Rapid identification of pathogens using molecular techniques

  • Theo P. Sloots
    Affiliations
    Queensland Paediatric Infectious Diseases Laboratory, Queensland Children’s Medical Research Institute, Children’s Health Queensland, Brisbane, Australia

    Microbiology Department, Pathology Queensland, Brisbane, Qld, Australia
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  • Michael D. Nissen
    Affiliations
    Queensland Paediatric Infectious Diseases Laboratory, Queensland Children’s Medical Research Institute, Children’s Health Queensland, Brisbane, Australia

    Microbiology Department, Pathology Queensland, Brisbane, Qld, Australia
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  • Andrew N. Ginn
    Affiliations
    Centre for Infectious Diseases and Microbiology, University of Sydney, Westmead Hospital, Westmead, Australia

    Centre for Research Excellence in Critical Infection, Marie Bashir Institute for Infectious Diseases and Biosecurity and Westmead Millennium Institute, University of Sydney, Sydney, NSW, Australia
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  • Jonathan R. Iredell
    Correspondence
    Address for correspondence: Centre for Infectious Diseases and Microbiology, University of Sydney, Westmead Hospital, Westmead, NSW 2145, Australia.
    Affiliations
    Centre for Infectious Diseases and Microbiology, University of Sydney, Westmead Hospital, Westmead, Australia

    Centre for Research Excellence in Critical Infection, Marie Bashir Institute for Infectious Diseases and Biosecurity and Westmead Millennium Institute, University of Sydney, Sydney, NSW, Australia
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      Summary

      Real-time PCR is the traditional face of nucleic acid detection in the diagnostic microbiology laboratory and is now generally regarded as robust enough to be widely adopted. Methods based on nucleic acid detection of this type are bringing increased accuracy to diagnosis in areas where culture is difficult and/or expensive, and these methods are often effective partners to other rapid molecular diagnostic tools such as matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS). This change in practice has particularly affected the recognition of viruses and fastidious or antibiotic-exposed bacteria, but has been also shown to be effective in the recognition of troublesome or specialised phenotypes such as antiviral resistance and transmissible antibiotic resistance in the Enterobacteriaceae. Quantitation and high-intensity sequencing (of multiple whole genomes) has brought new opportunities as well as new challenges to the microbiology community. Diagnostic microbiologists currently training might be expected to deal less with the culturebased techniques of the last half-century than with the high-volume data and complex analyses of the next.

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      References

        • Espy M.J.
        • Uhl J.R.
        • Sloan L.M.
        • et al.
        Real-time PCR in clinical microbiology: applications for routine laboratory testing.
        Clin Microbiol Rev. 2006; 19: 165-256
        • Prosperi M.C.F.
        • Yin L.
        • Nolan D.J.
        • et al.
        Empirical validation of viral quasispecies assembly algorithms: state-of-the-art and challenges.
        Sci Rep. 2013; 3: 3827
        • Reuter S.
        • Ellington M.J.
        • Cartwright E.J.
        • et al.
        Rapid bacterial whole-genome sequencing to enhance diagnostic and public health microbiology.
        JAMA Intern Med. 2013; 173: 1397-1404
        • Barzon L.
        • Lavezzo E.
        • Militello V.
        • et al.
        Applications of next-generation sequencing technologies to diagnostic virology.
        Int J Mol Sci. 2011; 12: 7861-7884
        • Prosperi M.C.
        • Salemi M.
        QuRe: Software for viral quasispecies reconstruction from next-generation sequencing data.
        Bioinformatics. 2012; 28: 132-133
        • Robinson E.R.
        • Walker T.M.
        • Pallen M.J.
        Genomics and outbreak investigation: from sequence to consequence.
        Genome Med. 2013; 5: 36
        • Dobrindt U.
        • Hacker J.
        Whole genome plasticity in pathogenic bacteria.
        Curr Opin Microbiol. 2001; 4: 550-557
        • Whiley D.M.
        • Bialasiewicz S.
        • Bletchly C.
        • et al.
        Detection of novel influenza A(H1N1) virus by real-time RT-PCR.
        J Clin Virol. 2009; 45: 203-204
        • Whiley D.M.
        • Jacob K.
        • Nakos J.
        • et al.
        Improved detection of genetic markers of antimicrobial resistance by hybridization probe-based melting curve analysis using primers to mask proximal mutations: examples include the influenza H275Y substitution.
        J Antimicrob Chemother. 2012; 67: 1375-1379
        • Webb S.A.
        • Pettila V.
        • Seppelt I.
        • et al.
