ABSTRACT
Staphylococcus pseudintermedius can easily be mistaken for Staphylococcus aureus using phenotypic and rapid biochemical methods. We began confirming the identification of all coagulase-positive staphylococci isolated from human wound cultures at our centralized laboratory, servicing both community and inpatients, with matrix-assisted laser desorption ionization–time of flight mass spectrometry instead of using phenotypic and rapid biochemical tests, and determined the prevalence of S. pseudintermedius since the change in identification procedure and at what cost. A retrospective review was performed on all wound swab cultures from which coagulase-positive staphylococci were isolated 7 months before and after the change in identification procedure. A total of 49 S. intermedius/pseudintermedius (SIP) isolates were identified, including 7 isolates from 14,401 wound cultures in the before period and 42 isolates from 14,147 wound cultures in the after period. The number of SIP isolates as a proportion of isolated coagulase-positive staphylococci increased significantly from the before, 7/6,351 (0.1%), to the after, 42/5,435 (0.7%), period (difference, 0.6% [95% confidence interval, 0.037 to 0.83%, P < 0.0001]). Antibiotic susceptibility testing was performed in 42 isolates; none had an oxacillin MIC of 1.0 to 2.0 μg/ml, the range in which, if the isolate was misidentified as S. aureus, a very major error in susceptibility interpretation would occur. The increase in cost of the change in identification procedure was Can$17,558 per year in our laboratory, performing microbiology testing for community and acute-care patients in a zone servicing nearly 1.7 million people. While we will only continue to learn more about this emerging pathogen if we make attempts to properly identify it in clinical cultures, the additional time and cost involved may be unacceptably high in some laboratories.
KEYWORDS: Staphylococcus pseudintermedius, human infection, methicillin resistance, quality improvement
INTRODUCTION
Staphylococcus pseudintermedius was defined as a novel coagulase-positive staphylococci species in 2005 (1) and has been established as a common commensal and major cause of skin and soft-tissue infections (SSTI) in dogs and cats (2, 3). It has since been found to be an emerging zoonotic infection in humans responsible for mild to moderate SSTI (4), including bullous skin lesions (5), sinonasal infections (6), and invasive diseases, including endocarditis, implanted cardioverter defibrillator infection, and spinal fixation device infection (7–9).
S. pseudintermedius is a member of the Staphylococcus intermedius group (SIG), which includes S. intermedius, S. delphini, and S. cornubiensis (2, 10). The members of this group are difficult to distinguish from each other and from the Staphylococcus aureus complex (which includes S. aureus, S. argenteus, and S. schweitzeri) biochemically (1, 11). Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) can discriminate between S. aureus and the SIG (12). The importance of distinguishing S. pseudintermedius from S. aureus lies in the discrepancy in oxacillin breakpoints and methods used for methicillin resistance screening (13). In 2016, the Clinical and Laboratory Standards Institute (CLSI) redefined the breakpoints for S. pseudintermedius (oxacillin susceptible, <0.25 μg/ml; oxacillin resistant, ≥0.5 μg/ml) to be separated from that of S. aureus and Staphylococcus lugdunensis (oxacillin susceptible, ≤2 μg/ml; oxacillin resistant, ≥4 μg/ml) (14, 15). At the same time, it was determined that S. pseudintermedius should be screened for methicillin resistance using only oxacillin MIC or disk diffusion, as cefoxitin screening methods and oxacillin salt agar used for S. aureus have been shown to be insensitive in identifying mecA-mediated methicillin-resistant S. pseudintermedius (MRSP) (16). In 2021, the oxacillin breakpoints for S. pseudintermedius were again redefined to susceptible at ≤0.5 μg/ml and resistant at ≥1.0 μg/ml (13). If S. pseudintermedius is wrongly identified as S. aureus, based on morphology and basic biochemical characteristics, the wrong breakpoints may be applied, resulting in the potential for very major errors in susceptibility reporting, i.e., an oxacillin-resistant S. pseudintermedius could be wrongly reported as an oxacillin-susceptible S. aureus.
