paper-10直接PCR

PCR was ten copies of each pathogen.82.5%(66/80).For 34patients with high to 91.2%(31/34)when repeated and smear (64.7%,22/34)(p <0.001),the diagnosis of infectious keratitis with 100%,respectively.The time required to with high sensitivity and specificity keratitis in the future.24January 2014Currently,microscopic examination and culture remains the reference standard for the aetiological diagnosis of FK [6].specific;however,this method has requires approximately 5–7days.of corneal scrapes was detection rate for fungi [4,7,8];requires experienced technicians.an attractive non-invasive technique,for the clinical diagnosis of keratitis [9,10].However,aside from the required equipment may not be it is imperative to develop a rapid,method for diagnosing infec-high sensitivity and specificity has the conventional techniques 2.A variety based on amplification,such as nested PCR [11,12],real-time PCR [13–15],loop-mediated isother-mal amplification [16,17],and dot hybridization [18],are being1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950ª2014The AuthorsClinical Microbiology and Infection ª2014European Society of Clinical Microbiology and Infectious Diseases10.1111/1469-0691.12571developed for fungal detection.These assays are better than normal PCR,because of higher sensitivity,greater rapidity,or more convenience;however,template DNA extraction is required before all of the above assays.The DNA extraction procedure is not only time-consuming,but is also restricted by the limited specimens obtained from pathogenic corneas.Moreover,some of the template DNA is lost during extrac-tion.Therefore,a direct PCR assay using a special polymerase was developed that can detect pathogens in corneal specimens without template DNA extraction.Materials and MethodsPatient selectionSixty-seven patients with suspected infectious keratitis were the defect stroma ability of the with clinical FK,and a mittee Eye A and from 3onto blood agar or Sabouraud’s agar for bacterial and fungal culture at 37°C and 28°C,respectively;another two scrapings were smeared slides for Gram ing;and then,a was directly and immediately where it was a suspicion of used only for simplex virus keratitis (AK)on non-nutrient was also used to Direct PCR Direct PCR was Ver.2(TaKaRa,fragments;herpes simplex virus-1HS2)targeted the conserved US4G;and Acanthamoeba primers the conserved 29region of 18S PCR was performed in a 25-l L 12.5l L of 29MightyAmp MightyAmp DNA Polymerase (1.25U/forward and reverse primer mixture 4Touchdown PCR was used for of an initial denaturation step at by ten cycles of denaturation at 98°C –60°C for 30s (decreasing by 0.5°C at 72°C for 30s,and then °C for 30s,60°C for 30s,and 72°C extension at 72°C for 10min.The were inserted into the plasmid positive nucleotides after sequencing analysis.The detection limit of with 0,100,101,102,103and 104(ATCC 90029),Pseudomonas aeru-HSV-1(McKrea strain),and Acantha-specimenswere subjected to direct PCR for of fungi,bacteria,HSV-1,and Acantha-sterile water was added to the suspend the specimens,and the to homogeneity by pipetting up and taken out as the template in a direct controls with plasmids containing positive nucleotides and negative controls without any tem-plate were consistently performed.ª2014The AuthorsClinical Microbiology and Infection ª2014European Society of Clinical Microbiology and Infectious Diseases,CMI2Clinical Microbiology and InfectionCMI1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950Statistical analysis Statistical comparison of direct PCR,culture,smear and confocal microscopy for fungal detection was performed with the chi-square test with SPSS 13.0software (SPSS,Chicago,IL,USA).The performance indices of culture and direct PCR tests for infectious keratitis diagnosis,including their sensitivity,specificity,positive predictive value (PPV),and negative predictive value (NPV),were calculated after discrepant analysis.Differences in any performance index between direct PCR and culture were analysed with Fisher’s exact test.A p-value of <0.05was considered to be statistically significant.ResultsSpecific fragments amplified by direct PCR with low Detection of After the direct to analyse 80of fungi,bacteria,samples.The obtained with fungus-specific one corneal was co-infected with of corneal ulcer detection rate specimens from ulcer,and non-microbial