Keywords
prosthesis - infections - culture - prolonged incubation -
Staphylococcus aureus
Introduction
The demand for total joint arthroplasty (TJA) is significantly rising in India because
of increased elderly population, trained orthopaedic surgeons, sedentary lifestyle,
booming economy, improved hospital infrastructures, and emerging medical tourism.
Moreover, the recent price capping of the medical devices by the central government
has made total knee arthroplasty (TKA) affordable for the common man.[1] Even with the best precautions, prosthetic joint infections (PJIs) do occur and
have been posing a significant burden on orthopaedic services due to the sheer number
of patients with hip and knee prostheses. The incidence of PJI after primary interventions
in total hip arthroplasty (THA) or TKA is 1.5 to 2.5% but may increase to higher rates
(2–20%) when revision procedures are performed. A single institutional study on PJIs
from northern India have reported a cumulative incidence of 1.1%.[1]
[2]
The clinical spectrum of PJIs is variable and clinical signs and symptoms include
pain, joint swelling or effusion, erythema, fever, drainage, or a discharging sinus.[1] The Musculoskeletal Infection Society (MSIS) proposed a definition for PJI in 2015
which later was revised in 2018; it can be universally adopted for PJI diagnosis.[3]
[4] As per the 2015 definition, definite PJI diagnosis could be made if one of two major
pathognomonic criteria or four of six minor criteria are met while later in 2018 it
was revised to scoring-based system where operative criteria were used to fulfill
the criteria for PJI in case of inconclusive scores in minor criteria ([Table 1]). It is important to identify the infecting organism responsible for successful
treatment outcomes in PJIs by administration of appropriate and specific antimicrobial
agents. Microbiological culture remains the standard technique for the identification
of the infecting microorganism, but the frequency of negative culture results ranges
from 7 to 23%.[3] The biofilm formation especially in low-grade arthroplasty infection on the implant
by sessile bacteria is one of the major pathogenic factors responsible for PJIs as
well as for culture negativity where bacteria enmeshed within the biofilm fail to
grow on culture. The biofilms further increase the problem of antibiotic drug resistance,
hence making it more important to isolate the infecting pathogen and determine its
antimicrobial susceptibility for optimal patient management. Routinely in most of
the laboratories in India, the culture reports are dispatched in 48 to 72 hours and
the chances are high that most of the prosthesis-related infections remain undiagnosed
microbiologically. In particular, it seems that the need for an appropriate period
of incubation has been underestimated. Many studies have suggested prolonging the
culture incubation periods up to 14 days in joint infections.[5]
[6] But keeping culture for prolonged times in a conventional laboratory set up will
further increase the isolation of contaminants rather than the pathogens. Hence this
study was planned to assess the optimal incubation times for cultures in PJIs in conventional
culture settings and to study the aerobic bacterial profile of PJIs so as to design
optimal antibiotic therapy for patients.
Table 1
MSIS criteria for PJI diagnosis[3]
[4]
|
2015 MSIS criteria
|
2018 MSIS criteria
|
|
Abbreviations: CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; LE, leucocyte
esterase; PJI, prosthetic joint infection; PMN, polymorphonuclear; WBC, white blood
cell.
aFor inconclusive minor criteria patients, operative scores may fulfill definition
of PJI.
bConsider for molecular diagnosis in these cases like next generation sequencing.
|
|
Major criteria
|
1. A sinus tract communicating with the prosthesis.
2. A pathogen is isolated by culture from two separate tissue or fluid samples obtained
from the affected prosthetic joint.
|
1. A sinus tract communicating with the prosthesis.
2. A pathogen is isolated by culture from two separate tissue or fluid samples obtained
from the affected prosthetic joint.
|
|
Interpretation
|
Any one of the above is definite PJI.
|
Any one of above is definite PJI.
|
|
Minor criteria
|
1. Elevated serum ESR and CRP concentration.
2. Elevated synovial leukocyte count.
3. Elevated synovial PMN%.
4. Presence of purulence in the affected joint.
5. Isolation of a microorganism in one culture of periprosthetic tissue or fluid.
6. Greater than five neutrophils per high-power field in five high-power fields observed
from histologic analysis of periprosthetic tissue at ×400 magnification.
