1 - Front Cover [Seite 1]
2 - Respiratory Infections [Seite 2]
3 - copyright [Seite copyright]
- 3 [Seite 3]
4 - Contributors [Seite 4]
5 - Contents [Seite 6]
6 - Clinics In Laboratory Medicine [Seite Clinics In Laboratory Medicine]
- 10 [Seite 10]
7 - Preface [Seite Preface]
- 12 [Seite 12]
8 - Infections in Patients with Cystic Fibrosis [Seite 14]
8.1 - Key points [Seite 14]
8.2 - Introduction, epidemiology, and clinical presentation [Seite 14]
8.3 - Microbiology, diagnostic considerations, and susceptibility testing of agents of chronic infection [Seite 16]
8.4 - Staphylococcus aureus [Seite 16]
8.5 - Pseudomonas aeruginosa [Seite 20]
8.6 - Burkholderia cepacia complex and related organisms [Seite 22]
8.7 - Stenotrophomonas and Achromobacter [Seite 24]
8.8 - Mycobacterium [Seite 25]
8.9 - Fungi [Seite 26]
8.10 - Respiratory viruses [Seite 27]
8.11 - Microbiome considerations [Seite 27]
8.12 - References [Seite 28]
9 - Urine Antigen Tests for the Diagnosis of Respiratory Infections [Seite 36]
9.1 - Key points [Seite 36]
9.2 - Introduction [Seite 36]
9.3 - Microbiology [Seite 37]
9.3.1 - Legionella [Seite 37]
9.3.2 - Pneumococcus [Seite 37]
9.3.3 - Histoplasma [Seite 37]
9.4 - Epidemiology, disease presentation, and pathogenesis [Seite 38]
9.4.1 - Legionella [Seite 38]
9.4.2 - Pneumococcus [Seite 38]
9.4.3 - Histoplasma [Seite 39]
9.5 - Diagnosis [Seite 39]
9.5.1 - Legionella Urinary Antigen Testing [Seite 40]
9.5.1.1 - ELISA [Seite 40]
9.5.1.2 - LFA [Seite 40]
9.5.2 - Pneumococcal Urinary Antigen Test [Seite 42]
9.5.2.1 - Effects of study design on the sensitivity of the pUAT [Seite 42]
9.5.2.2 - Colonization and the pUAT [Seite 44]
9.5.3 - Histoplasma Antigen Testing [Seite 44]
9.5.3.1 - Miravista Diagnostics enzyme immunoassay [Seite 44]
9.5.3.1.1 - Second generation [Seite 44]
9.5.3.1.2 - Third generation [Seite 45]
9.5.3.2 - Immuno-Mycologics EIA [Seite 45]
9.6 - Treatment [Seite 46]
9.6.1 - Empiric Therapy for LD/Pneumococcal Pneumonia [Seite 46]
9.6.2 - Targeted Therapy for LD/Pneumococcal Pneumonia [Seite 46]
9.6.3 - Treatment of Histoplasma [Seite 46]
9.7 - Summary and discussion [Seite 47]
9.8 - References [Seite 47]
10 - Pertussis [Seite 54]
10.1 - Key points [Seite 54]
10.2 - Introduction [Seite 54]
10.3 - Microbiology [Seite 55]
10.4 - Epidemiology [Seite 55]
10.5 - Clinical presentations [Seite 57]
10.6 - Pathogenesis, immunity, and vaccination [Seite 57]
10.7 - Diagnosis [Seite 59]
10.8 - Case definitions [Seite 59]
10.9 - Laboratory testing [Seite 60]
10.9.1 - Specimens [Seite 60]
10.9.2 - Test Methods [Seite 60]
10.9.2.1 - Direct fluorescent antibody [Seite 62]
10.9.2.2 - Culture [Seite 62]
10.9.2.3 - Nucleic acid amplification tests [Seite 62]
10.9.2.4 - Pretreatment and extraction [Seite 62]
10.9.2.5 - Targets for PCR [Seite 63]
10.9.2.6 - Low copy number and high CT values [Seite 64]
10.9.2.7 - NAATs other than PCR [Seite 64]
10.9.2.8 - Contamination [Seite 65]
10.9.2.9 - Interpretation [Seite 65]
10.9.2.10 - Serology [Seite 65]
10.10 - Treatment [Seite 66]
10.11 - Summary/discussion [Seite 66]
10.12 - References [Seite 67]
11 - Antibiotic Resistance in Nosocomial Respiratory Infections [Seite 74]
11.1 - Key points [Seite 74]
11.2 - Introduction [Seite 74]
11.3 - Etiology [Seite 75]
11.4 - Epidemiology [Seite 76]
11.5 - Clinical presentation [Seite 77]
11.6 - Pathogenesis [Seite 77]
11.7 - Diagnosis [Seite 78]
11.8 - Treatment [Seite 83]
11.9 - Summary [Seite 84]
11.10 - References [Seite 84]
