	0.36
Presence of extrarespiratory comorbidity	10 (53)	14 (39)	0.33

Values are n (%) or median (range)

Significantly different between groups

BALF = bronchoalveolar lavage fluid

Respiratory disease diagnosis and comorbidity

Respiratory disease diagnosis distribution was significantly different between groups (P = 0.02) (see Table S1B in the supplementary material), unlike comorbidity prevalence (P = 0.33) (Table 1).

Clinical findings

Clinical data are presented in Table 1. The duration of clinical signs before presentation was not significantly different between the two groups (P = 0.20). No significant differences in prevalence of respiratory signs and vomiting were found between the groups. The only significant difference detected in the clinical data was a higher prevalence of systemic signs in the M– group (n = 10/36, 28%) than in the M+ group (n = 0/19, P = 0.01).

Bloodwork findings

ALT activity was increased in 5/23 (21.7%) and 4/14 (28.6%) cats in the M– and M+ groups, respectively. This difference was not significant (P = 0.71). ALP activity was increased in none of the 23 cats of the M– group in which it was measured and in 1/14 (7.1%) cats of the M+ group. This difference was not significant (P = 0.38).

Radiographic findings

Thoracic radiographs were available for review in 14/19 (74%) and 25/36 (69%) cats in the M+ and M– groups, respectively. Radiographic data are summarised in Table 2. The only significant difference detected was a lower prevalence of alveolar pattern in the M– group (n = 6/25, 24%) than in the M+ group (n = 8/14, 57%; P = 0.04).

Table 2

Radiographic and endoscopic data of cats with M felis (M+) and without M felis (M–) detected in BALF

	M+	M–	P value
Number of radiographs available for review	14	25
Bronchial pattern	13 (92.9)	24 (96.0)	1.00
Bronchial collapse	1 (7.1)	4 (16.0)	0.64
Bronchiectasis	2 (14.3)	3 (12.0)	1.00
Alveolar pattern	8 (57.1)	6 (24.0)	0.04*
Diffuse interstitial pattern	4 (28.6)	12 (48.0)	0.32
Nodular interstitial pattern	2 (14.3)	2 (8.0)	0.61
Pleural lesion	1 (7.1)	1 (4.0)	1.00
Cardiovascular lesion	1 (7.1)	3 (12.0)	1.00
Atelectasis	5 (35.7)	6 (24.0)	0.48
Hyperinflation	0 (0)	4 (16.0)	0.28
Mineralised lesion	2 (14.3)	1 (4.2)	0.54
Number of endoscopic examinations available for review	18	36
Erythema	17 (94.4)	36 (100)	0.15
Mucus or pus	17 (94.4)	34 (94.4)	1.00
Obvious pus	5 (31.3)	5 (13.9)	0.25
Bronchiectasis	4 (22.2)	13 (36.1)	0.36
Airway collapse	5 (27.8)	2 (5.6)	0.03*
Nodules	1 (5.6)	4 (11.1)	0.66

Values are n (%)

Significantly different between groups

BALF = bronchoalveolar lavage fluid

Endoscopic findings

Endoscopic data are summarised in Table 2. The prevalence of bronchial collapse was significantly lower in the M– group (n = 2/36, 6%) than in the M+ group (n = 5/18, 28%; P = 0.03). No other significant differences in endoscopic lesions were found between the two groups.

BALF findings

BALF cytological and bacteriological data are summarised in Table 3. The TNCC was not significantly different between the two groups (P = 0.53). In the M+ group, the percentage of neutrophils was significantly higher (mean value: M– 35%, 95% CI 24–47; M+ 65%, 95% CI 50–80; P = 0.002) and percentage of eosinophils, significantly lower (mean value: M– 25%, 95% CI 16–34; M+ 9%, 95% CI 0–21; P = 0.03) than in the M– group. Prevalence of another infection was not significantly different between groups (P = 0.11) (see Table S2 in the supplementary material). The median (range) M felis PCR quantification value was 9*10⁴ copies per reaction (130–9*10⁷).