        Critical care services and 2009 H1N1 influenza in Australia and New Zealand.
        N Engl J Med. 2009; 361: 1925-1934
        • Blyth C.C.
        • Iredell J.R.
        • Dwyer D.E.
        Rapid-test sensitivity for novel swineorigin influenza A (H1N1) virus in humans.
        N Engl J Med. 2009; 361: 2493
        • Hackett H.
        • Bialasiewicz S.
        • Jacob K.
        • et al.
        Screening for H7N9 influenza A by matrix gene-based real-time reverse-transcription PCR.
        J Virol Methods. 2014; 195: 123-125
        • Ellem J.
        • Partridge S.R.
        • Iredell J.R.
        Efficient direct extended-spectrum β-lactamase detection by multiplex real-time PCR: accurate assignment of phenotype by use of a limited set of genetic markers.
        J Clin Microbiol. 2011; 49: 3074-3077
        • Morinaga Y.
        • Yanagihara K.
        Broad-range PCR in the identification of bacterial and fungal pathogens from positive blood culture bottles: a sequencing approach.
        Methods Mol Biol. 2015; 1237: 65-72
        • Tang Y.W.
        • Kilic A.
        • Yang Q.
        • et al.
        StaphPlex system for rapid and simultaneous identification of antibiotic resistance determinants and Panton-Valentine leukocidin detection of staphylococci from positive blood cultures.
        J Clin Microbiol. 2007; 45: 1867-1873
        • Werner G.
        • Serr A.
        • Schutt S.
        • et al.
        Comparison of direct cultivation on a selective solid medium, polymerase chain reaction from an enrichment broth, and the BD GeneOhm VanR Assay for identification of vancomycin-resistant enterococci in screening specimens.
        Diagn Microbiol Infect Dis. 2011; 70: 512-521
        • Fredricks D.N.
        • Relman D.A.
        Improved amplification of microbial DNA from blood cultures by removal of the PCR inhibitor sodium polyanetho-lesulfonate.
        J Clin Microbiol. 1998; 36: 2810-2816
        • Akane A.
        • Matsubara K.
        • Nakamura H.
        • et al.
        Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification.
        J Forensic Sci. 1994; 39: 362-372
        • Regan J.F.
        • Furtado M.R.
        • Brevnov M.G.
        • et al.
        A sample extraction method for faster, more sensitive PCR-based detection of pathogens in blood culture.
        J Mol Diagn. 2012; 14: 120-129
        • Edwards K.J.
        • Logan J.M.
        • Langham S.
        • et al.
        Utility of real-time amplification of selected 16S rRNA gene sequences as a tool for detection and identification of microbial signatures directly from clinical samples.
        J Med Microbiol. 2012; 61: 645-652
        • Kirkbright S.
        • Fatovich D.
        • Kee C.
        • et al.
        Quantitative rt-PCR holds promise as a screening tool for patients with severe sepsis.
        Emerg Med Australas. 2011; 23: 502-506
        • Kommedal O.
        • Simmon K.
        • Karaca D.
        • et al.
        Dual priming oligonucleotides for broad-range amplification of the bacterial 16S rRNA gene directly from human clinical specimens.
        J Clin Microbiol. 2012; 50: 1289-1294
        • Sontakke S.
        • Cadenas M.B.
        • Maggi R.G.
        • et al.
        Use of broad range 16S rDNA PCR in clinical microbiology.
        J Microbiol Methods. 2009; 76: 217-225
        • Lyra J.M.
        • Maruza M.
        • Verza M.
        • et al.
        Evaluation of four molecular methods for the diagnosis of tuberculosis in pulmonary and blood samples from immunocompromised patients.
        Mem Inst Oswaldo Cruz. 2014; 109: 805-813
        • Michou I.V.
        • Constantoulakis P.
        • Makarounis K.
        • et al.
        Molecular investigation of menstrual tissue for the presence of Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis collected by women with a history of infertility.
        J Obstet Gynaecol Res. 2014; 40: 237-242
        • Barbee L.A.
        • Dombrowski J.C.
        • Kerani R.
        • et al.
        Effect of nucleic acid amplification testing on detection of extragenital gonorrhea and chlamydial infections in men who have sex with men sexually transmitted disease clinic patients.
        Sex Transm Dis. 2014; 41: 168-172
        • Thomas L.C.
        • Gidding H.F.
        • Ginn A.N.
        • et al.
        Development of a real-time Staphylococcus aureus and MRSA (SAM-) PCR for routine blood culture.
        J Microbiol Methods. 2007; 68: 296-302
        • van Hal S.J.