and the positive 12results with culture,smear,or one case with corneal ulcer for bacteria with culture,and two results for fungi with smear.The with HSK,bacterial keratitis (BK),by direct PCR,improved when the drugs were given (HSK —both ganciclovir eye ointment were applied and acyclovir drug or injection 7was BK —levofloxacin or gatiflox-or gatifloxacin eye ointment,were applied for topical treatment susceptibility;once the report had drug was administered according to containing metronidazole com-or polyhexamethyl metformin were and metronidazole injection was when needed).However,in most of the patients with FK,PCR,improved when antifungal —natamycin and fluconazole eye —itraconazole/fluconazole or voric-one patient did not improve until as the drug administration for BK)did not improve until antibiotic membrane transplantation was applied.diagnostic tests for FKpatients with high suspicion of FK debridement was subjected to culture,and some of them were microscopy.Table 2summarizes all and other non-molecular tests forFIG.1.Detection limit of the direct PCR assay for fungi,bacteria,ª2014The AuthorsClinical Microbiology and Infection ª2014European Society of Clinical Microbiology and Infectious Diseases,CMICMIZhao et al.Direct PCR to diagnose fungal keratitis 31234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950subjected to direct PCR (Table 3).The first corneal debride-ment was usually performed on the first day of hospitalization.Inpatients who received the third corneal debridement after 12–14days of antifungal treatment,only one sample had a positiveª2014The AuthorsClinical Microbiology and Infection ª2014European Society of Clinical Microbiology and Infectious Diseases,CMI4Clinical Microbiology and InfectionCMI1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950result for fungus-specific DNA.During the period of treatment,the inflammatory infiltration of the corneal ulcer also decreased.Performance of the direct PCR for the diagnosis of infectious keratitisThe performance of culture and direct PCR with corneal scrapings from all patients except those with high suspicion of HSK during the first corneal debridement was analysed.If culture was considered as the reference method,the sensitivity and specificity of direct PCR were 95.8%(23/24)and 23.7%(9/38),respectively (Table 4).Among the 62scrapings,there were 32concordant results (23positive and nine negative)and 30discordant results (29direct PCR-positive but one culture-neg-ative).The concordance rate between culture and direct PCR assay was 51.6%(32/62)for all samples.Among the 29scrapings that generated positive results with direct PCR but negativefact that it can scrapings for merase,obviating known,the ulcer,especially limited,and DNA extraction,with direct PCR,be used as could be up to of real-time PCR,Moreover,only the assay.Thus,laboratory for cases caused by the basis of clinical ª2014The AuthorsClinical Microbiology and Infection ª2014European Society of Clinical Microbiology and Infectious Diseases,CMICMIZhao et al.Direct PCR to diagnose fungal keratitis 51234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950generally does not make a difference in the choice of antifungal agents [22,23].Thus,using direct PCR with fungal common primers for the rapid and sensitive diagnosis of FK has obvious potential value.Laboratory testing is very helpful for the correct diagnosis and effective treatment of FK and AK,the clinical appearance of which usually mimics that of BK [24]and HSK [25],respec-tively.For FK,culture is still considered to be the reference standard for identification of pathogens;however,it is not clear whether patients treated on the basis of culture results have better outcomes than those treated empirically with broad-spectrum antibiotics [26].Moreover,culture is relatively insensitive and time-consuming.In the present study,the positive detection rate of culture for FK (35.3%)was even lower than that described in our previous report (61.8%)[4],which may be because the cases in the previous report were study visit.that each PCR clinical copy this 64.7%,copy culture can be two widely from and,if and is 14four Nevertheless,there were two scrapings from patients with suspected FK that showed positive results when they wereexamined with showed positive results might be in the ulcer.The results in the not all