Table 3

Data of cats with M felis (M+) and without M felis (M–) detected in BALF

	M+ (n = 19)	M– (n = 34)	P value
Total nucleated cell count (/µl)	1873 (230–11,050)	1513 (160–40,490)	0.53
Neutrophils (%)	65	35	0.002*
Eosinophils (%)	9	25	0.03*
Lymphocytes (%)	1.26	1.62	0.63
Macrophages (%)	21	36	0.07
Other infection^† (%)	37	17	0.11

Values are % or median (range), obtained using the Margin command

Statistically different between groups

†

The presence of another bacteria in BALF culture

BALF = bronchoalveolar lavage fluid

Treatment

Antibiotics were used significantly more frequently to treat cats in the M+ group (n = 17/19, 89%) than in the M– group (n = 15/36, 42%; P = 0.001). Antibiotics that were prescribed after BAL included doxycycline (n = 13, 76%), marbofloxacin (n = 4, 23%) and azithromycin (n = 1, 6%) in the M+ group cats and amoxicillin–clavulanic acid (n = 7, 47%), doxycycline (n = 6, 40%), marbofloxacin (n = 3, 20%), azithromycin (n = 1, 7%), sulfonamide–trimethoprim (n = 1, 7%) and clindamycin (n = 1, 7%) in the M– group cats. The latter were more often treated with steroids (M–: n = 29/36, 80.6%; M+: n = 9/19, 47.4%; P = 0.01). No other significant differences in treatment received were found between the two groups (Table S3).

Follow-up

Follow-up data are summarised in Tables 4 and 5. The time between bronchoscopy and T1 or T2 was not significantly different between the groups (P = 0.26 and 0.25, respectively).

Table 4

Follow-up data at T1 and T2 of cats with M felis (M+) and without M felis (M–) detected in BALF

	M+ (n = 19)	M– (n = 36)	P value
Time from T0 to T1 (days)	27 (1–1140)	16 (1–458)	0.26
Outcome at T1			0.28
Complete response	7 (36.8)	13 (36.1)
Partial response	11 (57.9)	15 (41.7)
No response	1 (5.3)	8 (22.2)
Time from T0 to T2 (days)	555 (1–2312)	1041 (2–2407)	0.25
Outcome at T2			0.15
Complete response	5 (26.3)	16 (44.4)
Partial response	10 (52.6)	9 (25.0)
No response	4 (21.1)	11 (30.6)

Values are n (%) or median (range)

Significantly different between groups

BALF = bronchoalveolar lavage fluid; T0 = day of bronchoscopy; T1 = first follow-up after bronchoscopy; T2 = last follow-up after bronchoscopy