        • Stark D.
        • Lockwood B.
        • et al.
        Methicillin-resistant Staphylococcus aureus (MRSA) detection: comparison of two molecular methods (IDI-MRSA PCR assay and GenoType MRSA Direct PCR assay) with three selective MRSA agars (MRSA ID, MRSASelect, and CHROMagar MRSA) for use with infection-control swabs.
        J Clin Microbiol. 2007; 45 (): 2486-2490
        • Banks J.T.
        • Bharara S.
        • Tubbs R.S.
        • et al.
        Polymerase chain reaction for the rapid detection of cerebrospinal fluid shunt or ventriculostomy infections.
        Neurosurgery. 2005; 57: 1237-1243
        • Wang H.Y.
        • Kim S.
        • Kim J.
        • et al.
        Multiplex real-time PCR assay for rapid detection of methicillin-resistant staphylococci directly from positive blood cultures.
        J Clin Microbiol. 2014; 52: 1911-1920
        • Al-Mohri H.A.
        • Tadros M.A.
        • Louie L.
        • et al.
        Utility of direct, real-time PCR in the management of a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (vanB genotype).
        Eur J Clin Microbiol Infect Dis. 2008; 27: 321-322
        • Cekin Y.
        • Erman Daloglu A.
        • Ogunc D.
        • et al.
        Evaluation of vancomycin resistance 3 multiplexed PCR assay for detection of vancomycin-resistant enterococci from rectal swabs.
        Ann Lab Med. 2013; 33: 326-330
        • Zhou X.
        • Arends J.P.
        • Kampinga G.A.
        • et al.
        Evaluation of the Xpert vanA/vanB assay using enriched inoculated broths for direct detection of vanB vancomycin-resistant enterococci.
        J Clin Microbiol. 2014; 52: 4293-4297
        • Roisin S.
        • Laurent C.
        • Denis O.
        • et al.
        Impact of rapid molecular screening at hospital admission on nosocomial transmission of methicillin-resistant Staphylococcus aureus: cluster randomised trial.
        PLoS One. 2014; 9: e96310
        • Hombach M.
        • Pfyffer G.E.
        • Roos M.
        • et al.
        Detection of methicillin-resistant Staphylococcus aureus (MRSA) in specimens from various body sites: performance characteristics of the BD GeneOhm MRSA assay, the Xpert MRSA assay, and broth-enriched culture in an area with a low prevalence of MRSA infections.
        J Clin Microbiol. 2010; 48: 3882-3887
        • Laurent C.
        • Bogaerts P.
        • Schoevaerdts D.
        Evaluation of the Xpert MRSA assay for rapid detection of methicillin-resistant Staphylococcus aureus from nares swabs of geriatric hospitalized patients and failure to detect a specific SCCmec type IV variant.
        Eur J Clin Microbiol Infect Dis. 2010; 29: 995-1002
        • Patel P.A.
        • Ledeboer N.A.
        • Ginocchio C.C.
        • et al.
        Performance of the BD GeneOhm MRSA achromopeptidase assay for real-time PCR detection of methicillin-resistant Staphylococcus aureus in nasal specimens.
        J Clin Microbiol. 2011; 49: 2266-2268
        • Chan W.S.
        • Chan T.M.
        • Lai T.W.
        • et al.
        Complementary use of MALDI-TOF MS and real-time PCR-melt curve analysis for rapid identification of methicillin-resistant staphylococci and VRE.
        J Antimicrob Chemother. 2015; 70: 441-447
        • Clerc O.
        • Prod’hom G.
        • Senn L.
        • et al.
        Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and PCR-based rapid diagnosis of Staphylococcus aureus bacteraemia.
        Clin Microbiol Infect. 2014; 20: 355-360
        • Syrmis M.W.
        • Moser R.J.
        • Whiley D.M.
        • et al.
        Comparison of a multiplexed MassARRAY system with real-time allele-specific PCR technology for genotyping of methicillin-resistant Staphylococcus aureus.
        Clin Microbiol Infect. 2011; 17: 1804-1810
        • Leopold S.R.
        • Goering R.V.
        • Witten A.
        • et al.
        Bacterial whole-genome sequencing revisited: portable, scalable, and standardized analysis for typing and detection of virulence and antibiotic resistance genes.
        J Clin Microbiol. 2014; 52: 2365-2370
        • Salipante S.J.
        • SenGupta D.J.
        • Cummings L.A.
        • et al.
        Application of whole genome sequencing for bacterial strain typing in molecular epidemiology.
        J Clin Microbiol. 2015;
        • Gosiewski T.
