Despite some limitations, current molecular diagnostic methods have a great potential to include targets useful in the rapid identification of microorganisms and antimicrobial resistance, to analyse directly unprocessed samples and to obtain quantitative results in pneumonia, an entity of complex microbiological diagnosis due to the features of the pathogens commonly implicated.
by Dr A. Camporese
A change in culture without culture?
Developing accurate methods for diagnosing respiratory tract infections has long been a challenge for the clinical microbiology laboratory [1].
The current semi-quantitative agar-plate based culture method used in most clinical microbiology laboratories for analysing specimens from patients with suspected community-acquired pneumonia (CAP), hospital-acquired pneumonia (HAP), or ventilator-associated pneumonia (VAP), although adequate for recovering and identifying a wide variety of bacterial species from respiratory specimens, is slow, and cannot differentiate between colonization and infection. Moreover, results may be misleading, particularly if a Gram stain is not performed in parallel to ascertain the adequacy of expectorated sputum samples or endotracheal aspirates [2].
As the Infectious Diseases Society of America (IDSA) and American Thoracic Society (ATS) CAP guideline notes, one of the problems with diagnostic tests for respiratory tract infections “is driven by the poor quality of most sputum microbiological samples and the low yield of positive culture results” [3]. Moreover, the highest predictive value of a culture occurs only when Gram stain shows a predominant morphotype, and the culture yields predominant growth of a single recognized respiratory pathogen of that morphotype (e.g., Streptococcus pneumoniae) [2].
Unfortunately, such concordance decreases rapidly when specimens are collected after the initiation of antimicrobial therapy or when their arrival at the microbiology laboratory is significantly delayed.
One approach that may improve the diagnosis of respiratory tract infections and shorten the time necessary to place patients on appropriate therapy is the use of nucleic acid amplification methods.
Straight ahead toward molecular assays
Today clinical microbiologists appear to be on the threshold of a potentially important transition, with a substantial increase in the use of molecular diagnostic tests to replace or augment the century-old methods of culture, as many experts now view traditional microbiology as slow and antiquated, especially when compared with newer technologies used in other areas of laboratory medicine [4].
Traditional methods demonstrated poor sensitivity and specificity for detecting specific pathogens, particularly when the specimen being cultured is from a non-sterile anatomical compartment, such as the respiratory tract.
For this reason, molecular methods are becoming more widely used also for the detection of respiratory pathogens, in part because of their superior sensitivity, relatively rapid turnaround time, and ability to identify pathogens that are slow growing or difficult to culture.
However, to have a positive impact on patient management, molecular tests will need to be easy to use, and provide clear, definitive results that will give physicians the data necessary to start, or in some cases withhold, antimicrobial agents [5].
Further, to be really successful, industry must determine which combination of molecular targets [Table 1] and clinical specimens will produce results that will effectively guide anti-infective therapy regimens for patients with pneumonia or other respiratory tract diseases.
Another key challenge for industry will be to develop assays that are not only rapid, but also readily accessible, because development of an assay that is rapid, but unavailable on evening or night shifts, or at weekends, because of its technical complexity, limits the clinical value of the test.
Moreover, to be successful, molecular assays will need to be perceived by health care systems as cost-effective, but cost-effectiveness should be determined not only by comparison to the costs of performing slower, conventional methods in the laboratory, but also by consideration of the cost savings achieved from optimized antimicrobial therapy, decreased use of additional diagnostic tests, and shorter hospital stays [2].
To address issues on these topics, the IDSA and the Food and Drug Administration (FDA) co-sponsored a workshop on molecular diagnostic testing for respiratory tract infections in November 2009, with the participation of the FDA, industry, authorities in microbiology, statisticians and others. Respiratory tract infections were selected because this is the site of most infections treated with antibiotics in paediatric and adult practice, and they also represent a group of infections in which an etiologic agent is seldom identified in non-research settings [4].
The IDSA believes that patient care could be improved by accurate and rapid identification of pathogens, which would promote more judicious use of antibiotics, permit pathogen directed therapy, and provide potentially important
epidemiologic information.
Thus, the IDSA strongly desires development and implementation of molecular diagnostic tests that are easy, rapid, technically uncomplicated, applicable to specimens that are readily obtained, reasonably priced, sensitive and specific, because such tests will greatly improve antimicrobial stewardship, thereby helping to reduce the spread and impact of antibiotic resistance. Such tests will also facilitate conduct of clinical trials supporting the approval of new antibacterial agents [4].
Respiratory samples suitable for molecular assays
A variety of respiratory samples are amenable to molecular testing, including expectorated sputum, bronchoalveolar lavages (BALs), protected bronchial brushes, and endotracheal aspirates [2, 5, 6]. Of these, expectorated sputum samples are by far the most common respiratory samples submitted to the clinical microbiology laboratory, but are also the poorest in overall quality.
Endotracheal aspirates from ventilated patients are often of better quality than that of expectorated sputum obtained from patients with CAP/HAP, but may still be contaminated with upper respiratory tract flora.
Therefore, obtaining specimens from the site of infection that are not contaminated with upper respiratory tract flora remains to date a real and constant problem. BALs and protected brush samples seem more likely to yield samples from the site of infection, but require significantly more effort to obtain, and thus offer a much smaller market for a new molecular test.
Moreover, there is a significant gap in our knowledge as to how well molecular tests for bacterial pathogens would perform on expectorated sputum samples, compared with performance on BALs or protected brush samples from the same patient collected within a similar period [2].
This knowledge gap is also a barrier to test development, because a molecular test that cannot be performed on expectorated sputum (given all the problems with specimen quality) may not have broad enough appeal among physicians to make it a financially viable product (from the industry perspective).
Technology perspectives
There are a wide array of emerging technologies for the detection and quantification of respiratory pathogens directly from clinical specimens. Some of these technologies, such as real-time PCR, have potential for high-throughput testing, and others will allow rapid near patient testing, but more studies are needed to fully determine their performance characteristics and determine their ideal clinical application [6,7].
Molecular assays may target either a single pathogen or multiple respiratory pathogens in a single assay. There are merits to both single-pathogen and multiplex approaches. Certain bacterial respiratory pathogens cause such distinct clinical syndromes that assays that target them individually still have clinical utility. These include already many organisms, such as Chlamydophila pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila, or Bordetella pertussis [Table 1].
Moreover, some multiplex assays for respiratory tract disease already include many targets for a rapid diagnosis of CAP, HAP, and VAP, but, in designing new assays, it will be critical to understand whether an assay for a determined number of bacterial pathogens will meet physicians’ needs and provide adequate data for initiating or altering anti-infective therapy [7, 8].
Potential and currently available targets for multiplex or individual molecular assays for respiratory tract samples in immunocompetent and/or immunocompromised patients with CAP, HAP, or VAP are presented in Table 1 [7].
Further, in this age of multidrug resistance, expanding the target selection to include key antimicrobial resistance genes that would alter existing therapy or guide empirical therapy, should also be considered [Table 1].
Lastly, if molecular-based diagnostic methods currently available are helpful in detecting single and multiple bacterial pathogens simultaneously, including the most frequent cause of CAP/HAP/VAP, the real-time PCR is also well known for its ability to quantify targets.