While other areas of the clinical laboratory had transitioned to organism identification largely by MALDI-TOF MS, S. aureus isolated from wound specimens continued to be identified efficiently by phenotypic and rapid biochemical tests (17). Isolates were only sent for confirmatory identification by MALDI-TOF MS if a history of animal exposure was provided on the requisition or due to technologist suspicion of an atypical colony morphology or a delayed positive clumping factor (slide coagulase) test suggestive of S. lugdunensis. Methicillin-resistance S. aureus (MRSA) isolates were also confirmed by MALDI-TOF. As a result, S. pseudintermedius was rarely identified from wound specimens.
To determine the laboratory and clinical impact of S. pseudintermedius isolates we might have been misidentifying as S. aureus, we began confirming the identification of all coagulase-positive staphylococci from human wound cultures with MALDI-TOF MS. The objectives of this study were to determine the proportion of coagulase-positive staphylococci found to be S. pseudintermedius since changing our identification procedure, the overall prevalence of S. pseudintermedius and MRSP isolation, and to determine the increased cost of this change.
MATERIALS AND METHODS
We performed a retrospective review of all wound swab cultures from which coagulase-positive staphylococci (S. aureus, S. lugdunensis, SIG, and S. schleiferi) were isolated from 1 May 2018 to 31 July 2019. The confirmation of the identification of all coagulase-positive staphylococci isolated from wound cultures with MALDI-TOF MS (Vitek MS; bioMérieux, France) began on 12 December 2018. The period from 1 May to 11 December 2018 is referred to here as the “before period” and the period from 12 December 2018 to 31 July 2019 as the “after period.” At the beginning of the study period, the Vitek MS V3.0 Knowledge Base was in place and was upgraded to V3.2 during the study period on 5 February 2019. S. argenteus and S. schweitzeri were not identified separately from S. aureus as they are not in either database.
Our centralized laboratory performs microbiology testing for both community and acute care patients in the Calgary Health Zone, servicing nearly 1.7 million people (https://www.albertahealthservices.ca/assets/about/publications/ahs-ar-2019/zones.html). Specimens were submitted as swabs in Liquid Amies transport medium (Copan Venturi transystem transport swab 140CQ; Copan Diagnostics Inc., Murrietta, CA). Demographic and clinical data provided to the laboratory on the requisition, including age, sex, sample source, and clinical history, were analyzed descriptively. Basic statistical methods were used to calculate proportions. Difference in proportions was calculated using the two-proportion Z test. This study was approved by the Conjoint Health Research Ethics Board (CHREB), Alberta Health Services, and University of Calgary (REB19-1086).
Wound swabs were plated onto 5% sheep blood agar (Dalynn, AB), chocolate agar (Dalynn, AB), colistin nalidixic agar (Dalynn, AB), and MacConkey agar (Dalynn, AB) and incubated at 35°C, 5% CO2 for 16 to 18 h. In the before period, S. aureus was identified from wound swab cultures based on basic characteristics, including a beta-hemolytic colony, Gram stain showing Gram-positive cocci, positive catalase, and positive clumping factor (slide coagulase) (17), and confirmatory identification by MALDI-TOF MS to rule out other coagulase-positive staphylococci, including S. pseudintermedius, was only performed if a history of animal exposure was provided on the requisition due to technologist suspicion of atypical colony morphology or delayed positive clumping factor test.
While MALDI-TOF MS (Vitek MS, Biomereux) can discriminate between S. aureus and the S. intermedius group, it is unable to discriminate between S. intermedius and S. pseudintermedius, instead providing a split identification of S. intermedius/pseudintermedius (SIP) (12). In both database versions used during the study period, the manufacturer notes for S. intermedius/pseudintermedius state “Subspecies or species group is displayed as a low discrimination result. A choice should be made between the proposed subspecies or species.” Our laboratory reports the identification of these isolates as S. intermedius/pseudintermedius, although previous work done in our laboratory confirmed such isolates to be S. pseudintermedius by sequencing of cpn60 (4).
MALDI-TOF MS results were correlated with Gram stain, biochemical, and phenotypic tests (i.e., catalase results, slide coagulase, and, if performed, tube coagulase). Antibiotic susceptibility testing was performed using Vitek2 (bioMérieux, Marcy-l’Etoile, France) or Gram-positive MicroScan panel (Beckman Coulter, CA) based on medical microbiologist preference with interpretation of results in accordance with CLSI guidelines for S. pseudintermedius (18, 19).