|
Preoperative diagnosis
|
Score
|
|
Serum
|
Elevated CRP or D-dimer
Elevated ESR
|
2
1
|
|
Synovial
|
Elevated synovial WBC count or LE
Positive α-defensin
Elevated synovial PMN (%)
Elevated synovial CRP
|
3
3
2
1
|
|
Score
|
≥ 6—infected
2–5—possibly infecteda
0–1—not infected
|
|
Intraoperative diagnosis
a
|
|
|
Preoperative score
Positive histology
Positive purulence
Single positive culture
|
–
3
3
2
|
|
Interpretation
|
Four out of six criteria met is definite PJI.
|
Score
|
≥ 6—infected
4–5—inconclusiveb
≤ 3—not infected
|
Materials and Methods
This prospective observational study was conducted for a period of one year in the
Departments of Microbiology and Orthopaedics at Government Medical College Hospital,
Chandigarh after due approval from Institutional Research and Ethics Committee of
our hospital. The clinically suspected PJI patients meeting 2015 MSIS diagnostic criteria
(minimum one major criteria or four minor criteria) were included in the study after
taking due informed consent. From these patients, at least three samples that include
joint fluid or aspirate, bone fragments, pus, synovial tissue samples were aseptically
collected, transferred into the aerobic blood culture bottles (Hi-media, Mumbai, India),
and sent for culture and sensitivity. The samples were not tested for the presence
of anaerobes. The bone fragments and synovial tissue were cut into small bits/crushed
with the sterile pestle and mortar before inoculation into the culture bottles. All
clinical and laboratory procedures were performed as per standard guidelines.[7]
[8] In the laboratory, the culture bottles were incubated under aerobic conditions at
37°C and daily subcultures were made on 5% sheep blood agar and MacConkey for 7 days
until the microbial growth was obtained and the last subculture being performed at
tenth day of incubation before reporting the culture results as sterile. The maximum
incubation period of 10 days was taken based on our observation in a pilot study conducted
on 20 suspected PJI samples in which out of 20 samples, 12 grew pathogenic microorganism
within 10 days while eight remained sterile even after 15 days of incubation. The
cultural isolates obtained were identified using standard microbiological techniques.[9] The culture showing growth of similar pathogen with similar antimicrobial profile
isolated from at least two samples was taken as a definitive pathogen causing infection.
For all culture positive cases, the antimicrobial susceptibility testing of the bacterial
isolates obtained was performed and interpreted as per CLSI Guidelines, 2018. The
antimicrobials tested against gram-positive isolates were penicillin (10IU), cefoxitin
(30 μg), erythromycin (15 μg), clindamycin (2 μg), cotrimoxazole (1.25/23.7 μg), linezolid
(30 μg), ciprofloxacin (5 μg), teicoplanin (30 μg), gentamicin (10 μg), doxycycline
(30 μg) and for gram-negative bacteria, cefepime (30 μg), amoxycillin clavulanic acid
(30/20 μg), ciprofloxacin (5 μg), piperacillin tazobactam (100/10 μg), amikacin (30
μg), ceftazidime (30 μg), ceftriaxone (30 μg), imipenem (10 μg), tetracycline (30
μg), cefotaxime (30 μg), tobramycin (10 μg), ampicillin sulbactam (10/10 μg) were
tested (HiMedia, Mumbai, India). The multidrug resistant (MDR) isolates were phenotypically
characterized as per their antibiograms into methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci, metallo β-lactamase (MBL), extended spectrum
β lactamase (ESBL) producers. As per the CLSI guidelines, the methicillin resistance
in Staphylococcus was determined by cefoxitin disk (30 µg) method while vancomycin resistance was tested
by vancomycin screen agar. MBL production in gram-negative bacteria was tested by
disk synergy method and ESBL production was confirmed by disk potentiation test using
ceftazidime (30 µg), and cefotaxime (30 µg) antibiotic disks with or without clavulanic
acid (10 µg).[10]
Results
Infection and Bacterial Isolates
Out of 200 clinically suspected PJI patients, 105 patients met the MSIS criteria and
were included in the study. From these patients, samples that included 51 wound discharge
or pus, 33 joint fluid aspirates, 10 bone fragments, and 28 synovial tissue samples
were collected in triplicate and were processed by aerobic culture methods. Seventy-five
patients were male and the rest female. The age group of PJI patients ranged from
12 to 76 years with mean age being 44.5 years. Definitive evidence of PJI by culture
positivity was observed from 70 patients (66.67%) in which at least two out of three
samples grew same bacterial pathogen on culture while in 35 samples, no pathogen grew
on culture even after 10 days of incubation.