Where available, the application of quantitative molecular tests for the detection of key pathogens, such as S. pneumoniae, both in sputum and in blood, defining a threshold for classification, such as a colonizer or as an invasive pathogen, might be relevant in CAP patients, mainly in whom antibiotic therapy has been initiated, and might be a useful tool for severity assessment [9, 10].
Conclusion
Significant progress exists on the development and improvement of molecular-based methods feasible to be applied to the diagnosis of lower respiratory tract infection.
Multiplex assays, user-friendly formats, results in a few hours, high sensitivity and specificity in pathogen identification, detection of antibiotic resistance genes and target quantification, among others, are some of the contributions of novel molecular-based diagnosis approaches.
Developing new molecular tests for other bacterial respiratory pathogens, particularly microorganisms that can be both asymptomatic colonizers and overt pathogens of the respiratory tract, detection of pathogens and new key antimicrobial resistance genes in unprocessed samples, and determination of the microbial load by quantitative multi-pathogen tests will be some of the future challenges of molecular diagnosis in CAP/HAP/VAP.
References
1. Bartlett JG. Decline in microbial studies for patients with pulmonary infections. Clin Infect Dis 2004; 39: 170–172.
2. Tenover FC. Developing molecular amplification methods for rapid diagnosis of respiratory tract infections caused by bacterial pathogens. Clin Infect Dis 2011; 52(S4): S338–S345.
3. Mandell LA, Wunderink RG, Anzueto A, et al. Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults. Clin Infect Dis 2007; 44(S2): S27–S72.
4. Infectious Disease Society of America (IDSA). An unmet medical need: rapid molecular diagnostics tests for respiratory tract infections. Clin Infect Dis 2011; 52(S4): S384–S395.
5. Caliendo AM. Multiplex PCR and emerging technologies for the detection of respiratory pathogens. Clin Infect Dis 2011; 52(S4): S326–S330.
6. Lung M and Codina G. Molecular diagnosis in HAP/VAP. Curr Opin Crit Care 2012; 18: 487–494.
7. Camporese A. Impact of recent advances in molecular techniques on diagnosing lower respiratory tract infections (LRTIs). Infez Med 2012; 4: 237–244.
8. Johansson N, Kalin M, Tiveljung-Lindell A, et al. Etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods. Clin Infect Dis 2010; 50: 202–209.
9. Werno AM, Anderson TP, Murdoch DR. Association between pneumococcal load and disease severity in adults with pneumonia. J Med Microbiol 2012; 61: 1129–1135.
10. Woodhead M, Blasi F, Ewig S, et al. Guidelines for the management of adult lower respiratory tract infections-Full version. Clin Microbiol Infect 2011; 17(S6): E1–E59.
The author
Alessandro Camporese MD
Clinical Microbiology and Virology Department
S. Maria degli Angeli Regional Hospital, Pordenone, Italy
E-mail: alessandro.camporese@aopn.fvg.it
Your IVD partner for instruments and reagents
, /in Featured Articles /by 3wmediaSTA Compact Max
, /in Featured Articles /by 3wmediaTest smarter. Run faster.
, /in Featured Articles /by 3wmediaOsmomat 3000 – freezing point osmometer
, /in Featured Articles /by 3wmediaRecent advances and perspectives in the molecular diagnosis of pneumonia
, /in Featured Articles /by 3wmediaDespite some limitations, current molecular diagnostic methods have a great potential to include targets useful in the rapid identification of microorganisms and antimicrobial resistance, to analyse directly unprocessed samples and to obtain quantitative results in pneumonia, an entity of complex microbiological diagnosis due to the features of the pathogens commonly implicated.
by Dr A. Camporese
A change in culture without culture?
Developing accurate methods for diagnosing respiratory tract infections has long been a challenge for the clinical microbiology laboratory [1].
The current semi-quantitative agar-plate based culture method used in most clinical microbiology laboratories for analysing specimens from patients with suspected community-acquired pneumonia (CAP), hospital-acquired pneumonia (HAP), or ventilator-associated pneumonia (VAP), although adequate for recovering and identifying a wide variety of bacterial species from respiratory specimens, is slow, and cannot differentiate between colonization and infection. Moreover, results may be misleading, particularly if a Gram stain is not performed in parallel to ascertain the adequacy of expectorated sputum samples or endotracheal aspirates [2].
As the Infectious Diseases Society of America (IDSA) and American Thoracic Society (ATS) CAP guideline notes, one of the problems with diagnostic tests for respiratory tract infections “is driven by the poor quality of most sputum microbiological samples and the low yield of positive culture results” [3]. Moreover, the highest predictive value of a culture occurs only when Gram stain shows a predominant morphotype, and the culture yields predominant growth of a single recognized respiratory pathogen of that morphotype (e.g., Streptococcus pneumoniae) [2].
Unfortunately, such concordance decreases rapidly when specimens are collected after the initiation of antimicrobial therapy or when their arrival at the microbiology laboratory is significantly delayed.
One approach that may improve the diagnosis of respiratory tract infections and shorten the time necessary to place patients on appropriate therapy is the use of nucleic acid amplification methods.
Straight ahead toward molecular assays
Today clinical microbiologists appear to be on the threshold of a potentially important transition, with a substantial increase in the use of molecular diagnostic tests to replace or augment the century-old methods of culture, as many experts now view traditional microbiology as slow and antiquated, especially when compared with newer technologies used in other areas of laboratory medicine [4].
Traditional methods demonstrated poor sensitivity and specificity for detecting specific pathogens, particularly when the specimen being cultured is from a non-sterile anatomical compartment, such as the respiratory tract.
For this reason, molecular methods are becoming more widely used also for the detection of respiratory pathogens, in part because of their superior sensitivity, relatively rapid turnaround time, and ability to identify pathogens that are slow growing or difficult to culture.
However, to have a positive impact on patient management, molecular tests will need to be easy to use, and provide clear, definitive results that will give physicians the data necessary to start, or in some cases withhold, antimicrobial agents [5].
Further, to be really successful, industry must determine which combination of molecular targets [Table 1] and clinical specimens will produce results that will effectively guide anti-infective therapy regimens for patients with pneumonia or other respiratory tract diseases.
Another key challenge for industry will be to develop assays that are not only rapid, but also readily accessible, because development of an assay that is rapid, but unavailable on evening or night shifts, or at weekends, because of its technical complexity, limits the clinical value of the test.
Moreover, to be successful, molecular assays will need to be perceived by health care systems as cost-effective, but cost-effectiveness should be determined not only by comparison to the costs of performing slower, conventional methods in the laboratory, but also by consideration of the cost savings achieved from optimized antimicrobial therapy, decreased use of additional diagnostic tests, and shorter hospital stays [2].
To address issues on these topics, the IDSA and the Food and Drug Administration (FDA) co-sponsored a workshop on molecular diagnostic testing for respiratory tract infections in November 2009, with the participation of the FDA, industry, authorities in microbiology, statisticians and others. Respiratory tract infections were selected because this is the site of most infections treated with antibiotics in paediatric and adult practice, and they also represent a group of infections in which an etiologic agent is seldom identified in non-research settings [4].
The IDSA believes that patient care could be improved by accurate and rapid identification of pathogens, which would promote more judicious use of antibiotics, permit pathogen directed therapy, and provide potentially important
epidemiologic information.