Table 5

Univariate analysis of factors’ influence on the evolution of cats at T1 and T2

Variable	T1			T2
Variable	OR	95% CI	P value	OR	95% CI	P value
Group (reference: M–)	1.48	0.53–4.18	0.46	0.79	0.29-2.14	0.64
Respiratory disease diagnosis (reference: feline asthma)	0.85	0.63–1.14	0.27	0.58	0.42–0.82	0.002*
Presence of extrarespiratory comorbidity	0.94	0.35–2.57	0.91	1.06	0.40–2.81	0.91
Age	0.77	0.66–0.89	<0.001*	0.88	0.78–0.99	0.04*
Sex (reference: male)	1.10	0.40–3.06	0.84	0.81	0.29–2.22	0.68
Purebred	1.12	0.34–3.62	0.85	0.44	0.13–1.46	0.18
Duration of signs	1.00	1.00–1.00	0.19	1.00	1.00–1.00	0.59
Cough	0.65	0.17–2.52	0.53	0.31	0.07–1.42	0.13
Abnormal breathing	1.13	0.41–3.10	0.82	1.08	0.40–2.91	0.88
Upper respiratory signs	0.63	0.20–1.98	0.43	0.73	0.22–2.38	0.60
Abnormal auscultation	0.71	0.26–1.95	0.51	0.75	0.28–2.01	0.57
Systemic signs	0.53	0.13–2.21	0.39	0.74	0.20–2.78	0.66
Vomiting	1.71	0.59–5.02	0.33	1.48	0.52–4.21	0.46
Increased ALT	1.33	0.31–5.73	0.70	1.34	0.33–5.49	0.69
Increased ALP	0.43	0.01–13.76	0.64	0	0^–∞	0.99
Bronchial pattern – radiography	8.07	0.54–120.97	0.13	0	0^–∞	0.99
Bronchial severity – radiography	0.44	0.19–1.01	0.053	0.73	0.33–1.60	0.43
Bronchial collapse – radiography	0.79	0.13–4.68	0.80	0.36	0.07–1.99	0.24
Bronchiectasis – radiography	0.44	0.06–3.02	0.41	0.19	0.03–1.28	0.09
Alveolar pattern – radiography	1	0.29–3.42	1.00	0.35	0.10–1.19	0.09
Diffuse interstitial pattern – radiography	0.29	0.08–1.03	0.06	0.21	0.06–0.77	0.02*
Nodular interstitial pattern – radiography	0.79	0.13–4.68	0.80	4.56	0.45–45.90	0.20
Pleural lesion – radiography	0.12	0.01–1.86	0.13	0	0^–∞	0.99
Cardiovascular lesion – radiography	0.44	0.06–3.02	0.41	0.61	0.07–5.20	0.65
Atelectasis – radiography	0.35	0.09–1.36	0.13	0.11	0.03–0.46	0.003*
Hyperinflation – radiography	1.69	0.25–11.49	0.59	1.13	0.16–8.07	0.90
Mineralised lesion – radiography	3.32	0.29–37.71	0.33	3.22	0.29–35.28	0.34
Mucus/pus – bronchoscopy	4.05	0.49–33.56	0.19	3.70	0.28–48.20	0.32
Obvious pus – bronchoscopy	2.56	0.69–9.42	0.16	1.39	0.40–4.88	0.61
Bronchiectasis – bronchoscopy	1.26	0.42–3.79	0.68	0.60	0.21–1.74	0.35
Airway collapse – bronchoscopy	1.93	0.45–8.31	0.38	2.57	0.55–12.11	0.23
Nodules – bronchoscopy	0.18	0.03–1.03	0.054	0.41	0.06–2.92	0.37
Steroid treatment	0.45	0.15–1.34	0.15	1.21	0.43–3.40	0.72
Deworming treatment	0.84	0.31–2.30	0.74	1.53	0.57–4.14	0.40
Bronchodilator treatment	0.68	0.20–2.35	0.55	0.91	0.29–2.91	0.88
Antibiotic treatment	0.72	0.26–2.00	0.53	0.46	0.17–1.26	0.13
Anti-gastroesophageal reflux treatment	1.22	0.36–4.14	0.75	0.91	0.29–2.91	0.88
Other infection – BALF	0.83	0.26–2.65	0.76	0.28	0.09–0.91	0.04*
Logcopies of M felis PCR – BALF	1.03	0.84–1.28	0.74	1.07	0.87–1.31	0.54

Significantly different between groups

ALP = alkaline phosphatase; ALT = alanine aminotransferase; BALF = bronchoalveolar lavage fluid; CI = confidence interval; OR = odds ratio of improved clinical evolution; T1 = first follow-up after bronchoscopy; T2 = last follow-up after bronchoscopy