        • Flis A.
        • Sroka A.
        • et al.
        Comparison of nested, multiplex, qPCR; FISH; SeptiFast and blood culture methods in detection and identification of bacteria and fungi in blood of patients with sepsis.
        BMC Microbiol. 2014; 14: 2323
        • Trebesius K.
        • Leitritz L.
        • Adler K.
        • et al.
        Culture independent and rapid identification of bacterial pathogens in necrotising fasciitis and strepto-coccal toxic shock syndrome by fluorescence in situ hybridisation.
        Med Microbiol Immunol. 2000; 188: 169-175
        • Kok J.
        • Thomas L.C.
        • Olma T.
        • et al.
        Identification of bacteria in blood culture broths using matrix-assisted laser desorption-ionization Sepsityper and time of flight mass spectrometry.
        PLoS One. 2011; 6: e23285
        • Ferreira L.
        • Sanchez-Juanes F.
        • Gonzalez-Avila M.
        • et al.
        Direct identification of urinary tract pathogens from urine samples by matrix-assisted laser desorption ionization-time of flight mass spectrometry.
        J Clin Microbiol. 2010; 48: 2110-2115
        • Kok J.
        • Chen S.C.
        • Dwyer D.E.
        • et al.
        Current status of matrix-assisted laser desorption ionisation-time of flight mass spectrometry in the clinical microbiology laboratory.
        Pathology. 2013; 45: 4-17
        • Oliveira K.
        • Procop G.W.
        • Wilson D.
        • et al.
        Rapid identification of Staphylococcus aureus directly from blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes.
        J Clin Microbiol. 2002; 40: 247-251
        • Burckhardt I.
        • Zimmermann S.
        Using matrix-assisted laser desorption ionization-time of flight mass spectrometry to detect carbapenem resistance within 1 to 2. 5 hours.
        J Clin Microbiol. 2011; 49: 3321-3324
        • Hrabak J.
        • Walkova R.
        • Studentova V.
        • et al.
        Carbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spectrometry.
        J Clin Microbiol. 2011; 49: 3222-3227
        • Hrabak J.
        • Studentova V.
        • Walkova R.
        • et al.
        Detection of NDM-1, VIM-1, KPC, OXA-48, and OXA-162 carbapenemases by matrix-assisted laser desorption ionization-time of flight mass spectrometry.
        J Clin Microbiol. 2012; 50: 2441-2443
        • Sparbier K.
        • Schubert S.
        • Weller U.
        • et al.
        Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based functional assay for rapid detection of resistance against β-lactam antibiotics.
        J Clin Microbiol. 2012; 50: 927-937
        • Lee Y.H.
        • Cho B.
        • Bae I.K.
        • et al.
        Klebsiella pneumoniae strains carrying the chromosomal SHV-11 β-lactamase gene produce the plasmid-mediated SHV-12 extended-spectrum β-lactamase more frequently than those carrying the chromosomal SHV-1 β-lactamase gene.
        J Antimicrob Chemother. 2006; 57: 1259-1261
        • Nuesch-Inderbinen M.T.
        • Kayser F.H.
        • Hachler H.
        Survey and molecular genetics of SHV β-lactamases in Enterobacteriaceae in Switzerland: two novel enzymes, SHV-11 and SHV-12.
        Antimicrob Agents Chemother. 1997; 41: 943-949
        • Ginn A.N.
        • Wiklendt A.M.
        • Zong Z.
        • et al.
        Prediction of major antibiotic resistance in Escherichia coli and Klebsiella pneumoniae in Singapore, USA and China using a limited set of gene targets.
        Int J Antimicrob Agents. 2014; 43: 563-565
        • Ginn A.N.
        • Zong Z.
        • Wiklendt A.M.
        • et al.
        Limited diversity in the gene pool allows prediction of third-generation cephalosporin and aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae.
        Int J Antimicrob Agents. 2013; 42: 19-26
        • Kohl T.A.
        • Diel R.
        • Harmsen D.
        • et al.
        Whole-genome-based Mycobacterium tuberculosis surveillance: a standardized, portable, and expandable approach.
        J Clin Microbiol. 2014; 52: 2479-2486
        • Walker T.M.
        • Ip C.L.
        • Harrell R.H.
        • et al.
        Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study.
        Lancet Infect Dis. 2013; 13: 137-146
        • Deplano A.
        • Denis O.
        • Nonhoff C.
        • et al.
        Outbreakof hospital-adapted clonal complex-17 vancomycin-resistant Enterococcus faecium strain in a haematology unit: role of rapid typing for early control.