Thus, the IDSA strongly desires development and implementation of molecular diagnostic tests that are easy, rapid, technically uncomplicated, applicable to specimens that are readily obtained, reasonably priced, sensitive and specific, because such tests will greatly improve antimicrobial stewardship, thereby helping to reduce the spread and impact of antibiotic resistance. Such tests will also facilitate conduct of clinical trials supporting the approval of new antibacterial agents [4].
Respiratory samples suitable for molecular assays
A variety of respiratory samples are amenable to molecular testing, including expectorated sputum, bronchoalveolar lavages (BALs), protected bronchial brushes, and endotracheal aspirates [2, 5, 6]. Of these, expectorated sputum samples are by far the most common respiratory samples submitted to the clinical microbiology laboratory, but are also the poorest in overall quality.
Endotracheal aspirates from ventilated patients are often of better quality than that of expectorated sputum obtained from patients with CAP/HAP, but may still be contaminated with upper respiratory tract flora.
Therefore, obtaining specimens from the site of infection that are not contaminated with upper respiratory tract flora remains to date a real and constant problem. BALs and protected brush samples seem more likely to yield samples from the site of infection, but require significantly more effort to obtain, and thus offer a much smaller market for a new molecular test.
Moreover, there is a significant gap in our knowledge as to how well molecular tests for bacterial pathogens would perform on expectorated sputum samples, compared with performance on BALs or protected brush samples from the same patient collected within a similar period [2].
This knowledge gap is also a barrier to test development, because a molecular test that cannot be performed on expectorated sputum (given all the problems with specimen quality) may not have broad enough appeal among physicians to make it a financially viable product (from the industry perspective).
Technology perspectives
There are a wide array of emerging technologies for the detection and quantification of respiratory pathogens directly from clinical specimens. Some of these technologies, such as real-time PCR, have potential for high-throughput testing, and others will allow rapid near patient testing, but more studies are needed to fully determine their performance characteristics and determine their ideal clinical application [6,7].
Molecular assays may target either a single pathogen or multiple respiratory pathogens in a single assay. There are merits to both single-pathogen and multiplex approaches. Certain bacterial respiratory pathogens cause such distinct clinical syndromes that assays that target them individually still have clinical utility. These include already many organisms, such as Chlamydophila pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila, or Bordetella pertussis [Table 1].
Moreover, some multiplex assays for respiratory tract disease already include many targets for a rapid diagnosis of CAP, HAP, and VAP, but, in designing new assays, it will be critical to understand whether an assay for a determined number of bacterial pathogens will meet physicians’ needs and provide adequate data for initiating or altering anti-infective therapy [7, 8].
Potential and currently available targets for multiplex or individual molecular assays for respiratory tract samples in immunocompetent and/or immunocompromised patients with CAP, HAP, or VAP are presented in Table 1 [7].
Further, in this age of multidrug resistance, expanding the target selection to include key antimicrobial resistance genes that would alter existing therapy or guide empirical therapy, should also be considered [Table 1].
Lastly, if molecular-based diagnostic methods currently available are helpful in detecting single and multiple bacterial pathogens simultaneously, including the most frequent cause of CAP/HAP/VAP, the real-time PCR is also well known for its ability to quantify targets.
Where available, the application of quantitative molecular tests for the detection of key pathogens, such as S. pneumoniae, both in sputum and in blood, defining a threshold for classification, such as a colonizer or as an invasive pathogen, might be relevant in CAP patients, mainly in whom antibiotic therapy has been initiated, and might be a useful tool for severity assessment [9, 10].
Conclusion
Significant progress exists on the development and improvement of molecular-based methods feasible to be applied to the diagnosis of lower respiratory tract infection.
Multiplex assays, user-friendly formats, results in a few hours, high sensitivity and specificity in pathogen identification, detection of antibiotic resistance genes and target quantification, among others, are some of the contributions of novel molecular-based diagnosis approaches.
Developing new molecular tests for other bacterial respiratory pathogens, particularly microorganisms that can be both asymptomatic colonizers and overt pathogens of the respiratory tract, detection of pathogens and new key antimicrobial resistance genes in unprocessed samples, and determination of the microbial load by quantitative multi-pathogen tests will be some of the future challenges of molecular diagnosis in CAP/HAP/VAP.
References
1. Bartlett JG. Decline in microbial studies for patients with pulmonary infections. Clin Infect Dis 2004; 39: 170–172.
2. Tenover FC. Developing molecular amplification methods for rapid diagnosis of respiratory tract infections caused by bacterial pathogens. Clin Infect Dis 2011; 52(S4): S338–S345.
3. Mandell LA, Wunderink RG, Anzueto A, et al. Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults. Clin Infect Dis 2007; 44(S2): S27–S72.
4. Infectious Disease Society of America (IDSA). An unmet medical need: rapid molecular diagnostics tests for respiratory tract infections. Clin Infect Dis 2011; 52(S4): S384–S395.
5. Caliendo AM. Multiplex PCR and emerging technologies for the detection of respiratory pathogens. Clin Infect Dis 2011; 52(S4): S326–S330.
6. Lung M and Codina G. Molecular diagnosis in HAP/VAP. Curr Opin Crit Care 2012; 18: 487–494.
7. Camporese A. Impact of recent advances in molecular techniques on diagnosing lower respiratory tract infections (LRTIs). Infez Med 2012; 4: 237–244.
8. Johansson N, Kalin M, Tiveljung-Lindell A, et al. Etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods. Clin Infect Dis 2010; 50: 202–209.
9. Werno AM, Anderson TP, Murdoch DR. Association between pneumococcal load and disease severity in adults with pneumonia. J Med Microbiol 2012; 61: 1129–1135.
10. Woodhead M, Blasi F, Ewig S, et al. Guidelines for the management of adult lower respiratory tract infections-Full version. Clin Microbiol Infect 2011; 17(S6): E1–E59.
The author
Alessandro Camporese MD
Clinical Microbiology and Virology Department
S. Maria degli Angeli Regional Hospital, Pordenone, Italy
E-mail: alessandro.camporese@aopn.fvg.it
Respiratory infections due to non-diphtheriae Corynebacterium species
, /in Featured Articles /by 3wmediaSome species of non-diphtheriae Corynebacterium bacteria are opportunistic pathogens responsible for lower respiratory tract infections primarily in immunocompromised patients or in patients with chronic respiratory diseases. In the last years an increasing number of reports have demonstrated their role as emerging pathogens causing pneumonia or exacerbations of chronic pulmonary diseases. Thus, these species should not always be considered as mere colonizers.
by Dr M. Díez-Aguilar, Dr R. Cantón, Dr M. A. Meseguer
Non-diphtheriae Corynebacterium species are considered to be colonizers of the skin, nasopharyngeal tract and mucous membranes. However, in the last decade there have been an increasing number of reports that recognize these microorganisms as opportunistic pathogens that can cause disease in certain circumstances [1–3]. Since the population of immunocompromised patients is constantly growing, due to AIDS, age, use of invasive devices and immunosuppressive regimens, e.g. after transplantation, the clinical relevance of these opportunistic pathogens is rising.
A broad range of infectious diseases caused by non-diphtheriae Corynebacterium species have been reported including endocarditis, bacteriemia, pneumonia, tracheobronchitis, necrotizing tracheitis, exudative pharyngitis, rhinosinusitis, osteitis, conjunctivitis, and skin and urinary tract infections.