inflammation because three or eye evisceration rejection or The direct repeatability.corneal and the repeatability debridement was with culture as of PCR-based the Xxxxxxxx.8ª2014The AuthorsClinical Microbiology and Infection ª2014European Society of Clinical Microbiology and Infectious Diseases,CMI6Clinical Microbiology and InfectionCMI1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950Supporting InformationAdditional Supporting Information may be found in the online version of this article:Figure S1.Specificity evaluation of HSV-1and Acantha-moeba primers used in direct PCR.Fungi:Candida albicans.Bacteria:Pseudomonas aeruginosa .Table S1.Primers used in this study.References1.Whitcher JP,Srinivasan M,Upadhyay MP.Corneal blindness:a global perspective.Bull World Health Organ 2001;79:214–221.2.Thomas PA,Geraldine P.Infectious keratitis.Curr Opin Infect Dis 2007;20:129–141.3.J4.Xie5.6.26:7.GN.8.GN.9.10.of 11.in 12.Ten 13.Farhatullah S,S.Diagnosis of on corneal 14.Ikeda Y,polymerase determinants of 119:1111–1119.15.Itahashi M,andquantification of merase chain Arch Ophthalmol 16.Ge Z,Qing Y,ofAcanthamoeba Clin Microbiol Infect 17.Reddy AK,isothermal by herpes simplex 18.Kuo MT,Chang Ahighly sensitive dot hybridization fungal keratitis in Shandong province,China.260–265.Chen M,Zhao J.Therapeutic effect of deepfor active or quiescent herpetic stromal Exp Ophthalmol 2012;250:1187–1194.N,Zeyer J,Burgmann H.Simple absolutecorrecting for quantitative PCR efficiency community samples.Appl Environ Microbiol J,Krishnan T et parison of natamycintreatment of fungal keratitis.Arch Ophthalmol GB.Mycotic keratitis:an overview of2008;51:183–199.A,Wilhelmus KR.The clinical diagnosis ofOphthalmol 2007;143:940–944.Folberg R,Meier PA,Wenzel RP,Elgin RG.to be caused by Acanthamoeba .Am J –142.GK,Papadopoulos GE,PetropoulosJX.Does identification of the causal influence the outcome?Eur J Ophthalmol Srinivasan M et al.Prospective comparison ofchain reaction in the diagnosis of 2008;146:714–723.ª2014The AuthorsClinical Microbiology and Infection ª2014European Society of Clinical Microbiology and Infectious Diseases,CMICMIZhao et al.Direct PCR to diagnose fungal keratitis 71234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950Author Query FormJournal:CLMArticle:12571Dear Author,During the copy-editing of your paper,the following queries arose.Please respond to these by marking up your proofs with the necessary changes/additions.Please write your answers on the query sheet if there is insufficient space on the page proofs.Please write clearly and follow the conventions shown on the attached corrections sheet.If returning the proof by fax do not write too close to the paper’s edge.Please remember that illegible mark-ups may delay publication.Many thanks for your assistance.MARKED PROOFPlease correct and return this setInstruction to printerLeave unchangedunder matter to remainthrough single character, rule or underlineNew matter followed by ororor ororor or or orand/orand/ore.g.e.g.under character over character new character new charactersthrough all characters to be deleted through letter orthrough characters under matter to be changed under matter to be changed under matter to be changed under matter to be changed under matter to be changed Encircle matter to be changed (As above)(As above)(As above)(As above)(As above)(As above)(As above)(As above)linking charactersthrough character or where requiredbetween characters or words affectedthrough character or where required orindicated in the margin DeleteSubstitute character or substitute part of one or more word(s)Change to italics Change to capitalsChange to small capitals Change to bold type Change to bold italic Change to lower caseChange italic to upright type Change bold to non-bold type Insert ‘superior’ characterInsert ‘inferior’ character Insert full stop Insert commaInsert single quotation marksInsert double quotation marks Insert hyphenStart new paragraph No new paragraph Transpose Close upInsert or substitute spacebetween characters or words Reduce space between characters or wordsInsert in text the matter T extual markMarginal markPlease use the proof correction marks shown below for all alterations and corrections. If you in dark ink and are made well within the page margins.wish to return your proof by fax you should ensure that all amendments are written clearly。