Cost analysis was performed based on our current MALDI-TOF MS materials cost of Can$0.74 per isolate. Previous workflow analysis in our laboratory had shown that MALDI takes 100.8 s of microbiology laboratory technologist (MLT) time per isolate. Labor cost for 100.8 s of MLT time is Can$1.52. Conversions to U.S. dollars and European euros were based on Bank of Canada exchange rates the week of 30 March to 5 April 2021. Cost of clumping factor testing was not included, as it is still performed in the new workflow to identify which isolates require definitive identification by MALDI-TOF MS and so is cost neutral.
We estimated the sensitivity and specificity of SIP identification by the former criteria (i.e., history of animal exposure, technologist suspicion of an atypical colony morphology, or a delayed positive clumping test) by defining MALDI-TOF as the gold standard (assuming 100% sensitivity and specificity) in order to determine how many SIP isolates were missed in the before period. We estimated the annual number of isolates per year based on the number of SIP isolated over the after period and adjusted the rate over 12 months.
RESULTS
Proportion of SIP from wound cultures.
During the entire study period, 1 May 2018 to 31 July 2019, 28,548 wound cultures were processed in our laboratory, including 14,401 specimens in the before period and 14,147 in the after period (Table 1). Coagulase-positive staphylococci were isolated from 44.1% and 41.0% of wound cultures in the before and after periods, respectively. In the before period, 6,351 coagulase-positive staphylococci were isolated, including S. aureus (95.6%), S. lugdunensis (4.3%), and SIP (0.1%). In the after period, 5,807 coagulase-positive staphylococci were isolated, including S. aureus (93.6%), S. lugdunensis (5.7%), and S. pseudintermedius (0.7%). The absolute difference in the number of isolated SIP as a proportion of the isolated coagulase-positive staphylococci was 0.6% (95% confidence interval [CI], 0.04 to 0.83%; P < 0.00001). No S. delphini or S. schleiferi isolates were identified in the entire study period. A total of 49 SIP isolates were isolated during the entire study period from 47 unique encounters by 46 unique patients (Table 2).
TABLE 1.
Summary of prevalence of coagulase-positive staphylococci
| Parameter | Before period, no. (%)a | After period, no. (%)a | Total no. | % (95% CI) differencec |
|---|---|---|---|---|
| Total wound cultures | 14,401 | 14,147 | 28,548 | |
| Total coagulase-positive staphylococci isolated | 6,351 | 5,807 | 12,158 | |
| S. aureus b | 6,071 (95.6) | 5,435 (93.6) | 11,506 | 2.0 (1.19, 2.81), P < 0.00001 |
| S. lugdunensis | 273 (4.3) | 330 (5.7) | 603 | 1.4 (2.18, 0.62), P < 0.00001 |
| SIP | 7 (0.1) | 42 (0.7) | 49 | 0.6 (0.83, 0.037), P < 0.00001 |
Proportion of total coagulase-positive staphylococci.
Includes MRSA at 1,140 before period, 947 after period, and 2,087 total.
Significance was assessed with two-sided P value using z-test.
TABLE 2.
Summary of isolates
| Isolate no. | Study period | Specimen collection date (yr-mo-day) | Patient | Patient age (yr) | Gender | Encounter type | Specimen source | Clumping factor |
|---|---|---|---|---|---|---|---|---|
| 1 | Before | 2018-05-01 | A | 66 | Male | Community | Superficial wound swab, leg, lower | Positive |
| 2 | Before | 2018-05-01 | A | 66 | Male | Community | Ulcer, heel | Positive |
| 3 | Before | 2018-06-18 | B | 67 | Male | Community | Superficial wound swab, foot | Not recorded |
| 4 | Before | 2018-06-18 | C | 36 | Male | Community | Deep wound swab, leg | Negative |
| 5 | Before | 2018-08-01 | C | 36 | Male | Community | Superficial wound swab, leg | Negative |
| 6 | Before | 2018-09-21 | D | 64 | Male | Community | Superficial wound swab, toe | Positive |
| 7 | Before | 2018-09-27 | E | 50 | Male | Community | Swab, toe | Positive |
| 8 | After | 2018-12-11 | F | 52 | Female | Inpatient | Superficial wound swab, skin, postoperative wound | Positive |
| 9 | After | 2018-12-12 | G | 43 | Female | Community | Swab, arm | Positive |
| 10 | After | 2018-12-13 | H | 69 | Male | Community | Superficial wound swab, foot | Positive |