The most commonly involved joint in device-related infection was knee joint (54%;
57/105) followed by hip joint (40%; 42/105) while shoulder and ankle joint were involved
in rest (5.7%; 6/105) of the cases. Highest isolation rate of pathogenic bacteria
was from pus samples (82.35%; 42/51) followed by synovial tissue (53.58%; 15/28),
bone (40%; 4/10), and joint fluid or aspirate (27.27%; 9/33) ([Fig. 1]). Seventy-two bacterial strains were isolated from culture positive cases among
which Staphylococcus aureus was the most frequently isolated pathogen (40.28%; n−29), followed by Klebsiella pneumoniae (15.27%; n−11), Acinetobacter CBC (13.89%; n−10), and E. coli (13.89%; n−10) ([Fig. 2]).
Fig. 1 The profile of bacterial pathogens in prosthetic joint infections.
Fig. 2 Sample wise culture positivity in prosthetic joint infections.
Time of Diagnosis of Infection by Culture
The mean number of days until bacterial growth became detectable in infected samples
was 3.6 days in our study and it did not differ much among different clinical samples
except in bone fragments, the mean and median incubation period for culture positivity
was observed as 5 days. Overall, maximum culture positivity was seen on 3rd day of
incubation (32.85%; n−23/70) while it was 25.71% (18/70) on day 4 and 14.28% (10/70) on day 5. Cumulatively,
92.85% (65/70) of culture grew pathogens within 5 days of incubation period and 97.14%
(68/70) by day 7 ([Fig. 3]). Gram staining could reveal significant findings in only 25 cases out of 70 culture
positivity with overall sensitivity of only 35.71%.
Fig. 3 Time to culture positivity in prosthetic joint infections.
Antimicrobial Drug Susceptibility
S aureus was the most common organism isolated in our study and showed only 4% susceptibility
to penicillin, ciprofloxacin (44%), clindamycin (44%), erythromycin (48%), linezolid
(99.07%), gentamicin (78%), and 100% susceptibility toward vancomycin and teicoplanin.
MRSA constituted 24.13% (n−7) of total 29 S. aureus isolates and vancomycin resistance was not observed among gram-positive cocci. Acinetobacter CBC showed only 22% susceptibility toward ceftazidime while imipenem was found to be
susceptible in 56% cases. 78% susceptibility was observed for tobramycin, amikacin,
and ampicillin sulbactam and ciprofloxacin was susceptible in 44% cases. The percentage
susceptibility observed among other gram-negative bacilli isolated was 40% for tetracycline,
piperacillin-tazobactam (20%), imipenem (70%), amikacin (30%), ceftazidime (05%),
ceftriaxone (15%), cefepime (10%), amoxiclav (15%), and ciprofloxacin (30%). ESBL
production was seen in 54% (6/11) isolates of K. pneumoniae and 50% (5/10) E. coli, while 36.36% (4/11) of K. pneumoniae, 30% E. coli (3/10) and 30% Acinetobacter CBC (3/10) were MBL producers. Amp C production was observed in one isolate each of K. pneumoniae, E. coli, and Acinetobacter CBC. Among K. pneumoniae and E. coli isolates, co-expression of the three enzymes (ESBL, MBL, and Amp C) was seen in one
isolate each.
