Preliminary Application Of Next-generation Sequencing In Pathogens Detection Of Prosthetic Joint Infection

Background Prosthetic joint infection(PJI)is one of the complications that are difficult to completely avoid after joint replacement.The incidence rate is about 1% to 3%.Although the incidence is not high,due to difficult diagnosis and complicated treatment,it often brings serious burdens to the patient’s home and social court.Timely and accurate microbiological diagnosis is an important basis for selecting treatment methods.At present,clinically relying on culture technology to detect PJI disease-causing microorganisms,but due to the formation of pathogenic bacteria biofilm,the use of antibiotics before culture,some bacteria culture conditions are harsh,slow growth or even unable to culture,leading to culture positive rate is not high.The molecular diagnostic technology represented by PCR technology is often used as a laboratory supplement for the detection of pathogenic microorganisms in PJI.Some bacteria can be quickly detected,but it is limited by the influence of mixed bacteria in PJI infection that is difficult to discern by one-generation sequencing.Misdiagnosis of polyinfection pathogens.In recent years,Next Generation Sequencing(NGS)has gradually been applied to the detection of pathogens of infectious diseases.The features of NGS can measure a large number of sequences and can detect mixed bacterial infections in a single run.The potential value of detecting all pathogenic bacteria in PJI mixed infection.Purpose To explore the preliminary role of Next-generation sequencing technology in the detection of pathogens in Prosthetic joint infection with microbial culture positive synovial fluid.Methods Eighteen Synovial fluid specimens from patients with positive PJI culture were collected from our hospital from April 1,2016 to October 1,2017.Among them,Tsukayama II/III type 8 cases and Tsukayama IV type 10 cases;there were incisions in 6 cases,no incisions in 12 cases.Total synovial fluid DNA was extracted and PCR amplification was performed on the V3-V4 region of the 16 S r DNA gene.Illumina,Miseq platform,and 2 x 250 bp double-strand sequencing strategy were used to sequence the amplified products.The sequencing results were compared with the SILVA database.The species and abundance of bacteria in the specimen.Results A total of 5330283 high-quality sequences and 8449 operational taxonomic units(OTU)were obtained from the 16 S r DNA amplicon sequenced in the DNA of 18 artificial joint infection microbial culture joints.At the level of bacteria,18 DNA samples were obtained.A total of 8 different bacterial phylums were detected;at the level of bacteria,a total of 34 different bacterial genera were detected;18 DNA samples detected by this technique were identical to the traditional laboratory culture positive genus,12 of which were The DNA samples detected the bacteria outside the traditional laboratory culture using this technique;the bacterial species detected by a NGS sequencing(average of 4 species per sample)was significantly more than the bacterial species detected by conventional bacterial culture(average of 1 species per sample)(p<0.01);At the genus level,3.4 species and 4.5 species of bacteria were detected in each sample of Tsukayama II/III and IV respectively,and there was no significant difference between the two groups(P>0.05).At the bacterial level,6.33 species of bacteria were detected per case in the incision exudate group(sinus group),and 2.75 species of bacteria were detected in each case in the incision non-exudate group.There was a significant difference between the two groups(p <0.05).Conclusions At the bacterial level,a NGS technology can detect PJI pathogens that are consistent with laboratory culture;in most PJI cases,bacteria that are not cultivated in the laboratory can also be detected,but whether it is used to culture undetected bacteria Pathogens also require physicians to make clinical judgments;at bacterial level,the amount of bacteria detected by a NGS in the incision exudate and sinus tract was higher than that in the incision non-osmotic group and no sinus tract group.a NGS has certain reference significance for the diagnosis of pathogenic bacteria mixed with bacterial infection PJI.