12 - Nontuberculous Mycobacteria in Respiratory Infections [Seite 88]
12.1 - Key points [Seite 88]
12.2 - Microbiology [Seite 89]
12.3 - Epidemiology [Seite 89]
12.4 - Pathogenesis and clinical significance [Seite 92]
12.5 - Diagnosis [Seite 94]
12.6 - Specimen processing [Seite 95]
12.7 - Acid-fast microscopy [Seite 96]
12.8 - Direct NAA assays [Seite 97]
12.9 - Growth detection [Seite 97]
12.10 - Identification [Seite 99]
12.10.1 - Nucleic Acid Hybridization Methods [Seite 99]
12.10.2 - PCR and Restriction Fragment Length Polymorphism Analysis [Seite 100]
12.10.3 - Line Probe Assays [Seite 101]
12.10.4 - DNA Sequencing [Seite 101]
12.10.5 - MALDI-TOF MS [Seite 102]
12.11 - Genotyping [Seite 103]
12.12 - Antimicrobial susceptibility testing [Seite 103]
12.13 - Identified focus areas in NTM research [Seite 105]
12.14 - References [Seite 106]
13 - Molecular Diagnosis of Tuberculosis and Drug Resistance [Seite 114]
13.1 - Key points [Seite 114]
13.2 - Introduction [Seite 114]
13.3 - Microbiology [Seite 115]
13.4 - Epidemiology [Seite 115]
13.5 - Clinical presentation [Seite 115]
13.6 - Pathogenesis of pulmonary tuberculosis [Seite 116]
13.7 - Molecular diagnosis [Seite 116]
13.7.1 - Genes Associated with Drug Resistance [Seite 116]
13.7.1.1 - Isoniazid [Seite 118]
13.7.1.2 - Rifampin and rifabutin [Seite 119]
13.7.1.3 - Ethambutol [Seite 120]
13.7.1.4 - Pyrazinamide [Seite 120]
13.7.1.5 - Fluoroquinolones [Seite 120]
13.7.1.6 - Injectable drugs [Seite 121]
13.8 - Molecular assays [Seite 121]
13.8.1 - Molecular Beacon Assays [Seite 121]
13.8.2 - Line-Probe Assays [Seite 124]
13.8.3 - Sanger Sequencing [Seite 124]
13.8.4 - Pyrosequencing [Seite 124]
13.8.5 - Next-Generation Sequencing [Seite 125]
13.9 - Practical use of molecular diagnostic assays [Seite 125]
13.10 - Impact on patient management, TB control, and infection control [Seite 125]
13.11 - Summary [Seite 126]
13.12 - References [Seite 127]
14 - Nonmolecular Methods for the Diagnosis of Respiratory Fungal Infections [Seite 132]
14.1 - Key points [Seite 132]
14.2 - Introduction [Seite 132]
14.3 - Microbiology/Epidemiology [Seite 133]
14.4 - Clinical presentation [Seite 134]
14.5 - Pathogenesis [Seite 135]
14.6 - Diagnosis [Seite 135]
14.6.1 - Invasive Aspergillosis (IA) [Seite 136]
14.6.1.1 - GM assay [Seite 136]
14.6.1.2 - BG assay [Seite 139]
14.6.1.3 - Aspergillus lateral-flow devices [Seite 140]
14.6.1.4 - Anti-Aspergillus antibodies [Seite 140]
14.6.2 - Chronic Pulmonary Aspergillosis [Seite 141]
14.6.3 - Pneumonia Caused by Non-Aspergillus Molds [Seite 141]
14.6.4 - Pneumocystis jirovecii Pneumonia [Seite 141]
14.6.5 - Pulmonary Cryptococcosis [Seite 142]
14.7 - Treatment [Seite 143]
14.8 - Discussion/Summary [Seite 143]
14.9 - References [Seite 145]
15 - Interferon-Gamma Release Assays [Seite 154]
15.1 - Key points [Seite 154]
15.2 - Introduction [Seite 154]
15.3 - Microbiology [Seite 155]
15.4 - Epidemiology [Seite 155]
15.5 - Clinical presentation [Seite 156]
15.6 - Pathogenesis [Seite 157]
15.7 - Diagnosis [Seite 158]
15.7.1 - Interferon-Gamma Release Assays [Seite 158]
15.7.2 - Test Performance of IGRAs [Seite 159]
15.7.3 - IGRAs in Targeted Populations [Seite 160]
15.8 - Treatment [Seite 162]
15.9 - Summary/Discussion [Seite 162]
15.10 - References [Seite 163]
16 - Respiratory Fungal Infections [Seite 168]
16.1 - Key points [Seite 168]
16.2 - Introduction [Seite 168]
16.3 - Aspergillosis [Seite 170]
16.3.1 - Diagnosis [Seite 170]
16.3.2 - Combination Testing [Seite 171]
