The Application Of Broad-range PCR And Metagenomic Next-Generation Sequencing In Microbiological Diagnosis Of Prosthetic Joint Infection

Prosthetic joint infection(PJI)is one of the most serious complications after total joint replacement.PJI requires multiple surgical procedures and long-term antimicrobial treatment,which bring heavy burden to patients and society.Timely and accurate microbiological diagnosis is crucial to the success of PJI treatment.Due to formation of biofilm on the implant surface,the small bioburden of planktonic microorganisms,presence of fastidious pathogens,and prior use of antibiotic before sampling,the conventional microbiological culture method has a low positive rate for pathogenic organisms and requiring long incubation time.The broad-spectrum molecular diagnostic methods commonly used in clinical practice include broad-range PCR(BR-PCR)and metagenomic next-generation sequencing(mNGS),which can detect pathogenic microorganisms from PJI samples without culture procedures.The ability of these two methods in detecting pathogenic microorganisms from synovial fluid,periprosthetic tissue and sonicate fluid and the PJI diagnostic value remains unclear.Part 1 Comparison of culture and broad-range polymerase chain reaction methods for diagnosing periprosthetic joint infection: analysis of joint fluid,periprosthetic tissue,and sonicated fluidObjective To compare the capabilities of culture and broad-range polymerase chain reaction(PCR)using joint fluid(JF),periprosthetic tissue(PT),and sonicated fluid(SF)for the detection of causative pathogens and diagnosis of periprosthetic joint infection(PJI).Methods Sixty-seven subjects underwent knee or hip revision surgery,with 53 PJI and 14 aseptic failure(AF)cases included retrospectively.JF,PT,and SF samples were collected after a suspension of antibiotics more than two weeks,and culture and broadrange PCR were performed for all samples.The amplification product of PCR was sequenced by Sanger methods,and aligned by BLAST.Results A total of 53 strains of microorganisms was detected by culture,and 47 strains of bacteria were detected by BR-PCR.BR-PCR was unable to accurately detect six polymicrobial infections and two fungal infections.The sensitivities of SF culture(83.0%),JF-PCR(83.0%),and SF-PCR(84.9%)were similar(P > 0.05),but each was significantly more sensitive than JF culture(69.8%),PT culture(71.7%),and PT-PCR(34.0%)(P < 0.05).The specificities of JF culture,PT culture,SF culture,JF-PCR,PTPCR,and SF-PCR were similar(100,100,85.7,85.7,100,and 78.6%,respectively)(P > 0.05).Conclusions SF culture,JF-PCR,and SF-PCR were more sensitive than JF culture,PT culture,and PT-PCR for diagnosing PJI among patients who have stopped taking antibiotics for two weeks or more.Compared with PCR methods,SF culture has the advantage of detecting polymicrobial or fungal infections.PT-PCR proved to be insufficiently sensitive for providing correct diagnoses.Part 2 The application of metagenomic next-generation sequencing in microbiological diagnosis of prosthetic joint infectionSection 1 The application of metagenomic next-generation sequencing from joint fluid in periprosthetic joint infection diagnosticsObjective To investigate the capabilities of metagenomic next-generation sequencing(mNGS)for the detection of causative pathogens and diagnosis of periprosthetic joint infection(PJI)from joint fluid.Methods Seventy subjects underwent revision or debridement surgery after index arthroplasty,with 49 PJI and 21 aseptic failure(AF)cases included prospectively.Ten subjects underwent primary total joint arthroplasty(PTJA)were enrolled as negative controls.Joint fluid,periprosthetic tissue,and sonicate fluid samples were collected for culture,joint fluid samples were obtained for mNGS tests.The mNGS assays were sequenced using the BGISEQ-500 platform.The optimal threshold of relative abundance of genus-level(RAG)was calculated for final interpretation of mNGS results.The concordance and diagnostic value for PJI of culture and mNGS result were compared.Results The diagnostic thresholds of RAG were 15% and 30% for bacterial and fungal identification respectively.Among 39 culture-positive PJI subjects,37 subjects(94.9%)were mNGS-positive,of which 27 subjects achieved concordance in specieslevel,32 subjects achieved concordance in genus-level.The mNGS test was able to identify more potential pathogens from 6 culture-positive subjects,and to identify potential pathogens from all 10 culture-negative PJIs.However,mNGS test was incapable of identifying 8 strains of culturable pathogens from 6 PJIs and 1 AFs.The positive rate of mNGS was 94.4% among subjects receiving antibiotics within 2 weeks prior to sampling,significantly higher than joint fluid culture(20.7%,P<0.01)and comprehensive culture(61.1%,P<0.05).The culture and mNGS results were all negative in 10 PTJAs.The diagnostic sensitivity of mNGS was 95.9%,significantly higher than joint fluid culture and comprehensive culture(61.2% and 76.6%,respectively,P<0.05).The specificity of mNGS,joint fluid culture and comprehensive culture were similar.Conclusions The mNGS is able to effectively identify pathogens from joint fluid of PJI patients,especially for culture-negative cases due to prior antimicrobial therapy before sampling.mNGS could detect more potential pathogens from culture-positive PJIs.The mNGS method has high sensitivity