        J Antimicrob Chemother. 2007; 60: 849-854
        • Yu F.
        • Ying Q.
        • Chen C.
        • et al.
        Outbreak of pulmonary infection caused by Klebsiella pneumoniae isolates harbouring blaIMP-4 and blaDHA-1 in a neonatal intensive care unit in China.
        J Med Microbiol. 2012; 61: 984-989
        • Sabat A.
        • Krzyszton-Russjan J.
        • Strzalka W.
        • et al.
        New method for typing Staphylococcus aureus strains: multiple-locus variable-number tandem repeat analysis of polymorphism and genetic relationships of clinical isolates.
        J Clin Microbiol. 2003; 41: 1801-1804
        • Chin C.S.
        • Sorenson J.
        • Harris J.B.
        • et al.
        The origin of the Haitian cholera outbreak strain.
        N Engl J Med. 2011; 364: 33-42
        • Grad Y.H.
        • Lipsitch M.
        • Feldgarden M.
        • et al.
        Genomic epidemiology of the Escherichia coli O104:H4 outbreaks in Europe, 2011.
        Proc Natl Acad Sci USA. 2012; 109: 3065-3070
        • Sabat A.J.
        • Budimir A.
        • Nashev D.
        • et al.
        Overview of molecular typing methods for outbreak detection and epidemiological surveillance.
        Euro Surveill. 2013; 18: 20380
      1. Outbreak of Escherichia coli O104:H4 infections associated with sprout consumption - Europe and North America, May-July 2011. MMWR Morb Mortal Wkly Rep 2013; 62: 1029–31.

        • Cantey J.B.
        • Sreeramoju P.
        • Jaleel M.
        • et al.
        Prompt control of an outbreak caused by extended-spectrum β-lactamase–producing Klebsiella pneumoniae in a neonatal intensive care unit.
        J Pediatr. 2013; 163: 672-679
        • Snitkin E.S.
        • Zelazny A.M.
        • Thomas P.J.
        • et al.
        Tracking a hospital outbreak of carbapenem-resistant Klebsiella pneumoniae with whole-genome sequencing.
        Sci Transl Med. 2012; 4: 148ra16
        • Smit E.
        Antiviral resistance testing.
        Curr Opin Infect Dis. 2014; 27: 566-572
        • Andrei G.
        • Georgala A.
        • Topalis D.
        • et al.
        Heterogeneity and evolution of thymidine kinase and DNA polymerase mutants of herpes simplex virus type 1: implications for antiviral therapy.
        J Infect Dis. 2013; 207: 1295-1305
        • Andrei G.
        • Snoeck R.
        Herpes simplex virus drug-resistance: new mutations and insights.
        Curr Opin Infect Dis. 2013; 26: 551-560
        • Andrei G.
        • Topalis D.
        • Fiten P.
        • et al.
        In vitro-selected drug-resistant varicella-zoster virus mutants in the thymidine kinase and DNA poly-merase genes yield novel phenotype-genotype associations and highlight differences between antiherpesvirus drugs.
        J Virol. 2012; 86: 2641-2652
        • Sauerbrei A.
        • Bohn K.
        Phenotypic and genotypic testing of HSV-1 resistance to antivirals.
        Methods Mol Biol. 2014; 1144: 149-165
        • Capobianchi M.R.
        • Giombini E.
        • Rozera G.
        Next-generation sequencing technology in clinical virology.
        Clin Microbiol Infect. 2013; 19: 15-22
        • Wang C.
        • Mitsuya Y.
        • Gharizadeh B.
        • et al.
        Characterization of mutation spectra with ultra-deep pyrosequencing: application to HIV-1 drug resistance.
        Genome Res. 2007; 17 (): 1195-1201
        • Burke D.S.
        Recombination in HIV: an important viral evolutionary strategy.
        Emerg Infect Dis. 1997; 3: 253-259
        • Shao W.
        • Boltz V.F.
        • Spindler J.E.
        • et al.
        Analysis of 454 sequencing error rate, error sources, and artifact recombination for detection of Low-frequency drug resistance mutations in HIV-1 DNA.
        Retrovirology. 2013; 10: 18
        • Stekler J.D.
        • Ellis G.M.
        • Carlsson J.
        • et al.
        Prevalence and impact of minority variant drug resistance mutations in primary HIV-1 infection.
        PLoS One. 2011; 6: e28952
        • Gianella S.
        • Richman D.D.
        Minority variants of drug-resistant HIV.
        J Infect Dis. 2010; 202: 657-666
        • Schwaber M.J.