On univariate analysis, no significant difference was observed between groups and clinical response at T1 (odds ratio [OR] 1.5, 95% CI 0.5–4.2; P = 0.46) and T2 (OR 0.8, 95% CI 0.3–2.1; P = 0.64). Only increasing age was significantly associated with a worse clinical response at T1 (OR 0.8, 95% CI 0.7–0.9; P <0.001). Increasing age (OR 0.9, 95% CI 0.8–0.99; P = 0.04) and presence of another respiratory infection (OR 0.3, 95% CI 0.1–0.9; P = 0.03), or a radiographic diffuse interstitial pattern (OR 0.2, 95% CI 0.06–0.8; P = 0.02) and/ or atelectasis (OR 0.1, 95% CI 0.03–0.5; P = 0.003) were significantly associated with a worse clinical response at T2. Clinical response at T2 also was impacted by final respiratory diagnosis (OR 0.6, 95% CI 0.4–0.8; P = 0.002).

On multivariable analysis, detecting M felis in BALF remained non-significantly associated with clinical response at T1 (OR 1.1, 95% CI 0.1–17.3; P = 0.94) and T2 (OR 3.8, 95% CI 0.3–42.4; P = 0.28). Increasing radiographic bronchial pattern severity (OR 0.24, 95% CI 0.1–0.9; P = 0.04) was significantly and independently associated with a worse clinical response at T1. Increasing age (OR 0.8, 95% CI 0.7–0.9; P = 0.006) and presence of atelectasis on thoracic radiographs (OR 0.09, 95% CI 0.02–0.5; P = 0.006) were significantly and independently associated with a worse clinical response at T2.

Survival analysis

Survival data are summarised in Table S4 in the supplementary material. On bivariate analysis (to adjust on the confounding effect of increasing age on survival), there was no significant difference in risk of death between the two groups (hazard ratio [HR] 1.2, 95% CI 0.5–3.0; P = 0.66). Increasing age (HR 1.3, 95% CI 1.1–1.5; P <0.001), presence of radiographic pleural lesion (HR 13.4, 95% CI 2.2–81.5; P = 0.005) and atelectasis (HR 2.9, 95% CI 1.0–8.4; P = 0.045) were significantly associated with an increased risk of death. The presence of a radiographic bronchial pattern was significantly associated with a decreased risk of death (HR 0.07, 95% CI 0.01–0.5; P = 0.005).

On multivariable analysis, detecting M felis in the BALF remained non-significantly associated with the risk of death (HR 2.3, 95% CI 0.3–17.4; P = 0.42). The variables significantly and independently associated with the risk of death are presented in Table 6.

Table 6

Multivariate survival analysis

Variable	Adjusted HR	95% CI	P value
Group (reference: M–)	2.3	0.3–17.4	0.42
Age	1.6	1.2–2.2	0.001*
Purebred	0.01	0.001–0.18	0.003*
Bronchial pattern – radiography	0.005	0.0001–0.18	0.004*
Atelectasis – radiography	19.7	2.6–147	0.004*
Bronchodilator treatment	17.1	2.1–140	0.008*
Other infection – BALF	17.8	2.5–126	0.004*

A hazard ratio significantly higher than 1 means that the variable is associated with a higher risk of death. A hazard ratio significantly lower than 1 means that the variable is associated with a lower risk of death

Significantly different from 1

BALF = bronchoalveolar lavage fluid; HR = hazard ratio

Discussion

The study reported here describes a population of cats with LRT disease in which M felis was researched. Overall, few differences were found between cats with M felis detected in the BALF and those without. Interestingly, the detection of M felis was significantly associated with the presence of a radiographic alveolar pattern and a neutrophilic inflammation in BALF.