and specificity for diagnosis of PJI,can be used as an effective supplement to the culture method.Section 2 Comparison of broad-range polymerase chain reaction and metagenomic next-generation sequencing for diagnosing prosthetic joint infectionObjective To compare the concordance of microbiology result and prosthetic joint infection(PJI)diagnostic performance by broad-range polymerase chain reaction(BRPCR)and metagenomic next-generation sequencing(mNGS).To determine if BR-PCR could be a routine validation method for mNGS assay.Methods After excluding subjects with insufficient volume of joint fluid or inhibited PCR from subjects enrolled in part 1,49 PJI and 21 aseptic failure(AF)cases included prospectively.Previously established mNGS and BR-PCR methods were performed on joint fluid samples.The concordance of microbiology result and PJI diagnostic sensitivity and specificity by BR-PCR and mNGS were analyzed and compared.Results Taking positivity result as criterion,the consistency of BR-PCR and mNGS was fair(kappa value=0.675).Single pathogen was detected by BR-PCR in 2 subjects,whose mNGS results showed multiple pathogens.BR-PCR was unable to detect pathogens in 8 subjects,whose mNGS result were positive.mNGS failed to detect pathogens in 2 subjects whose BR-PCR result were positive.Of 36 subjects with positive mNGS and BR-PCR result,only genus-level identifications were achieved in 17 subjects by BR-PCR method,which were consistent with mNGS in 15 subjects;species-level identifications were achieved in 18 subjects by BR-PCR,which were consistent with mNGS in 15 subjects;BR-PCR failed to identify in 1 subject.BR-PCR was unable to detect pathogens in 3 culture-negative PJIs,whose mNGS results were all positive.The sensitivity of BR-PCR(82.2%)were lower than mNGS method(95.6%)with insignificantly differences(P>0.05).The specificity of BR-PCR and mNGS was similar(94.4% and 94.4%,respectively,P>0.05).Conclusions The mNGS is able to detect more pathogens from joint fluid of PJI subjects,mainly include polymicrobial pathogens and fungus.Due to fair consistency with mNGS and moderate positivity,BR-PCR could not be a routine validation method for mNGS assay.For culture-negative highly suspected PJI cases,mNGS could be the preferred molecular diagnostic method.Section 3 The application of metagenomic next-generation sequencing from sonicate fluid in periprosthetic joint infection diagnosticsObjectives To investigate the capabilities of metagenomic next-generation sequencing(mNGS)for the detection of causative pathogens and diagnosis of periprosthetic joint infection(PJI)from sonicate.To determine the optimized type of sample for mNGS assay.Methods Thirty-five consecutive patients who underwent revision arthroplasty from May 2017 to November 2018 were included prospectively,and divided into the PJI group and aseptic failure(AF)group.Joint fluid and sonicate fluid of the explanted prostheses were obtained for microbiological culture and mNGS.Periprosthetic tissues were only collected for culture.Synovial fluid of three patients undergoing primary arthroplasty,and been treated by sonication either,were included as the negative control group concurrently.Comparisons of microbiological result and diagnostic sensitivity and specificity of mNGS and culture tests were performed.Results A total of 20 PJI cases and 15 AF cases were included.Of the 13 culturepositive PJI patients,mNGS results of synovial fluid were positive in 12 cases,while culture and mNGS results were completely consistent at species level in 7 cases,consistent at the genus level in 1 case;mNGS results of sonicate fluid were positive in 13 cases,while culture and mNGS results were completely consistent at species level in 9 cases,consistent at the genus level in 1 case.Of 7 culture-negative PJI patients,6 cases had consistent mNGS results at species level both from synovial fluid and sonicate fluid,however 1 case had positive mNGS result only from sonication fluid.All Culture results and mNGS result of synovial fluid were negative in all 15 AF patients,but mNGS result of sonicate fluid was positive in 1 AF case.Cultures and mNGS results were negative in all three pairs of negative-control samples.Of all 70 samples,mNGS detected 24 pathogens in sonication fluids and 22 pathogens in synovial fluids.Differences of number of raw reads and human reads ratio were not significant between mNGS of sonicate fluid and synovial fluid(P < 0.001),but mNGS of sonicate fluid generated significantly higher number of microbial reads and of strictly-mapped reads at species level than that of synovial fluids(P < 0.001).There was no significant difference in diagnostic sensitivity of PJI between mNGS of sonication fluids and synovial fluids(90.0% vs.100.0%),but both of them were significantly higher than that of culture of synovial fluid,periprosthetic tissues and sonicate fluids(40.0%,60.0% and 65.0%,respectively,P < 0.05).The specificities were similar among various microbiological testing methods(P > 0.05).Conclusions mNGS of sonicate fluid can detect the presence of pathogens effectively and diagnose PJI accurately.mNGS can identify more pathogens and generate a higher number of pathogenic reads from sonication fluids than joint fluid.mNGS of joint fluid has met the clinical diagnostic demands for most PJI patients.mNGS of sonicate fluid could be applied to some intractable cases.