        • Carmeli Y.
        Mortality and delay in effective therapy associated with extended-spectrum β-lactamase production in Enterobacteriaceae bacteraemia: a systematic review and meta-analysis.
        J Antimicrob Chemother. 2007; 60: 913-920
        • Cortes J.A.
        • Garzon D.C.
        • Navarrete J.A.
        • et al.
        Impact of inappropriate antimicrobial therapy on patients with bacteremia in intensive care units and resistance patterns in Latin America.
        Rev Argent Microbiol. 2010; 42: 230-234
        • Lupei M.I.
        • Mann H.J.
        • Beilman G.J.
        • et al.
        Inadequate antibiotic therapy in solid organ transplant recipients is associated with a higher mortality rate.
        Surg Infect (Larchmt). 2010; 11: 33-39
        • Peralta G.
        • Sanchez M.B.
        • Garrido J.C.
        • et al.
        Impact of antibiotic resistance and of adequate empirical antibiotic treatment in the prognosis of patients with Escherichia coli bacteraemia.
        J Antimicrob Chemother. 2007; 60: 855-863
        • Bourdon N.
        • Berenger R.
        • Lepoultier R.
        • et al.
        Rapid detection of vanco-mycin-resistant enterococci from rectal swabs by the Cepheid Xpert vanA/ vanB assay.
        Diagn Microbiol Infect Dis. 2010; 67: 291-293
        • Jiang X.
        • Espedido B.A.
        • Partridge S.R.
        • et al.
        Paradoxical effect of Klebsiella pneumoniae OmpK36 porin deficiency.
        Pathology. 2009; 41: 388-392
        • Wachino J.
        • Arakawa Y.
        Exogenously acquired 16S rRNA methyltransferases found in aminoglycoside-resistant pathogenic Gram-negative bacteria: an update.
        Drug Resist Updat. 2012; 15: 133-148
        • Partridge S.R.
        • Tsafnat G.
        • Coiera E.
        • et al.
        Gene cassettes and cassette arrays in mobile resistance integrons.
        FEMS Microbiol Rev. 2009; 33: 757-784
        • Ramirez M.S.
        • Tolmasky M.E.
        Aminoglycoside modifying enzymes.
        Drug Resist Updat. 2010; 13: 151-171
        • Shaw K.J.
        • Rather P.N.
        • Hare R.S.
        • et al.
        Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes.
        Microbiol Rev. 1993; 57: 138-163
        • Karim A.
        • Poirel L.
        • Nagarajan S.
        • et al.
        Plasmid-mediated extended-spectrum β-lactamase (CTX-M-3 like) from India and gene association with insertion sequence ISEcp1.
        FEMS Microbiol Lett. 2001; 201: 237-241
        • Zong Z.
        • Partridge S.R.
        • Thomas L.
        • et al.
        Dominance of blaCTX-M within an Australian extended-spectrum β-lactamase gene pool.
        Antimicrob Agents Chemother. 2008; 52: 4198-4202
        • Nordmann P.
        • Poirel L.
        • Walsh T.R.
        • et al.
        The emerging NDM carbapenemases.
        Trends Microbiol. 2011; 19: 588-595
        • Chu Y.W.
        • Afzal-Shah M.
        • Houang E.T.
        • et al.
        IMP-4, a novel metallo-β-lactamase from nosocomial Acinetobacter spp. collected in Hong Kong between 1994 and 1998.
        Antimicrob Agents Chemother. 2001; 45: 710-714
        • Hawkey P.M.
        • Xiong J.
        • Ye H.
        • et al.
        Occurrence of a new metallo-β-lactamase IMP-4 carried on a conjugative plasmid in Citrobacter youngae from the People’s Republic of China.
        FEMS Microbiol Lett. 2001; 194: 53-57
        • Espedido B.A.
        • Partridge S.R.
        • Iredell J.R.
        blaIMP-4 in different genetic contexts in Enterobacteriaceae isolates from Australia.
        Antimicrob Agents Chemother. 2008; 52: 2984-2987
        • Peleg A.Y.
        • Franklin C.
        • Bell J.
        • et al.
        Emergence of IMP-4 metallo-β-lactamase in a clinical isolate from Australia.
        J Antimicrob Chemother. 2004; 54: 699-700
        • Espedido B.
        • Iredell J.
        • Thomas L.
        • et al.
        Wide dissemination of a carba-penemase plasmid among Gram-negative bacteria: implications of the variable phenotype.
        J Clin Microbiol. 2005; 43: 4918-4919
        • Roy Chowdhury P.