Discussion
PJIs are uncommon and occur in only 1 to 2% of patients with hip and knee replacements
and in up to 6% of patients with internal fixation of closed fractures.[6] Acquisition of PJI can occur by different mechanisms. Most common is the direct
seeding of microorganisms during surgery whereby after the initial contact (either
direct or aerosolized contamination of the prosthesis or periprosthetic tissue [PPT]),
microorganisms colonize the surface of the implant. As per many studies, the bacterial
concentration needed to induce an infection is significantly reduced in the presence
of the prosthetic material. Moreover, the presence of prosthesis can lessen the neutrophil
activity thereby increasing the infection susceptibility. Other mechanism of infection
initiation is through contiguous spread of infection from an adjacent site or by hematogenous
seeding.[1]
The management of PJI cases is difficult and a reliable microbiological diagnosis
is crucial for determining appropriate treatment. It is a gold standard test for PJI
diagnosis, but conventional PTT cultures have low sensitivity due to many factors
like prior antibiotic exposure, the presence of viable but uncultivable organisms,
slow growing organisms, inadequate incubation periods for culture, and the presence
of biofilms rendering cultures sterile. The culture-negative PJI is a very perplexing
condition to manage for the surgeon, patient, and infectious disease team.[6]
[11] In recent years, the prevalence of culture-negative PJI has been on the rise and
the traditional modalities for isolation of an infecting organism have failed in as
many as 45% of patients.[12] Withholding the antibiotic administration until specimens for culture has been obtained
may help in culture isolation of the infecting organism, and if the arthroplasty is
resected, the sonication fluid obtained after vortexing and sonication of the implant
components may be an appropriate sample to be sent for culture.[8]
[13]
Infectious Diseases Society of America (IDSA) recommends the collection of a minimum
of three and optimally five or six PPT samples for both aerobic and anaerobic cultures
at the time of revision surgery and each specimen should be obtained with a new sterile
instrument.[7] Despite withholding the preoperative antibiotics in cases of revision arthroplasty,
the sensitivity of PPT culture has been determined to be 63% and the optimal incubation
period for prosthetic joint samples remains controversial. One study has shown a 13-day
incubation period to be optimal for recovery of slow growing organisms like Cutibacterium (Propionibacterium) species, while no supporting evidence was gathered from other studies.[14]
Very few studies have focused on finding out the optimal incubation time for orthopaedic
surgical specimens and there are none so far from India. Most commonly, 5 to 7 days
of incubation time in culture is used for PJI diagnosis with a recent proposal of
prolonging it up to 14 days. Schäfer et al found a positive association of prolonged
incubation period with increased proportion of positive samples and diversity of bacterial
isolates, especially recovery of aerobic gram-positive rods, Propionibacterium spp., and Peptostreptococcus spp. were increased with 14-day incubation protocol.[5] Similar findings were observed by Gunthard et al and Butler-Wu et al where prolonging
the incubation period for more than 7 days resulted in increased culture positivity.[15]
[16] Keeping the culture bottles for prolonged period is a difficult thing to follow
in settings where conventional culture methods are being followed and with high throughput
of clinical samples. Also, the risk of growing contaminants rather pathogens increase
during frequent subculturing in conventional practices. Automated blood culture systems
offer a great help in such a scenario. The present study showed that 97.14% culture
positivity was observed within 7 days of incubation with a mean incubation period
of 3.6 days. Incubation periods did not differ much among different types of samples
except the mean and median incubation period was 5 days in bone fragment culture.