| 11 | After | 2019-01-10 | I | 65 | Male | Community | Superficial wound swab, metatarsals | Positive |
| 12 | After | 2019-01-18 | J | 46 | Male | Community | Superficial wound swab, elbow | Positive |
| 13 | After | 2019-01-21 | K | 68 | Female | Community | Superficial wound swab, thigh | Not recorded |
| 14 | After | 2019-01-24 | L | 23 | Male | Community | Superficial wound swab, biliary drain site | Not recorded |
| 15 | After | 2019-01-29 | M | 87 | Female | Community | Swab, leg | Negative |
| 16 | After | 2019-01-30 | N | 9 | Male | Community | Superficial wound swab, back | Not recorded |
| 17 | After | 2019-02-21 | O | 38 | Female | Community | Superficial wound swab, scalp | Positive |
| 18 | After | 2019-02-25 | P | 29 | Female | Community | Superficial wound swab, toe | Positive |
| 19 | After | 2019-03-11 | Q | 55 | Male | Community | Superficial wound swab, abdomen | Positive |
| 20 | After | 2019-03-12 | R | 74 | Female | Community | Subcutaneous abscess, thumb | Positive |
| 21 | After | 2019-04-03 | S | 55 | Male | Emergency | Superficial wound swab, skin, finger infection/postfinger crush wound infection | Not recorded |
| 22 | After | 2019-04-06 | T | 54 | Male | Emergency | Superficial wound swab, skin, leg cellulitis | Positive |
| 23 | After | 2019-04-10 | U | 55 | Male | Community | Superficial wound swab, foot | Positive |
| 24 | After | 2019-04-30 | V | 69 | Male | Community | Superficial wound swab, ankle | Positive |
| 25 | After | 2019-05-02 | W | 55 | Female | Community | Superficial wound swab, toe | Not recorded |
| 26 | After | 2019-05-17 | X | 48 | Female | Community | Superficial wound swab, foot | Not recorded |
| 27 | After | 2019-05-22 | Y | 71 | Male | Emergency | Superficial wound swab, foot | Positive |
| 28 | After | 2019-06-06 | Z | 55 | Female | Community | Superficial wound swab, breast | Positive |
| 29 | After | 2019-06-13 | AA | 20 | Female | Emergency | Superficial wound swab, foot | Not recorded |
| 30 | After | 2019-06-13 | AB | 14 | Female | Community | Superficial wound swab, knee | Not recorded |
| 31 | After | 2019-06-14 | AC | 55 | Male | Community | Superficial wound swab, palm | Negative |
| 32 | After | 2019-06-14 | AC | 55 | Male | Community | Deep wound swab, hand | Positive |
| 33 | After | 2019-06-14 | AD | 23 | Female | Emergency | Superficial wound swab, finger, vesicular palmoplantar dermatitis | Positive |
| 34 | After | 2019-06-19 | AE | 41 | Female | Emergency | Superficial wound swab, toe, postoperative complications | Positive |
| 35 | After | 2019-06-20 | AF | 60 | Male | Community | Subcutaneous abscess, finger | Indeterminate |
| 36 | After | 2019-06-21 | AG | 41 | Female | Emergency | Superficial wound swab, leg, cellulitis | Positive |
| 37 | After | 2019-06-22 | AH | 62 | Male | Emergency | Superficial wound swab, skin, diabetic foot infection | Negative |
| 38 | After | 2019-06-24 | AI | 41 | Male | Emergency | Superficial wound swab, heel | Positive |
| 39 | After | 2019-06-25 | AJ | 58 | Female | Community | Superficial wound swab, scalp | Negative |
| 40 | After | 2019-07-07 | AK | 54 | Male | Community | Superficial wound swab, forearm | Positive |
| 41 | After | 2019-07-07 | AL | 71 | Female | Community | Superficial wound swab, wrist | Positive |
| 42 | After | 2019-07-08 | AM | 83 | Male | Community | Superficial wound swab, foot | Positive |
| 43 | After | 2019-07-08 | AN | 72 | Male | Inpatient | Superficial wound swab, shin | Positive |
| 44 | After | 2019-07-12 | AO | 88 | Female | Community | Superficial wound swab, leg | Positive |
| 45 | After | 2019-07-17 | AP | 72 | Female | Community | Superficial wound swab, shin | Negative |
| 46 | After | 2019-07-18 | AQ | 57 | Female | Community | Superficial wound swab, toe | Negative |
| 47 | After | 2019-07-21 | AR | 79 | Female | Nursing Home | Superficial wound swab, ankle | Not recorded |
| 48 | After | 2019-07-24 | AS | 53 | Female | Community | Superficial wound swab, foot | Negative |
| 49 | After | 2019-07-29 | AT | 61 | Male | Emergency | Superficial wound swab, skin, cellulitis | Positive |
Characterization of patients.