        • Ingold A.
        • Vanegas N.
        • et al.
        Dissemination of multiple drug resistance genes by class 1 integrons in Klebsiella pneumoniae isolates from four countries: a comparative study.
        Antimicrob Agents Chemother. 2011; 55: 3140-3149
        • Xiao Y.
        • Hu Y.
        The major aminoglycoside-modifying enzyme AAC(3)-II found in Escherichia coli determines a significant disparity in its resistance to gentamicin and amikacin in China.
        Microb Drug Resist. 2012; 18: 42-46
        • Doi Y.
        • Arakawa Y.
        16S ribosomal RNA methylation: emerging resistance mechanism against aminoglycosides.
        Clin Infect Dis. 2007; 45: 88-94
        • Yong D.
        • Toleman M.A.
        • Giske C.G.
        • et al.
        Characterization of a new metallo-β-lactamase gene, blaNDM-1, and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India.
        Antimicrob Agents Chemother. 2009; 53: 5046-5054
        • Yigit H.
        • Queenan A.M.
        • Anderson G.J.
        • et al.
        Novel carbapenem-hydrolyzing β-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae.
        Antimicrob Agents Chemother. 2001; 45: 1151-1161
        • Coatsworth N.R.
        • Huntington P.G.
        • Hardiman R.P.
        • et al.
        A case of carbapenemase-producing Klebsiella pneumoniae in Australia.
        Pathology. 2012; 44: 42-44
        • Poirel L.
        • Lagrutta E.
        • Taylor P.
        • et al.
        Emergence of metallo-β-lactamase NDM-1-producing multidrug-resistant Escherichia coli in Australia.
        Antimicrob Agents Chemother. 2010; 54: 4914-4916
        • Sidjabat H.
        • Nimmo G.R.
        • Walsh T.R.
        • et al.
        Carbapenem resistance in Klebsiella pneumoniae due to the New Delhi metallo-β-lactamase.
        Clin Infect Dis. 2011; 52: 481-484
        • Jain A.
        • Hopkins K.L.
        • Turton J.
        • et al.
        NDM carbapenemases in the United Kingdom: an analysis of thefirst 250 cases.
        J Antimicrob Chemother. 1777; 69: 84
        • Woodford N.
        • Zhang J.
        • Warner M.
        • et al.
        Arrival of Klebsiella pneumoniae producing KPC carbapenemase in the United Kingdom.
        J Antimicrob Chemother. 2008; 62: 1261-1264
        • van Hal S.J.
        • Wiklendt A.
        • Espedido B.
        • et al.
        Immediate appearance of plasmid-mediated resistance to multiple antibiotics upon antibiotic selection: an argument for systematic resistance epidemiology.
        J Clin Microbiol. 2009; 47: 2325-2327
        • Rockett R.J.
        • Tozer S.J.
        • Peatey C.
        • et al.
        A real-time, quantitative PCR method using hydrolysis probes for the monitoring of Plasmodium falciparum load in experimentally infected human volunteers.
        Malar J. 2011; 10: 48
        • Nwakanma D.C.
        • Gomez-Escobar N.
        • Walther M.
        • et al.
        Quantitative detection of Plasmodium falciparum DNA in saliva, blood, and urine.
        J Infect Dis. 2009; 199: 1567-1574
        • Ferreira I.D.
        • Rosario V.E.
        Real-time quantitative PCR with SYBR Green I detection for estimating copy numbers of nine drug resistance candidate genes in Plasmodium falciparum.
        Malar J. 2006; 5: 1
        • Gullett J.C.
        • Nolte F.S.
        Quantitative nucleic acid amplification methods for viral infections.
        Clin Chem. 2015; 61: 72-78
        • Tsubota A.
        • Arase Y.
        • Someya T.
        • et al.
        Early viral kinetics and treatment outcome in combination of high-dose interferon induction vs. pegylated interferon plus ribavirin for naive patients infected with hepatitis C virus of genotype 1b and high viral load.
        J Med Virol. 2005; 75: 27-34
        • Kotton C.N.
        • Kumar D.
        • Caliendo A.M.
        • et al.
        Updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation.
        Transplantation. 2013; 96: 333-360
        • Ferenci P.
        • Laferl H.
        • Scherzer T.M.
        • et al.
        Peginterferon alfa-2a and ribavirin for 24 weeks in hepatitis C type 1 and 4 patients with rapid virological response.
        Gastroenterology. 2008; 135: 451-458
        • Ghany M.G.
        • Strader D.B.
        • Thomas D.L.
        • et al.