Gram stain is one of the most useful techniques for rapid diagnosis in the clinical
microbiology laboratory and allows detecting bacteria and performing an initial classification
of microbes based on their shape and staining characteristics. Its use in synovial
fluid has been claimed to be highly specific, although sensitivity is only 40 to 45%.[17] In our study too, Gram staining did not offer great sensitivity and correlated well
with culture in only 35.71%. Apart from the diagnostic challenges, the management
of these patients is a far more challenging issue, with scanty literature on the subject.[18]
The goals of PJI treatment involve the eradication of infection and restoration of
the pain-free function of the infected joint. However, it may not be possible to achieve
all these goals in every patient. For optimal treatment of PJI, both surgical intervention
and antimicrobial therapy are needed and antimicrobial treatment without surgical
intervention has not been routinely recommended. However, antibiotic suppression alone
is reserved for patients with multiple comorbidities and not eligible for any surgical
interventions, or for those patients who are unwilling for any surgery, and causative
organisms are susceptible to oral antibiotics. Moreover, the antimicrobial therapy
for PJI should be specific and based on the causative microorganism and its antimicrobial
susceptibility pattern.[1]
The most common etiological agents involved in PJIs are S. aureus and S. epidermidis that account for close to 65% of PJIs. They are the most commonly reported microorganisms
both in early and late infections as well as in TKA and THA.[19] Salgado et al have reported S. aureus as causative organism in 33% PJIs, out of which 24% were methicillin-sensitive S. aureus (MSSA) and 9% were MRSA.[20] We observed similar results in our study with S. aureus implicated in 40.28% of PJI cases among whom 9.7% were MRSA and 30.5% were MSSA infections.
In a review by Peel et al, MRSA contributed a much higher proportion (45%) as the
causative agent of PJIs.[21] As per the global data, gram-negative bacteria are less commonly associated with
PJI and account for only 6 to 23% of all episodes but the Indian studies on the other
hand report high frequency of gram-negative PJIs that mostly include members of family
Enterobacteriaceae and Pseudomonas aeruginosa.[1]
[13] In our study, Klebsiella pneumoniae, E. coli, Klebsiella aerogenes contributed to 30% of PJIs followed by 13% by Acinetobacter CBC and P. aeruginosa in 2.78% infections.
The antimicrobial dug resistance is a common phenomenon observed in biofilm formation
as the enmeshed bacteria in the extracellular matrix remain protected from antibiotics
and evade the host defense mechanisms. So is the case in PJI where the implant frequently
gets colonized with adherent microorganisms forming biofilms. In a study by Sebastian
et al, 64% of the gram-negative isolates were multidrug resistant and methicillin
resistance being observed among 34% of gram-positive isolates.[1]
[13] In our study, 24% (7/29) of S. aureus isolates were MRSA and MDR gram-negative pathogens were isolated from PJIs that include
29.94% ESBL (11/38), 26.31% MBL (10/38), and 7.89% (3/38) Amp C producers. A detailed
list of suggested antimicrobial agents and their dosing for specific pathogens is
available in IDSA guidelines for the management of PJIs.[7] At our institute, vancomycin and teicoplanin showed 100% susceptibility profile
against S. aureus while linezolid showed 96.55% susceptibility against gram-positive cocci (S. aureus and E. faecalis). A high degree of drug resistance was observed among gram-negative bacilli where
less than 50% susceptibility was observed for cephalosporins while imipenem has shown
better susceptibility profile (70%). For empirical treatment, the Gram staining of
clinical samples may provide the information about the presence and type of bacteria,
thus guide the antimicrobial treatment based on institutional antimicrobial susceptibility
pattern of pathogens. Nevertheless, the definitive antibiotic treatment should be
culture driven and based on actual antibiotic susceptibility pattern of the cultural
isolate.
The limitation of the present study was the unavailability of automated blood culture
system at our institute that would have been an ideal testing tool for prolonged cultures
and also would have helped in isolation of anaerobic pathogens causing PJIs so as
to provide a comprehensive picture for the infecting pathogens of PJI.
Conclusion
PJI is one of the most common cause of implant failure. With an increase in the number
of primary TJAs being performed each year, the total number of PJI cases will greatly
increase, significantly impacting health care system and patients. Despite the developments
of various techniques and diagnostic criteria, early and accurate diagnosis of PJI
is still challenging. Knowledge of the local microbiological spectrum of infection
and antibiogram of pathogens causing PJI’s is essential for choosing appropriate perioperative
antimicrobial agents and empirical antimicrobial. To reduce the culture negativity,
prolonged culture incubation periods for PJI diagnosis are needed that help in increasing
the sensitivity of microbiological culture thus optimizing the patient management.