Of the 46 unique patients with SIP isolated from wound cultures, 25 were male and 21 were female, with an age range from 9 to 88 years (average, 55 years; median, 56 years). The most common specimen source was the extremities (31 lower extremity, 10 upper extremity, 6 other superficial location, and 2 skin not otherwise specified). Two patients were admitted to the hospital, one with a surgical site infection, the other with cellulitis of the leg. The remainder were outpatients with 10 patients seeking care in the emergency department, one from long-term care and the remaining in community physician offices. There was no documentation of animal exposure provided to the laboratory in any case.
Characterization of specimens.
Of the 47 specimens from unique encounters, 20 were monomicrobial, 10 were polymicrobial with SIP in addition to other primary pathogens, 7 were polymicrobial, including SIP and opportunistic pathogens, and 10 were polymicrobial with SIP and skin flora. Of the 10 wound cultures that included SIP coisolated with other primary pathogens, the following were isolated: S. aureus (n = 5), S. pyogenes (n = 1), group G streptococci (n = 2), S. lugdunensis (n = 1), Pasteurella canis (n = 1), and Pseudomonas aeruginosa (n = 1). Wound cultures with potential or opportunistic pathogens included various enteric Gram-negative bacilli and Enterococcus faecalis.
Biochemical characterization of SIP.
Biochemical test results were recorded in the laboratory information system (LIS) in 47 of the 49 isolates. Catalase was positive in 47/47 (100%) cases. Clumping factor (slide coagulase) was recorded for 39 isolates; of these, 29 were positive (74.4%), 9 were negative (23.1%), and 1 was indeterminate (2.6%). Only 1 isolate that was clumping factor positive had a recorded tube coagulase that was negative. No isolates that were clumping factor negative or indeterminate had recorded tube coagulase results, as MALDI-TOF MS was used to identify these isolates in both the before and after periods.
Antimicrobial susceptibility profiles of SIP isolates.
Antimicrobial susceptibility testing was performed in 44 SIP isolates, 43 by VITEK2 and one by MicroScan panel; by 2021 CLSI guidelines, 40 were oxacillin susceptible (MIC, ≤0.50 μg/ml), and 4 were oxacillin resistant (MIC, ≥1.0 μg/ml). One isolate (from the before period, tested by MicroScan panel) had an oxacillin MIC of 0.5 μg/ml, which was reported as oxacillin resistant per CLSI 2018 guidelines but is susceptible by 2021 guidelines (13). Therefore, by 2021 CLSI guidelines, no potential very major errors in susceptibility interpretation were identified. The prevalence of MRSP was 11.4% (5/44). By comparison, our regional MRSA prevalence during the entire study period was 18.1% (2,087 MRSA of 11,506 S. aureus isolates). Of the 4 MRSP isolates, 1 was multidrug resistant (resistant to clindamycin, trimethoprim-sulfamethoxazole, and tetracycline), 2 were resistant to one additional antimicrobial class (one each resistant to tetracycline and erythromycin), and 1 isolate did not exhibit any other resistance (Table 3). While all the results available to the laboratory are reported in Table 3, only cefazolin (per oxacillin testing), clindamycin, erythromycin, and trimethoprim-sulfamethoxazole are routinely reported to the clinician on oxacillin-susceptible staphylococcal isolates from superficial wounds. For oxacillin-resistant isolates, vancomycin and tetracycline are additionally reported.
TABLE 3.