        Diagnosis, management, and treatment of hepatitis C: an update.
        Hepatology. 2009; 49: 1335-1374
        • Jensen D.M.
        • Morgan T.R.
        • Marcellin P.
        • et al.
        Early identification of HCV genotype 1 patients responding to 24 weeks peginterferon α-2a (40 kd)/ribavirin therapy.
        Hepatology. 2006; 43: 954-960
        • World Health Organization (WHO)
        Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection. WHO, Geneva2013
        • Baggaley R.F.
        • Griffin J.T.
        • Chapman R.
        • et al.
        Estimating the public health impact of the effect of herpes simplex virus suppressive therapy on plasma HIV-1 viral load.
        AIDS. 2009; 23: 1005-1013
        • Sahu G.K.
        Potential implication of residual viremia in patients on effective antiretroviral therapy.
        AIDS Res Hum Retroviruses. 2015; 31: 25-35
        • Alvarez Estevez M.
        • Chueca Porcuna N.
        • Guillot Suay V.
        • et al.
        Quantification of viral loads lower than 50 copies per milliliter by use of the Cobas AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0, can predict the likelihood of subsequent virological rebound to >50 copies per milliliter.
        J Clin Microbiol. 2013; 51: 1555-1557
        • Doyle T.
        • Smith C.
        • Vitiello P.
        • et al.
        Plasma HIV-1 RNA detection below 50 copies/ml and risk of virologic rebound in patients receiving highly active antiretroviral therapy.
        Clin Infect Dis. 2012; 54: 724-732
        • Pascual-Pareja J.F.
        • Martinez-Prats L.
        • Luczkowiak J.
        • et al.
        Detection of HIV-1 at between 20 and 49 copies per milliliter by the Cobas TaqMan HIV-1 v2.0 assay is associated with higher pretherapy viral load and less time on antiretroviral therapy.
        J Clin Microbiol. 2010; 48: 1911-1912
        • Dellinger R.P.
        • Levy M.M.
        • Carlet J.M.
        • et al.
        Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008.
        Crit Care Med. 2008; 36: 296-327
        • Kumar A.
        • Roberts D.
        • Wood K.E.
        • et al.
        Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock.
        Crit Care Med. 2006; 34: 1589-1596
        • Rivers E.
        • Nguyen B.
        • Havstad S.
        • et al.
        Early goal-directed therapy in the treatment of severe sepsis and septic shock.
        N Engl J Med. 2001; 345 (): 1368-1377
        • Bhat S.V.
        • Peleg A.Y.
        • Lodise Jr., T.P.
        • et al.
        Failure of current cefepime breakpoints to predict clinical outcomes of bacteremia caused by Gramnegative organisms.
        Antimicrob Agents Chemother. 2007; 51: 4390-4395
        • Ibrahim E.H.
        • Sherman G.
        • Ward S.
        • et al.
        The influence of inadequate antimicrobial treatment of bloodstream infections on patient outcomes in the ICU setting.
        Chest. 2000; 118: 146-155
        • Kumar A.
        • Haery C.
        • Paladugu B.
        • et al.
        The duration of hypotension before the initiation of antibiotic treatment is a critical determinant of survival in a murine model of Escherichia coli septic shock: association with serum lactate and inflammatory cytokine levels.
        J Infect Dis. 2006; 193: 251-258
        • Rangel-Frausto M.S.
        • Pittet D.
        • Costigan M.
        • et al.
        The natural history of the systemic inflammatory response syndrome (SIRS). A prospective study.
        JAMA. 1995; 273: 117-123
        • Hackett S.J.
        • Guiver M.
        • Marsh J.
        • et al.
        Meningococcal bacterial DNA load at presentation correlates with disease severity.
        Arch Dis Child. 2002; 86: 44-46
        • Rello J.
        • Lisboa T.
        • Lujan M.
        • et al.
        Severity of pneumococcal pneumonia associated with genomic bacterial load.
        Chest. 2009; 136: 832-840
        • Martínez J.A.
        • Soto S.
        • Fabrega A.
        • et al.
        Relationship of phylogenetic background, biofilm production, and time to detection of growth in blood culture vials with clinical variables and prognosis associated with Escherichia coli bacteremia.
        J Clin Microbiol. 2006; 44: 1468-1474
        • Liao C.H.
        • Lai C.C.
        • Hsu M.S.
        • et al.
        Correlation between time to positivity of blood cultures with clinical presentation and outcomes in patients with Klebsiella pneumoniae bacteraemia: prospective cohort study.
        Clin Microbiol Infect. 2009; 15: 1119-1125