Lower respiratory tract infection, typically occurs in the context of underlying immunosuppressive conditions (such as diabetes, malignancy, corticoid therapy) and in patients with pre-existing pulmonary diseases such as chronic obstructive pulmonary disease (COPD), bronchiectasis and cystic fibrosis. In these patients non-diphtheriae Corynebacterium species can cause pneumonia and acute exacerbations of COPD. Previous hospitalization, wide-ranged antibiotic therapy and presence of multiple medical devices are risk factors for acquiring non-diphtheriae corynebacterial infection. Nosocomial outbreak of infection or colonization has been also observed [4]. Nevertheless, community acquired bronchitis in elderly patients with COPD have been reported.
Typically, Corynebacterium pseudodiphtheriticum, Corynebacterium striatum, and Corynebacterium propinquum are the species more frequently involved in lower respiratory tract infections [1–4]. The role of other Corynebacterium species in lower respiratory tract infections could have been underestimated, as only a few cases have been reported. The various non-diphtheriae Corynebacterium species that have been involved as responsible for respiratory tract infections are shown in Table 1. After appropriate antibiotic treatment a favourable outcome was achieved in most patients.
Pathogenesis
The respiratory tract damage caused by these microorganisms is probably the result of their opportunistic overgrowth and their possible virulence factors in patients with immune impairment and/or compromised pulmonary function.
Patients with chronic respiratory infections, such as obstructive pulmonary disease and bronchiectasis are predisposed to a persistent and non-innocent colonization of the lower respiratory tract by several non-pathogenic microorganisms. The high density of microorganisms covering the surface of the bronchial mucosa results in consistent pathogenic effects throughout the respiratory epithelium. Such effects include reduction of the supply of oxygen, water and organic nutrients to cells of the bronchial epithelium, as well as the liberation of potentially bioactive molecules which induce pro-inflammatory processes leading to accumulation of immune inflammatory cells. Defective pulmonary defences (impaired mucociliary clearance, airway inflammation and permanent dilatation within the bronchial wall), periodic infectious exacerbations caused by other respiratory infecting pathogens, and local immune disorders can cause a ‘vicious cycle’ of infection and inflammation of the airway. In these conditions the replacement of the pharyngeal resident microbiota with the opportunistic overgrowth and predominance of corynebacterial organisms in the respiratory tract can take place resulting in disease.
However, these microorganisms could express virulence factors that would contribute to the infection. Still, the virulence factors of non-diphtheriae Corynebacterium infection remain poorly understood, but recent in vitro studies on Corynebacterium pseudodiphtheriticum behaviour with epithelial cells have demonstrated the capacity for adherence, internalization, intracellular survival and persistence of the organism [5]. Therefore, in vivo C. pseudodiphtheriticum not only multiplies at and remains on the surface of the epithelial host cells, but also could reach the cytoplasm. This ability of C. pseudodiphtheriticum to survive within host cells highlights the potential capacity of other non-diphtheriae Corynebacterium to act as opportunistic pathogens.
Microbiological diagnosis
The key for the microbiological diagnosis of respiratory tract infection caused by non-diphtheriae Corynebacterium species is the microscopic observation of the predominant presence of Corynebacterium morphotype in a Gram stained purulent respiratory sample [Fig. 1], together with an abundant growth in the culture [6]. To determine the quality of the sputum it is important to follow the scoring system of Washington and Murray, which assesses a good quality of samples when there are more than 25 leukocytes and less than 10 squamous epithelial cells per field.
Identification of Coryneform bacteria
It is important to correctly identify Coryneform bacteria to the species level in order to reach the microbiological diagnosis, but also to detect unsuspected species, investigate potential pathogenicity and describe new species that could be clinically relevant.
Phenotypic characteristics such as colony size, pigmentation, catalase, and motility are useful for establishing the genus. For identification to the species level, biochemical testing performed using commercially available identification systems such as API Coryne, API CH50 plus, API 20 E and Rap IDCB Plus method, as well as automated systems such as Vitek2 (bioMèriux) and Biology systems could be employed. However, these methods are unreliable for some species (Corynebacterium accolens, C. striatum).
Nowadays an accurate and definitive identification is reached by the use of sequence-based identification techniques: 16s RNA and rpoB gene are the two approaches used for the characterization of non-diphtheriae Corynebacterium species. In fact, in recent years, many new species of the Corynebacterium genus have been described thanks to molecular biology techniques [7]. The use of mass spectrometry technology like MALDI-TOF MS is acquiring an increasingly important role in identifying and detecting these microorganisms [8]. This technology requires neither extensive training nor cost and it has been reported that it provides identification to genus and species level with an accuracy that approaches that of genetic methods.
Antimicrobial susceptibility
It is essential to test the antimicrobial susceptibility in all clinically relevant isolates due to the variable susceptibility of these microorganisms. Overall, non-diphtheriae Corynebacterium species are constitutively resistant to macrolides, lincosamides and type B streptogramins; susceptible to cefotaxime, amoxicillin/clavulanate, rifampin, and vancomycin (the recommended drug to treat severe infections) and have variable susceptibility to other antibiotics. C. striatum is the species which exhibits the highest resistance pattern.
According to CLSI (Clinical and laboratory Standard Institute) guidelines the reference method is the broth microdilution technique. This committee provides interpretive criteria for penicillin and erythromycin based on minimum inhibitory concentration (MIC) values following testing by this method, and for cephalosporin and linezolid the criteria are currently adapted from those from Streptoccocus and Enteroccocus, respectively, and remaining criteria are adapted from those from Staphyloccocus.
Although some laboratories use the disc diffusion method for susceptibility testing, the interpretative categories for zone diameters need to be established. The diffusion gradient tests (i.e. Etest) showed a good correlation of MICs with the broth microdilution method.
Conclusion and future perspectives
It is clear that due to the increasing number of immunocompromised patients and those with pre-existing pulmonary diseases, non-diphtheriae Corynebacterium species should be considered as an emerging cause of lower respiratory tract infection. A rapid and accurate laboratory detection, identification and assessment of these opportunistic microorganisms are critical for the correct diagnosis, taking into consideration that some of them are resistant to multiple antibiotics. Although more studies are need to enhance the understanding of the clinical significance of these microorganisms, clinicians should be aware of the potential pathogenic role of these species in the context of immunosuppression or chronic respiratory disease and they should not be always considered as mere colonizers.
References
1. Díez-Aguilar M, Ruiz-Garbajosa P, Fernández-Olmos A, Guisado P, Del Campo R, Quereda C, Cantón R, Meseguer MA. Non-diphtheriae Corynebacterium species: an emerging respiratory pathogen. Eur J Clin Microbiol Infect Dis 2012; doi: 10.1007/s10096-012-1805-5.
2. Nhan TX, Parienti JJ, Badiou G, Leclercq R, Cattoir V. Microbiological investigation and clinical significance of Corynebacterium spp. in respiratory specimens. Diagn Microbiol Infect Dis 2012; 74(3): 236–241.
3. Otsuka Y, Ohkusu K, Kawamura Y, Baba S, Ezaki T, Kimura S. Emergence of multidrug-resistant Corynebacterium striatum as a nosocomial pathogen in long-term hospitalized patients with underlying diseases. Diagn Microbiol Infect Dis 2006; 54(2): 109–114.