Isolate antibiotic MICa (interpretation)
| Isolate no. | Panel | MIC (μg/ml) for: |
||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| OXA | CIP | CLI | CLI induc | ERY | GEN | LVX | LZD | MXF | PEN | RIF | SXT | TET | TIG | VAN | ||
| 1 | Not performed | |||||||||||||||
| 2 | Not performed | |||||||||||||||
| 3 | Not performed | |||||||||||||||
| 4 | Sta MS | 0.5 (S) | Not recorded | ≤0.25 (S) | Not recorded | ≤0.25 (S) | Not recorded | Not recorded | Not recorded | Not recorded | Not recorded | Not recorded | >2 (R) | Not recorded | Not recorded | 0.5 (S) |
| 5 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S)* | Pos | ≥8 (R) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≥16 (R) | ≤0.12 (S) | ≤0.5 (S) |
| 6 | Not performed | |||||||||||||||
| 7 | Sta VI | ≥4 (R) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | 1 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 8 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 9 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | 0.5 (S) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 10 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | 0.5 (S) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | ≤0.03 (S) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 11 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | 0.5 (S) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 12 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | 0.5 (S) | ≤0.5 (S) | 0.25 (S) | 2 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 13 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 14 | Sta VI | ≤0.25 (S) | ≥8 (R) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≥8 (R) | 1 (S) | 4 (R) | ≤0.03 (S) | ≤0.5 (S) | ≤10 (S) | ≥16 (R) | ≤0.12 (S) | ≤0.5 (S) |
| 15 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | 0.5 (S) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | 0.25 | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 16 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 17 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≥8 (R) | Neg | ≥8 (R) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 18 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | 0.12 | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 19 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | 0.5 (S) | Neg | ≥≥8 (R) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 20 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 21 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | 0.5 (S) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 22 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | ≤0.03 (S) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 23 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | 0.5 (S) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 24 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | ≤0.03 (S) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 25 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≥8 (R) | Neg | ≥8 (R) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 26 | Sta VI | ≥4 (R) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≥8 (R) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | 20 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 27 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S)* | Pos | ≥8 (R) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | 2 (S) | ≤0.12 (S) | 1 (S) |
| 28 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S)* | Pos | ≥8 (R) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 29 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 30 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | 0.5 (S) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | 0.25 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 31 | Not performed | |||||||||||||||
| 32 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | 0.5 (S) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | ≤0.03 (S) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 33 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | 0.5 (S) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≥16 (R) | ≤0.12 (S) | ≤0.5 (S) |
| 34 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | 0.25 (R) | ≤0.5 (S) | ≤10 (S) | ≥16 (R) | ≤0.12 (S) | ≤0.5 (S) |
| 35 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≥16 (R) | ≤0.12 (S) | ≤0.5 (S) |
| 36 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 37 | Sta VI | ≥4 (R) | ≥8 (R) | ≥8 (R) | Neg | ≥8 (R) | 8 (I) | ≥8 (R) | 1 (S) | 4 (R) | ≥0.5 (R) | ≤0.5 (S) | ≥320 (R) | ≥16 (R) | ≤0.12 (S) | ≤0.5 (S) |
| 38 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | 4 (R) | Neg | ≥8 (R) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | ≤0.03 (S) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 39 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | 0.5 (S) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 40 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | ≤0.03 (S) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 41 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | 0.5 (S) | ≤0.5 (S) | 0.25 (S) | 2 (S) | ≤0.25 (S) | ≤0.03 (S) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 42 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | 0.12 | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 43 | Sta VI | ≥4 (R) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | 1 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≥16 (R) | ≤0.12 (S) | ≤0.5 (S) |
| 44 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | 2 (I) | ≤0.5 (S) | ≤0.12 (S) | 4 (S) | ≤0.25 (S) | 0.25 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 45 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 46 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | 0.5 (S) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
| 47 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 1 (S) | ≤0.25 (S) | ≤0.03 (S) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 48 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | ≥0.5 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | ≤0.5 (S) |
| 49 | Sta VI | ≤0.25 (S) | ≤0.5 (S) | ≤0.25 (S) | Neg | ≤0.25 (S) | ≤0.5 (S) | ≤0.12 (S) | 2 (S) | ≤0.25 (S) | 0.25 (R) | ≤0.5 (S) | ≤10 (S) | ≤1 (S) | ≤0.12 (S) | 1 (S) |
Sta MS, Gram-positive MicroScan panel (Beckman Coulter, CA). Sta VI, Vitek 2 (bioMérieux, Inc., Durham, NC). An asterisk indicates reported as resistant due to positive clindamycin inducibility testing. Abbreviations: OXA, oxacillin; CIP, ciprofloxacin; CLI, clindamycin; ClI Induc, clindamycin inducibility test; ERY, erythromycin; LVX, levofloxacin; LZD, linezolid; MOX, moxifloxacin; PEN, penicillin; RIF, rifampin; SXT, trimethoprim-sulphamethoxazole; TET, tetracycline; TIG, tigecycline; VAN, vancomycin; S, susceptible; R, resistant; Pos, positive; Neg, negative.