The aim of this study was to appraise the role of M felis in LRT disease in cats. Epidemiological, clinical and blood parameters evaluated in this study were not significantly different between the M– and M+ groups, which suggests that detecting M felis in the LRT of cats is not specifically associated with a population subtype, unlike in humans, where M pneumoniae is more often detected in children and young adults.^2,3,13,14 As cats with an infectious respiratory disease are typically younger than those with a non-infectious one, these results may be more suggestive of an incidental M felis detection in the LRT rather than of a primary pathogenic role of M felis.¹⁵

Nevertheless, a radiographic alveolar pattern was significantly more frequently observed in cats in which M felis was detected in the LRT. Lobar or segmental lung consolidation is also reported in paediatric patients experiencing M pneumoniae pneumonia.¹⁶ In cats, the presence of alveolar lesions concomitant to M felis detection in the LRT has been reported sporadically.¹⁷ Cats with M felis detected in the LRT also had a significantly higher percentage of neutrophils in the BALF. A previous study supported this association.¹⁸ In humans, M pneumoniae PCR load in BALF is associated with increased neutrophil and total cell counts in BALF¹⁹ and peripheral neutrophil activation.²⁰ These results plead against an incidental M felis detection in the LRT and suggest a significant association between M felis and LRT inflammation.

This association could be causal if M felis is the primary pathogen responsible for the LRT neutrophilic inflammation. Although this hypothesis cannot be strictly refuted by our study, we would have expected a more favourable outcome in the M+ group compared with the M– group, as most of the cats in the M+ group received antibiotics that were effective against M felis. Only one-third and one-quarter of the cats with M felis detected in the LRT fully responded to treatment in the short term and long term, respectively, which was not significantly different from the cats in the M– group. The prognosis would also have been expected to be better in the group where the primary pathogen could be treated. However, M felis could still represent a primary pathogen if it leads to irreversible remodelling of the LRT that perpetuates inflammation. Pulmonary lesions, such as pneumatocele, bronchiectasis, abscess, lung cavitation and bronchiolitis obliterans, have been reported in humans after mycoplasmal infection.²¹ In mice, M pneumoniae pulmonary infection may also lead to chronic pulmonary diseases characterised by airway hyperreactivity, airway obstruction and histological inflammation.²²

Perhaps a more likely explanation could be that the association between M felis and BALF neutrophilia and radiographic alveolar patterns is consequential (ie, M felis colonises damaged airways) or contextual (ie, the presence of M felis and neutrophilic inflammation in the LRT arise from the same event). One study of cats with lethal pneumonias suggested that consequential M felis LRT colonisation was more likely than infection because M felis was never isolated as a lone pathogen.²³ In our study, however, coinfection was found in a minority of cats in the M+ group. Alternatively, translocation of M felis from the oral cavity to the LRT could be caused by aerodigestive reflux, which may also result in LRT neutrophilic inflammation or aspiration pneumonia.^24,25 Aerodigestive disease is poorly documented in cats and its prevalence might be underestimated. We tried to link the detection of M felis in the BALF and aerodigestive reflux by assessing liver enzymes and prevalence of vomiting. No significant associations were found but several cats were missing bloodwork data, altering the statistical power of our tests. Very few cats had folates and cobalamin blood levels available, precluding analysis on these parameters. Evaluating folates and cobalamin blood levels might have been interesting in those cats to further investigate aerodigestive disorders.

Considering the association between M felis and LRT disease more likely consequential or contextual, should antibiotics be used when M felis is detected from the BALF? The results of our survival analysis showed no significant difference between groups, suggesting that the detection of M felis in BALF does not impact survival. However, as most cats in the M+ group were treated with antibiotics, it cannot be determined whether not treating M felis would impact survival. A randomised study involving cats in which M felis is detected in the LRT and comparing the outcome of one group receiving antibiotics and the other group receiving a placebo would be useful to answer this important question.

Bronchial collapse was the only endoscopic finding significantly associated with the presence of M felis. Airway collapse has been identified in 48% of cats with spontaneous LRT disease.²⁶ Although it was often observed concurrently with pneumonia or LRT inflammation, no significant association was found.²⁶ In humans, airway collapse, such as tracheobronchomalacia or excessive dynamic airway collapse, has been associated with age,²⁷ LRT diseases such as asthma^27,28 and chronic obstructive pulmonary disease.^28–30

Several factors were found to be associated with increased risk of death. Age is an expected negative prognostic factor. Atelectasis, presence of another infection and use of a bronchodilator may be associated with more advanced or severe chronic respiratory disease, which may explain the increased risk of death. A radiographic bronchial pattern was associated with decreased risk of death, but this association was biased. Only two cats did not have a radiographic bronchial pattern and these cats had pleural effusion and alveolar lesions caused by life-threatening diseases.