4. Renom F, Garau M, Rubí M, Ramis F, Galmés A, Soriano JB. Nosocomial outbreak of Corynebacterium striatum infection in patients with chronic obstructive pulmonary disease. J Clin Microbiol 2007; 45(6): 2064–2067.
5. Souza MC, Santos LS, Gomes DL, Sabbadini PS, Santos CS, Camello TC, Asad LM, Rosa AC, Nagao PE, Hirata Júnior R, Guaraldi AL. Aggregative adherent strains of Corynebacterium pseudodiphtheriticum enter and survive within HEp-2 epithelial cells. Mem Inst Oswaldo Cruz 2012;107(4): 486–93.
6. Funke G, von Graevenitz A, Clarridge JE 3rd, Bernard KA. Clinical microbiology of coryneform bacteria.Clin Microbiol Rev 1997; 10(1): 125–159.
7. Bernard K. The genus corynebacterium and other medically relevant coryneform-like bacteria. J Clin Microbiol. 2012; 50(10): 3152–3158.
8. Gomila M, Renom F, Gallegos Mdel C, Garau M, Guerrero D, Soriano JB, Lalucat J. Identification and diversity of multiresistant Corynebacterium striatum clinical isolates by MALDI-TOF mass spectrometry and by a multigene sequencing approach. BMC Microbiol 2012;12: 52.
The authors
María Díez-Aguilar* MD; Rafael Cantón MD, PhD; and María Antonia Meseguer MD, PhD
Department of Clinical Microbiology, Ramón y Cajal University Hospital, Madrid, Spain
*Corresponding author
E-mail: maria_diez_aguilar@hotmail.com
Biomarker panels for the diagnosis of sepsis
, /in Featured Articles /by 3wmediaSepsis is a complex syndrome associated with significant morbidity and mortality. If detected and treated early, septic patients have better prognoses. Unfortunately, identification of sepsis is challenging because its pathophysiology is complex and its clinical signs and symptoms overlap with other inflammatory diseases. This review discusses emerging biomarker panels and their ability to predict sepsis in critically ill patients.
by Dr A. Woodworth and Dr J. Colon-Franco
Sepsis and SIRS: International definitions
Definitions of sepsis and related conditions date back to the 1991 consensus conference held by the American College of Chest Physicians and the Society of Critical Care Medicine [1].
This consensus group introduced the term SIRS to describe the systemic inflammatory response syndrome, a normal response to infection and non-infectious insults like trauma, pancreatitis, and burns [Figure 1]. In SIRS two or more of the following clinical signs manifest: abnormal body temperature (fever or hypothermia), tachypnea, tachycardia and abnormal white blood cell count (leukocytosis or leukopenia).
The consensus group defined sepsis as the presence of SIRS along with a documented infection. Left untreated, septic patients develop severe sepsis, characterized by organ dysfunction, and ultimately septic shock, characterized by organ failure, hypotension and decreased peripheral perfusion. Revision of these definitions in 2001, added ‘suspected infection’ to the classification of sepsis to address numerous clinical cases where microorganisms cannot be confirmed.
For over 20 years, these definitions have provided uniformity in clinical disease recognition and better characterized patient populations for sepsis research. Although recognition of SIRS is relatively straightforward, identification of patients with sepsis among those with SIRS remains challenging. This is due, in part, to overlapping clinical signs and symptoms between SIRS and sepsis as well as inherent difficulty in confirming infectious causes of SIRS.
Sepsis and SIRS: Pathobiology and related syndromes
Sepsis pathobiology is complex and not well characterized [Figure 2] [1]. Historical models describing an overactive proinflammatory response to infection likely oversimplify the process. Sepsis experts now support two distinct pathogenesis models for the progression of sepsis. The sequential response model describes an initial proinflammatory response to a pathogen, SIRS, followed by a compensatory anti-inflammatory response syndrome (CARS). In the second model, known as the mixed antagonist response syndrome, SIRS and CARS occur simultaneously and achieve homeostasis. Severe sepsis and septic shock are associated with an imbalance in the SIRS/CARS equilibrium. Although recent research supports the second model [2], larger studies exploring the underlying pathobiology, including expression of pro- and anti-inflammatory molecules throughout the course of sepsis pathogenesis are needed.
Sepsis diagnosis
Rapid diagnosis and treatment of sepsis reduces mortality. The ‘gold standard’ for sepsis diagnosis is identification of an infectious microorganism in patients with SIRS. Traditionally, pathogens in blood, urine or other body fluids were detected in the laboratory by culturing. Unfortunately, cultures have limited utility because some pathogens are slow growing and contamination is common, leading to a high number of false negative and positive results. Despite their disadvantages, identification of the infecting agent as well as its antibiotic susceptibility and resistance patterns remains crucial to administer or adjust antimicrobial treatment. Direct identification of a pathogen through molecular and proteomic-based approaches may help overcome these disadvantages [3].
Because of its non-specific clinical symptoms and the limited utility of bacterial cultures, researchers have looked to biomarkers to diagnose sepsis. Currently, the diagnostic utility of sepsis biomarkers is limited to confirming, ruling out sepsis or stratifying patients based on disease severity. Lactate, C-reactive protein (CRP) and procalcitonin (PCT) assist in the work-up of patients with suspected sepsis. Lactate, the end product of anaerobic glycolysis, is increased in septic shock and other conditions as a result of excessive energy demand, tissue hypoxia, and/or impaired metabolic pathways.
The Surviving Sepsis Campaign, an international collaboration developed to improve the management, diagnosis, and treatment and reduce mortality rates of sepsis, advocates measuring blood lactate within 6 h of presentation in patients with suspected sepsis [1]. A lactate concentration >4 mmol/L (36 mg/dL) is associated with increased morbidity and mortality and is used to guide sepsis resuscitation protocols. Lactate concentrations increase with severity of sepsis and are most useful for diagnosing septic shock, but lack diagnostic strength to discriminate early sepsis from SIRS.
Expression of proinflammatory molecules is markedly up-regulated in early sepsis. CRP and PCT expression is stimulated by proinflammatory cytokines. CRP is an acute phase reactant that is up-regulated in inflammatory processes, and is not specific for sepsis. PCT, the precursor of calcitonin in thyroidal C-cells, is systemically produced in non-thyroidal tissue in response to inflammation and infection. Compared to CRP, PCT more accurately distinguishes SIRS from sepsis [1, 4]. In critically ill adults, the diagnostic strength of PCT to distinguish sepsis from SIRS is low [1, 4]. This may be due to the fact that like CRP, PCT is overexpressed in non-infectious inflammatory states like surgery or trauma. Unlike CRP, PCT concentrations correlate with sepsis severity. Both CRP and PCT can predict prognosis and response to therapy in septic patients. PCT is also useful in ruling out bacterial infections and is used in algorithms guiding antimicrobial therapy in critically-ill patients [4]. However, because of its questionable diagnostic utility, PCT testing is not universally used in clinical practice.
Thousands of studies have investigated the clinical and diagnostic utility of hundreds of sepsis biomarkers. A recent review of relevant clinical and experimental studies identified 178 proposed sepsis biomarkers [4]. Besides PCT and CRP, 34 others were investigated as diagnostic markers for sepsis. None had sufficient diagnostic strength to differentiate septic patients from those with non-infectious SIRS.