Cost analysis.
In the before period, only coagulase-positive isolates that were atypical of S. aureus, e.g., nonpigmented or morphologically consistent with S. lugdunensis, had a history of animal exposure or MRSA isolates would have had identification confirmed by MALDI-TOF MS. Excluding MRSA, this was less than 10% of the remaining coagulase-positive staphylococci isolated per year. Over the 14-month study period, 10,071 non-MRSA coagulase-positive staphylococci were isolated from the wound bench in our laboratory. Assuming approximately 8,632 non-MRSA coagulase-positive staphylococci isolates per year continue to be identified by the wound bench in our laboratory, and that 90% of isolates would not previously have required confirmation of identification with MALDI-TOF MS, the increase in cost of identifying the additional 7,769 isolates per year by MALDI-TOF MS is at approximated Can$17,558 per year (approximately US$14,267 or €11,949), including reagent cost (Can$0.74 per isolate) and technologist time ($1.52 for 100.8 s MLT time per isolate). If SIP continues to account for 0.7% of coagulase-positive staphylococci identified in our laboratory (approximately 73 isolates per year), the additional cost per SIP isolate to confirm identification by MALDI is Can$240.52 (US$195, €164).
Sensitivity and specificity of before period method of SIP identification.
Again assuming 8,632 non-MRSA coagulase-positive staphylococci are isolated annually, and that in the before period approximately 10% of the non-MRSA isolates would have had final identification by MALDI-TOF, with a total of 0.1% being identified as SIP, approximately 9 SIP isolates per year could be expected to be isolated using the before period method. Following implementation of MALDI-TOF identification of all coagulase-positive staphylococci, we determined 0.7% of isolates are SIP, or approximately 60 SIP isolates per year at our current volumes. We can estimate then that the number of false-negative isolates from the former method would be 51, with a sensitivity of 14.3% and a specificity of 90.0% (Table 4).
TABLE 4.
Sensitivity and specificity calculations of before method based on MALDI-TOF as gold standard
| Identification of isolate | Identification of coagulase-positive staphylococci by MALDI-TOF (estimated no.) |
Sensitivity (%) | Specificity (%) | |
|---|---|---|---|---|
| Positive | Negative | |||
| SIP | 8.6 | 51.8 | 14.30 | 90.00 |
| Non-SIPa | 854.6 | 7,717 | ||
Non-SIP includes S. aureus, S. lugdunensis, SIG, and S. schleiferi.
DISCUSSION
Although the overall prevalence of SIP-positive wound cultures remained low, the total number of isolates as a proportion of all isolated coagulase-positive cocci from wound cultures increased significantly from the before to the after period at an increased cost in reagents and technologist time. Our results show that the rates of MRSP remain stable from 2013 to 2019 in our health region at 11.4% (4). In contrast, the prevalence of MRSP from a variety of clinical samples in two studies from the United States was much higher (19 to 27%) (6, 20).
None of the laboratory requisitions documented animal exposure, demonstrating that relying on referring physician history to direct the workup of cultures is insufficient as a trigger to look carefully for this pathogen. Although there are several studies looking into the carriage rates of S. pseudintermedius in individuals in close contact with dogs and cats (i.e., households with dogs or cats, individuals working in veterinary clinics or small-animal hospitals) (21–24), the prevalence of human infections is largely unknown. In a recent case series of S. pseudintermedius-positive wound cultures, over 90% of cases had contact with dogs prior to infection (4). Physicians may not realize that dog or cat ownership is a risk factor for S. pseudintermedius infection and may not indicate this exposure history on the requisition, except perhaps in the case of an infection secondary to a bite. Similar to our study, Yarbrough et al. found that less than 7% of cases with cultures positive for SIG had an association with dogs and speculated this may be due to not being documented (20). Based on this, selectively working up isolates for potential S. pseudintermedius based on provided history was shown to be insufficient, and this practice has stopped in our laboratory.