Our study has several limitations, many of which are inherent to its retrospective nature. Some data could have been missing from the medical records, such as exhaustive treatments before presentation, types of environment (ie, shelter, multi-cat or single-cat household) or bloodwork results, precluding comparison between groups on this data. Diagnostic investigations were performed at the discretion of the attending clinician and were not standardised. Treatments were also not standardised, which precluded the authors to investigate whether a targeted antibiotic treatment is indicated when M felis is detected from the BALF. Endoscopic reviews were performed based on endoscopic reports and pictures as videos were not available for most cases. Therefore, some endoscopic lesions could have been missed if not present on available endoscopic pictures and not described in the report. Follow-up timing, although not statistically different between groups, was also not standardised. Thus, the first follow-up was sometimes >60 days, which made the assessment of short-term treatment response less accurate in some cats. To avoid misclassification bias when comparing the two groups, cats whose Mycoplasma species PCR results were under the quantification level were excluded from rather than integrated into the M– group. Finally, treatment response was based on owner or referring veterinarian assessment for some cats, and was therefore subjective.

Conclusions

This study revealed that radiographic alveolar lesions and more pronounced neutrophilic inflammation in BALF were significantly associated with the detection of M felis in the BALF. This association might be causal, but it seems more likely consequential or contextual (ie, sharing the same cause). The detection of M felis in the BALF did not negatively impact prognosis; however, as most cats in the M+ group received antibiotics, the question of whether antibiotics should be used when M felis is isolated from the BALF remains open.

Supplemental Material

sj-docx-1-jfm-10.1177_1098612X241297870 – Supplemental material for Clinical and prognostic relevance of Mycoplasma felis PCR detection in feline lower respiratory tract disease

Table S1A: Definitions used to classify each cat in the study depending on the underlying respiratory disease.

Table S1B: Distribution of respiratory disease diagnosis for cats with M felis (M+) and without M felis (M–) detected in BALF.

Table S2: Prevalence of identified bacteria by bacterial culture on BALF for cats with M felis (M+) and without M felis (M–) detected in BALF.

Table S3: Treatment data of cats with M felis (M+) and without M felis (M–) detected in BALF.

Table S4: Bivariate survival analysis.

Footnotes

Accepted: 16 October 2024

Conflict of interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

Funding for the open access publication charge was provided by IVC Evidensia Research Fund.

Supplementary material

The following files are available as supplementary material:

Table S1A: Definitions used to classify each cat in the study depending on the underlying respiratory disease.

Table S1B: Distribution of respiratory disease diagnosis for cats with M felis (M+) and without M felis (M–) detected in BALF.

Table S2: Prevalence of identified bacteria by bacterial culture on BALF for cats with M felis (M+) and without M felis (M–) detected in BALF.

Table S3: Treatment data of cats with M felis (M+) and without M felis (M–) detected in BALF.

Table S4: Bivariate survival analysis.

Ethical approval

The work described in this manuscript involved the use of non-experimental (owned or unowned) animals. Established internationally recognised high standards (‘best practice’) of veterinary clinical care for the individual patient were always followed and/or this work involved the use of cadavers. Ethical approval from a committee was therefore not specifically required for publication in JFMS. Although not required, where ethical approval was still obtained, it is stated in the manuscript.

Informed consent

Informed consent (verbal or written) was obtained from the owner or legal custodian of all animals described in this work (experimental or non-experimental animals, including cadavers, tissues and samples) for all procedure(s) undertaken (prospective or retrospective studies). No animals or people are identifiable within this publication, and therefore additional informed consent for publication was not required.

ORCID iD

Kevin Le Boedec

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