Multiple biomarker panels for sepsis diagnosis
As our understanding of the underlying mechanisms of sepsis evolved, it became evident that a single biomarker could not identify all patients with this heterogeneous syndrome. Instead, a panel of biomarkers, consisting of molecules secreted in the blood throughout the disease process, may better predict sepsis among patients with systemic inflammation [3].
Recent studies [Table 1] explored the utility of novel multimarker panels to predict sepsis. In a prospective cohort study of 151 emergency department (ED) patients with SIRS, both panels of 3 and 6 biomarkers showed superior diagnostic utility for detection of bacterial infection compared to any single biomarker [Table 1; Study #1] [5]. In a separate cohort of 342 ED patients with SIRS, adding 1 to 3 biomarkers and/or clinical parameters did not improve upon PCT alone to predict bacteremia [Table 1; #2] [6]. In the latter study, only patients with documented positive blood cultures were included, excluding possible infection in other sites or fluids and patients with false negative cultures.
In a retrospective pilot study at our institution, 10 inflammatory biomarkers, chosen because of their expression pattern during SIRS and/or CARS, were measured in 63 critically-ill patients with SIRS [Table 1; #3]. Panels of 2 to 6 inflammatory biomarkers measured in multiplex were better able to identify sepsis among patients with SIRS compared to single markers. Because of the small sample size, a 2-marker panel was most predictive of sepsis. PCT and CRP showed limited diagnostic utility alone or in combination with other biomarkers [7]. In a second retrospective cohort study we evaluated the diagnostic utility of 5 inflammatory biomarkers up-regulated in SIRS and/or CARS in 169 ICU patients with SIRS [Table 1; #4]. The 5-biomarker panel outperformed any single biomarker to predict sepsis on the day that patients developed SIRS [8]. Studies are ongoing to validate these findings in a larger population and to compare these results with the diagnostic performance of PCT.
A sepsis risk score, generated from results of multiple biomarkers, may allow easy adoption of these panels into clinical practice [3, 9]. A study evaluating the plasma concentrations of 5 proinflammatory molecules demonstrated that, compared to individual markers, a sepsis score consisting of at least 2 biomarkers elevated above their respective cut-offs better discriminated between SIRS and sepsis in ICU patients [Table 1; #5] [10]. Gibot and colleagues investigated the concentration of three biomarkers in 300 patients consecutively admitted into the ICU [Table 1; #6] [9]. A bioscore of 0, 1, 2 or 3 was assigned based on the number of positive biomarkers (above pre-defined cut-off values). The bioscore surpassed the diagnostic strength of any of the individual biomarker results for the prediction of sepsis. This model was validated in a separate cohort of 228 patients presenting with clinical signs of sepsis. In these studies, the sepsis score strategy was practical with superior diagnostic utility.
Conclusions
Early identification and treatment of septic patients reduces mortality, however, signs and symptoms of early sepsis are similar to non-infectious SIRS. To date, no single biomarker has ample diagnostic strength to identify septic patients among a critically-ill population. A panel of biomarkers may better distinguish patients with sepsis from those with non-infectious SIRS. Most findings are from preliminary studies with small patient cohorts and require additional validation studies. These should be conducted in larger, multicentre populations with distinct validation cohorts. Rapid, automated, multiplexing platforms and/or point-of-care technologies may be necessary to obtain timely results for these multimarker sepsis panels. Combining biomarkers into equations or sepsis-scores that yield an interpretable and meaningful result is paramount for their clinical adoption.
In conclusion, using combinations of biomarkers to predict sepsis is an attractive strategy that may improve real time assessments and reduce morbidity and mortality in septic patients.
References
1. Faix JD. Established and novel biomarkers of sepsis. Biomark Med 2011; 5(2): 117–130.
2. Osuchowski MF, et al. Sepsis chronically in MARS: systemic cytokine responses are always mixed regardless of the outcome, magnitude, or phase of sepsis. J Immunol 2012; 189(9): 4648–56.
3. Casserly B, Read R, Levy MM. Multimarker panels in sepsis. Crit Care Clin 2011; 27(2): 391–405.
4. Pierrakos C, Vincent JL. Sepsis biomarkers: a review. Crit Care 2010; 14(1): R15.
5. Kofoed K, et al. Use of plasma C-reactive protein, procalcitonin, neutrophils, macrophage migration inhibitory factor, soluble urokinase-type plasminogen activator receptor, and soluble triggering receptor expressed on myeloid cells-1 in combination to diagnose infections: a prospective study. Crit Care 2007; 11(2): R38.
6. Tromp M, et al. Serial and panel analyses of biomarkers do not improve the prediction of bacteremia compared to one procalcitonin measurement. J Infect 2012; 65(4): 292–301.
7. Pyle AL, et al. Multiplex cytokine analysis for the differentiation of SIRS and sepsis. Am J Clin Pathol 2010; 134: 509.
8. Pyle AL, et al. A multi-marker approach to differentiate sepsis from SIRS. Am J Clin Pathol 2011; 136: 468–469.
9. Gibot S, et al. Combination biomarkers to diagnose sepsis in the critically ill patient. Am J Respir Crit Care Med 2012; 186(1): 65–71.
10. Selberg O, et al. Discrimination of sepsis and systemic inflammatory response syndrome by determination of circulating plasma concentrations of procalcitonin, protein complement 3a, and interleukin-6. Crit Care Med 2000; 28(8): 2793–2798.
The authors
Alison Woodworth, PhD, DABCC, FACB
Assistant Professor, Pathology, Microbiology and Immunology
Director, Esoteric Chemistry
Vanderbilt University Medical Center
Nashville, TN, USA
Jessica M. Colón-Franco, PhD
Clinical Chemistry Fellow
Department of Pathology, Microbiology and Immunology
Vanderbilt University Medical Center
Nashville, TN, USA
E-mail:
Alison.Woodworth@Vanderbilt.Edu
Proteomics of cerebrospinal fluid for biomarker discovery in multiple sclerosis
, /in Featured Articles /by 3wmediaThe discovery of reliable biomarkers, which are eligible for the prediction of both disease progression and response to treatment, means a great challenge in the management of multiple sclerosis (MS), a devastating disease of the central nervous system. The results of recent proteomic findings from the cerebrospinal fluid of MS patients hold promise of finding ideal biomarkers in the near future.
by Dr J. Füvesi, Dr C. Rajda, Dr D. Zádori, Dr K. Bencsik, Prof. Dr L. Vécsei and Prof. Dr J. Bergquist
Multiple Sclerosis
Multiple sclerosis is a demyelinative disorder of the central nervous system that affects mainly young adults. It has a great impact on quality of life, social and family life, and the careers of the patients.
In the majority of cases the disease starts with a relapsing–remitting (RR) phase. After a variable period of time it turns into a secondary progressive (SP) phase characterized by the gradual accumulation of residual symptoms. In 10–15% of cases a continuous progression is observed from the very beginning, this is the primary progressive (PP) form. In very rare fulminant cases frequent relapses with incomplete remissions cause severe disability or even death in a short duration of time.
The diagnosis of multiple sclerosis is still mainly clinical, supported by MRI and cerebrospinal fluid (CSF) analysis findings. The revised McDonald Criteria [1] allow earlier diagnosis, especially in PP MS. The routine diagnostic CSF analysis in MS includes the detection of oligoclonal bands and quantitative IgG analysis. Isoelectric focusing (IEF) on agarose gels followed by immunoblotting is considered the ‘gold standard’ for detecting the presence of oligoclonal bands [2]. The sensitivity of the method is above 95% and the specificity is more than 86%. An increased IgG index and the presence of oligoclonal bands in the CSF support an MS diagnosis.