Of isolates where biochemical test results were recorded in the LIS, only 74.4% of isolates were clumping factor positive, in contrast to the original species description, which describes these isolates as tube coagulase positive but clumping factor negative (1). All isolates in the previous study by our laboratory confirmed to be S. pseudintermedius by sequencing of cpn60 were also clumping factor positive (4). Although there is a potential that our study included some S. intermedius isolates, we presume this is uncommon. Given this discrepancy, further molecular and biochemical description of the SIG is warranted.
Although some studies have shown promise for MALDI-TOF MS to discriminate between SIG species (25, 26), split S. intermedius/pseudintermedius species-level identification within the SIG remains a limitation of the Vitek MS platform (12) necessitating molecular methods, namely, sequencing of the rpoB or cpn60 gene (27) to definitively distinguish between species, which is not practical in the clinical laboratory (10). Fortunately, oxacillin interpretive category MIC breakpoints are the same for both S. intermedius and S. pseudintermedius, although cefoxitin screening methods for mecA-mediated resistance differ (13). To be conservative, our laboratory assumes the species identification of S. pseudintermedius for the purposes of antimicrobial susceptibility testing.
Alternative identification methods could have been used to definitively identify S. aureus isolates without using MALDI-TOF MS. Tube coagulase testing could be performed instead of clumping factor testing but was determined not to be feasible as the 4-h incubation time to obtain a result causes significant workflow interruptions, complicated by the fact that our wound bench is only staffed by technologists from 07:30 to 15:35. Negative PYR testing of catalase-positive, clumping-factor positive Gram-positive cocci would be one strategy but was discounted due to cost (Can$2.42 materials plus Can$1.81 labor for 2 min MLT time for a total of Can$4.23 per isolate versus Can$2.26 per isolate for MALD-TOF MS). Commercial latex agglutination tests for S. aureus identification are not presently used in our laboratory and would have been more expensive to introduce than MALDI-TOF in reagent cost (for example, StaphTEX blue and latex agglutination cards [Hardy Diagnostics], US$3.20 per test; https://catalog.hardydiagnostics.com/cp_prod/product/st1000-staphtex-blue-a-rapid-latex-agglutination-test-for-istaphylococcus-aureusi-blue-beads-on-a-white-card-sold-separately-for-easiest-read-out-1000-tests-by-hardy-diagnostics-rapid-id-test-kits) though likely equivalent in technologist time to perform.
A further limitation of our study is a lack of chart review for further clinical history and treatment details. Most patients were seen in the community, and limited clinical information was provided to the laboratory. It is presumed that cultures were collected from clinically infected wounds. Most wound cultures with SIP were either monomicrobial or polymicrobial with skin flora (63.8%), supporting the role of S. pseudintermedius as a pathogen.
S. pseudintermedius is an emerging human pathogen, and we will only continue to learn more about it if we make attempts to properly identify it from clinical cultures. Phenotypic characteristics and rapid biochemical tests have proven inadequate in reliably distinguishing SIP from S. aureus. Our prior method was insensitive, with an estimated sensitivity of 14.3% for detection of SIP. However, even with careful observation, the overall prevalence of SIP isolated from wound cultures is presently very low in our health region. Acceptable accuracy of an identification method is considered to be >90% agreement with a reference method (28, 29). Given that the proportion of reported S. aureus isolates changed by only 2.0% (95% CI, 1.19 to 2.81%, P < 0.00001) with our change in identification method (Table 1), it would be justifiable given the cost and minimal impact on patient care to return to identification of S. aureus isolates using phenotypic and rapid biochemical tests at the cost of misidentification of some isolates. No SIP isolates over the total 14-month study period had an oxacillin MIC of 1.0 to 2.0 μg/ml. All SIP isolates in our study, if misidentified as S. aureus, would have resulted in no difference in antibiotic susceptibility reporting. The additional time and cost of Can$17,558 per year to identify isolates accurately to the species with no clinical impact may be deemed unacceptably high in some laboratories (29). With continual pressures to minimize laboratory costs, we must closely examine our practices and not let the perfect be the enemy of the very good.
Contributor Information
Kristen L. Brown, Email: klbrown@ucalgary.ca.
Alexander J. McAdam, Boston Children's Hospital
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