Although the diagnosis is quite straightforward in most cases, taking into account clinical findings and paraclinical tests, there are still no specific biomarkers to confirm the diagnosis nor do we have any validated prognostic markers to follow the progression of the disorder.
At the time of diagnosis, major problems include the identification of the different clinical forms of the disease and the identification of patients with a potential rapid progression before disability evolves; the differential diagnosis of clinically isolated syndrome (CIS) with optic neuritis as the presenting symptom, where neuromyelitis optica (NMO) spectrum disorder may be an alternative diagnosis. Markers of disease progression are needed to distinguish CIS patients with a high probability to develop clinically definite MS.
There is also a need for biomarkers of response to treatment and biomarkers for better understanding the underlying pathological processes of the disease. This is especially important with the growing variety of treatment options: now it is possible to change therapy in the case of an inadequate treatment response and to escalate MS treatment to more aggressive alternatives. In the near future individualized treatment choices need better classification of patient characteristics.
In order to discover new biomarkers in MS, one should analyse the whole protein content of body fluids, preferentially CSF. Because of its proximity to the central nervous system (CNS), CSF may reflect changes in the CNS that may help differentiate normal and pathological conditions.
Proteomics
Proteomics is the study of protein expression in an organism. There are excellent reviews on proteomic approaches [3–5], so we will discuss here only certain aspects of these methods relevant to multiple sclerosis biomarker research. Mass-spectrometry (MS in Italic to distinguish from multiple sclerosis in this paper) based protein identification strategies include whole-protein analysis (‘top-down’ proteomics) and analysis of enzymatically produced peptides (‘bottom-up’ proteomics) [4]. The latter is the standard for large-scale or high-throughput analysis of highly complex samples, and digestion with trypsin is the most common method. The separation of peptides and proteins is an important element of both approaches.
Mass spectrometry measures the mass-to-charge ratio (m/z) of ionized molecules, and, as multiple distinct peptides can have very similar or identical molecular masses, it can be difficult to identify the overlapping peptides [3]. The use of separation techniques, therefore, reduces the cases of coincident peptide masses simultaneously introduced into the mass spectrometer. One of the most commonly used separation techniques is high-performance liquid chromatography (HPLC) with a capillary column. Peptides of similar molecular mass but different hydrophobicity elute from the LC column and enter the mass spectrometer at different time points, no longer overlapping in the initial MS analysis. Liquid chromatography coupled to mass spectrometry reduces the complexity of the sample and allows more precise protein identification.
In order to limit the risk of systematic errors and achieve a high sample throughput, labelling by means of isobaric tags for relative and absolute quantification (iTRAQ) may be used [6]. Multiple samples may be processed in parallel with this multiplexed approach. The main advantage is that the samples are analysed under exactly the same conditions. The relative abundance of labelled peptides indicates relative changes in protein expression.
LC-MS experiments generate an enormous amount of data, making data analysis one of the most challenging parts of proteomic analysis. Protein identification and quantification is achieved by database searching. Programs, such as Mascot etc., compare observed spectra to predicted spectra for candidate peptides from a protein database. In a recent study Schutzer et al. established a database of the normal human CSF proteome [7].
Proteomics in multiple sclerosis
In recent years a number of papers appeared describing proteomic analysis of CSF or brain tissue of multiple sclerosis patients [8–12]. The first papers in the field analysed pooled samples from a relatively small group of patients [8, 9]. Hammack et al. [8] reported the analysis of a pooled sample of three relapsing–remitting MS patients and a pooled sample of three patients with non-MS inflammatory CNS disorders using two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting. They identified four proteins in the gels containing MS CSF that were not reported previously in normal human CSF: CRTAC-1B (cartilage acidic protein), tetranectin (a plasminogen-binding protein), SPARC-like protein (a calcium binding cell signalling glycoprotein) and autotaxin t (a phosphodiesterase).
In the study of Dumont et al. [9] CSF samples from five MS patients (4 RR, one SP) were analysed by 2-DE followed by liquid chromatography tandem mass spectrometry. With this method 15 proteins have been identified that were not previously observed in non-multiple sclerosis CSF 2-DE gels. These proteins were: psoriasin, calmodulin-related protein NB-1, annexin 1, EWI-2, Niemann–Pick disease type C2 protein (NPC-2), semenogelin 1 (SEM1), semenogelin 2 (SEM2), complement factor H-related protein 1 (FHR-1), procollagen C-proteinase enhancer protein (PCPE), aldolase A, N-acetyllactosaminide β-1,3-N-acetylglucosaminyl-transferase, tetranectin, cystatin A, superoxide dismutase 3 and glutathione peroxidase.
Later, publications started to focus on the differentiation of the clinical forms of the disease. Lehmensiek et al. compared CSF samples from RR MS and clinically isolated syndrome (CIS) patients with controls using two-dimensional difference gel electrophoresis (2-D-DIGE) and matrix-assisted laser desorption/ionization – time of flight (MALDI-TOF) mass spectrometry [10]. In RR MS Ig kappa chain NIG93 protein was increased in concentration, while transferrin isoforms, alpha 1 antitrypsin isoforms, alpha 2-HS glycoprotein, Apo E and transthyretin decreased. In a study of Stoop et al. [11] significant differences were observed comparing the peak lists of spectra from CSF of MS patients and patients with other neurological diseases (OND), and also clinically isolated syndrome (CIS) vs OND. Three differentially expressed proteins were identified in the CSF of MS patients compared to CSF of patients with OND: chromogranin A, clusterin and complement C3.
The same group compared proteome profiles of CSF from RR and PP multiple sclerosis and found that they overlap to a large extent [13]. The main detected difference was that protein jagged-1 was less abundant in PP MS compared to RR MS, whereas vitamin D-binding protein was only detected in the RR MS CSF samples. Ottervald et al. found an increased CSF level of vitamin-D-binding protein in SP MS compared to the control [14]. Recently, impaired vitamin D homeostasis has been linked to multiple sclerosis [15]: high serum levels of 25-hydroxyvitamin D correlated with a reduced risk of MS [16] and vitamin D supplementation was proposed as an add-on therapy [17].
Biomarkers of disease progression are emerging as new targets of proteomics. In our recently published paper we analysed the CSF of a rare fulminant case of MS and compared it with RR MS and control samples [18]. The aim of this study was to identify proteins related to rapid progression. The presented bottom-up strategy, based on isobaric tag labelling in conjunction with enzymatic digestion followed by nanoLC coupled off-line to MALDI TOF/TOF MS resulted in the identification of 78 proteins. Seven proteins were found to be upregulated in both fulminant MS samples but not in the relapsing–remitting case compared to the control. These proteins included Ig kappa and gamma-1 chain C region, complement C4-A, fibrinogen beta chain, serum amyloid A protein, neural cell adhesion molecule 1 and beta-2-glycoprotein 1. These proteins are involved in the immune response, blood coagulation, cell proliferation and cell adhesion.
Disease progression may be examined by analysing CSF samples from CIS patients who remain CIS and CIS patients who convert to clinically definite multiple sclerosis. Comabella et al. [19, 20] analysed pooled CSF samples with
isobaric labelling and mass spectrometry. They found that chitinase 3-like 1, ceruloplasmin and vitamin D-binding protein were upregulated in CSF of patients converted to clinically definite MS. In order to validate their results, the authors determined the levels of these selected proteins by enzyme-linked immunosorbent assay (ELISA) in individual CSF samples. Only chitinase 3-like 1 was validated. In a second validation step CSF chitinase 3-like 1 levels were measured in an independent CIS cohort and its level was again significantly increased in CIS patients who later converted to MS, compared to patients who remained as CIS. High CSF levels of this protein significantly correlated with the number of gadolinium enhancing and T2 lesions on baseline brain MRI scans and disability progression during follow-up.
The search for biomarkers that are able to identify patients at high risk of rapid progression becomes increasingly important with the appearance of more aggressive treatment possibilities. In another ongoing study we currently analyse LC-Fourier transform ion cyclotron resonance (FTICR) MS [20–22] data of a larger set of CSF samples from a variety of clinical forms of MS and matched controls.
Despite the increasing number of studies investigating potential biomarkers of MS disease progression and response to therapy, there is still no protein that is repeatedly identified and validated by different groups. This may be due to the relatively small sample sizes and the heterogeneity of the methods applied. Large scale multi-centre projects using standard methods for collecting, storing and analysing the samples are necessary to validate these preliminary results and integrate candidate biomarkers into the pathomechanism of the disease.
A great step in this direction is the BIOMS project, which aims a standardized sample collection, storage and processing during the preanalytical steps to rule out the differences occurred by sample preparation [23–25] and test the different methods and hypotheses on a great sample number in multiple centres to shed light on the sources of errors using different methods. One of these initiatives was the neurofilament validation study, which is a candidate biomarker in multiple sclerosis [26]. Another validation study tested two different methods of detecting the neutralizing antibodies against interferon-beta therapy, which is a biomarker of therapy in MS [27].
In the future multi-centre studies on standardized samples and methods can bring us closer to solve the questions regarding the pathological processes and the classification of patients to the most appropriate therapy.
Acknowledgement
TÁMOP-4.2.2.A-11/1KONV/-2012-0052 and The Swedish Research Council 621-2011-4423 are gratefully acknowledged for financial support.
References
1. Polman CH, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol 2011; 69: 292–302.
2. Freedman MS, et al. Recommended standard of cerebrospinal fluid analysis in the diagnosis of multiple sclerosis: a consensus statement. Arch Neurol 2005; 62: 865–870.
3. Karpievitch YV, et al. Liquid Chromatography Mass Spectrometry-Based Proteomics: Biological and Technological Aspects. Ann Appl Stat 2010; 4: 1797–1823.
4. Han X, et al. 3rd Mass spectrometry for proteomics. Curr Opin Chem Biol 2008; 12: 483–490.
5. Becker CH, Bern M. Recent developments in quantitative proteomics. Mutat Res 2011; 722: 171–182.
6. Ross PL, et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 2004; 3: 1154–1169.
7. Schutzer SE, et al. Establishing the proteome of normal human cerebrospinal fluid. PLoS One 2010; 5: e10980.
8. Hammack BN, et al. Proteomic analysis of multiple sclerosis cerebrospinal fluid. Mult Scler 2004; 10: 245–260.
9. Dumont D, et al. Proteomic analysis of cerebrospinal fluid from multiple sclerosis patients. Proteomics 2004; 4: 2117–2124.
10. Lehmensiek V, et al. Cerebrospinal fluid proteome profile in multiple sclerosis. Mult Scler 2007; 13: 840–849.
11. Stoop MP, et al. Multiple sclerosis-related proteins identified in cerebrospinal fluid by advanced mass spectrometry. Proteomics 2008; 8: 1576–1585.
12. Han MH, et al. Proteomic analysis of active multiple sclerosis lesions reveals therapeutic targets. Nature 2008; 451: 1076–1081.
13. Stoop MP, et al. Proteomics comparison of cerebrospinal fluid of relapsing remitting and primary progressive multiple sclerosis. PLoS One 2010; 5: e12442.
14. Ottervald J, et al. Multiple sclerosis: Identification and clinical evaluation of novel CSF biomarkers. J Proteomics 2010; 73: 1117–1132.
15. Cantorna MT, Mahon BD. Mounting evidence for vitamin D as an environmental factor affecting autoimmune disease prevalence. Exp Biol Med 2004; 229: 1136–1142.
16. Raghuwanshi A, et al. Vitamin D and multiple sclerosis. J Cell Biochem 2008; 105: 338–343.
17. §Myhr KM. Vitamin D treatment in multiple sclerosis. J Neurol Sci 2009; 286: 104–108.
18. Füvesi J, et al. Proteomic analysis of cerebrospinal fluid in a fulminant case of multiple sclerosis. Int J Mol Sci 2012; 13: 7676–7693.
19. Comabella M, et al. Cerebrospinal fluid chitinase 3-like 1 levels are associated with conversion to multiple sclerosis. Brain 2010; 133: 1082–1093.
20. Bergquist J. FTICR mass spectrometry in proteomics. Curr Opin Mol Ther 2003; 5: 310–314.
21. Ramstrom M, et al. Protein identification in cerebrospinal fluid using packed capillary liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry. Proteomics 2003; 3: 184–190.
22. Ramstrom M, et al. Cerebrospinal fluid protein patterns in neurodegenerative disease revealed by liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry. Proteomics 2004; 4: 4010–4018.
23. Teunissen CE, et al. A consensus protocol for the standardization of cerebrospinal fluid collection and biobanking. Neurology 2009; 73: 1914–1922.
24. Teunissen CE, et al. Short commentary on ‘a consensus protocol for the standardization of cerebrospinal fluid collection and biobanking’. Mult Scler 2010; 16: 129–132.
25. Tumani H, et al. Cerebrospinal fluid biomarkers in multiple sclerosis. Neurobiol Dis 2009; 35: 117–127.
26. Petzold A, et al. Neurofilament ELISA validation. J Immunol Methods 2010; 352: 23–31.
27. Bertolotto A, et al. Development and validation of a real time PCR-based bioassay for quantification of neutralizing antibodies against human interferon-beta. J Immunol Methods 2007; 321: 19–31.
The authors
Judit Füvesi1 PhD, MD; Cecilia Rajda1 PhD, MD; Dénes Zádori1 PhD, MD; Krisztina Bencsik1 PhD, MD; László Vécsei1,2 PhD, MD; and Jonas Bergquist3,4* PhD, MD
1 Department of Neurology, Faculty of Medicine, Albert Szent-Györgyi Clinical Center, University of Szeged, Szeged, Hungary
2 Neuroscience Research Group of Hungarian Academy of Sciences and University of Szeged, Szeged, Hungary
3 Analytical Chemistry, Department of Chemistry-Biomedical Center, Uppsala University, Uppsala, Sweden
4 Science for Life Laboratory (SciLife Lab), Uppsala University, Uppsala, Sweden
*Corresponding author
E-mail: jonas.bergquist@kemi.uu.se
…we can customize a Rapid DOA solution for you
, /in Featured Articles /by 3wmediaIntroducing Uni-Gold Enterics Rapids
, /in Featured Articles /by 3wmedia