16.3.3 - The Need for Prospective Clinical Trials [Seite 172]
16.4 - Pneumocystis jirovecii pneumonia [Seite 173]
16.4.1 - Introduction [Seite 173]
16.4.2 - Microbiology [Seite 173]
16.4.3 - Epidemiology [Seite 173]
16.4.4 - Clinical Presentation [Seite 174]
16.4.5 - Pathogenesis [Seite 174]
16.4.6 - Laboratory Diagnosis [Seite 174]
16.4.7 - PCR or Antigen Tests [Seite 176]
16.4.8 - Treatment [Seite 176]
16.5 - Summary [Seite 176]
16.6 - References [Seite 177]
17 - Rapid Diagnosis of Influenza [Seite 182]
17.1 - Key points [Seite 182]
17.2 - Influenza viruses [Seite 182]
17.2.1 - Pathogenesis [Seite 183]
17.2.2 - Clinical Presentation [Seite 183]
17.2.3 - Treatment [Seite 183]
17.3 - General principles of laboratory diagnosis of influenza infection [Seite 184]
17.4 - Sample collection and transport [Seite 186]
17.5 - Diagnostic methods [Seite 187]
17.5.1 - Viral Culture [Seite 187]
17.5.2 - Viral Antigen Detection [Seite 187]
17.5.2.1 - Immunofluorescence [Seite 187]
17.5.2.2 - Lateral flow IC [Seite 188]
17.5.2.3 - Performance of RIDTs [Seite 188]
17.5.3 - Nucleic Acid Detection [Seite 190]
17.5.3.1 - Conventional PCR [Seite 190]
17.5.3.2 - Real-time PCR [Seite 191]
17.5.3.3 - Multiplex methods [Seite 192]
17.6 - Rapid NAAT for the detection of influenza viruses [Seite 192]
17.6.1 - Description of the Systems [Seite 193]
17.6.2 - Assay Performance [Seite 195]
17.6.3 - Limitations and Future Developments [Seite 196]
17.7 - Factors to consider [Seite 196]
17.8 - Summary [Seite 198]
17.9 - References [Seite 198]
18 - Antiviral Resistance in Influenza Viruses [Seite 204]
18.1 - Key points [Seite 204]
18.2 - Introduction [Seite 204]
18.3 - Microbiology [Seite 205]
18.4 - Epidemiology and transmission [Seite 206]
18.5 - Pathogenesis [Seite 206]
18.6 - Clinical presentation [Seite 206]
18.7 - Pharmacologic agents [Seite 207]
18.7.1 - Adamantanes [Seite 207]
18.7.2 - NA Inhibitors [Seite 207]
18.7.3 - Other Antiviral Targets and Drugs [Seite 208]
18.8 - Resistance genetics [Seite 210]
18.9 - Influenza diagnosis [Seite 211]
18.10 - Antiviral drug susceptibility testing [Seite 212]
18.10.1 - Genotypic Methods [Seite 212]
18.10.2 - Phenotypic Methods [Seite 214]
18.10.3 - Patient Testing and Surveillance Programs [Seite 215]
18.11 - Treatment and prognosis [Seite 216]
18.12 - Summary [Seite 217]
18.13 - Acknowledgments [Seite 217]
18.14 - References [Seite 217]
19 - Emerging Respiratory Viruses Other than Influenza [Seite 226]
19.1 - Key points [Seite 226]
19.2 - Introduction [Seite 226]
19.3 - Microbiology [Seite 227]
19.3.1 - MERS-CoV [Seite 227]
19.3.2 - Ad14 [Seite 228]
19.3.3 - RV-C [Seite 229]
19.3.4 - HBoV1 [Seite 229]
19.4 - Epidemiology [Seite 230]
19.4.1 - MERS-CoV [Seite 230]
19.4.2 - Ad14 [Seite 230]
19.4.3 - RV-C [Seite 231]
19.4.4 - HBoV1 [Seite 232]
19.5 - Clinical presentation [Seite 232]
19.5.1 - MERS-CoV [Seite 232]
19.5.2 - Ad14 [Seite 232]
19.5.3 - RV-C [Seite 233]
19.5.4 - HBoV1 [Seite 233]
19.6 - Pathogenesis [Seite 234]
19.6.1 - MERS-CoV [Seite 234]
19.6.2 - Ad14 [Seite 234]
19.6.3 - RV-C [Seite 234]
19.6.4 - HBoV1 [Seite 236]
19.7 - Diagnosis [Seite 237]
19.8 - Treatment and prognosis [Seite 238]
19.9 - Summary [Seite 239]
19.10 - References [Seite 240]
20 - Index [Seite 248]
Infections in Patients with Cystic Fibrosis
Diagnostic Microbiology Update
Peter H. Gilligan, PhD, DABMMgilliganncphd@gmail.com, Pathology-Laboratory Medicine and Microbiology-Immunology, Clinical Microbiology-Immunology Laboratories, UNC Health Care, UNC Hospitals, UNC School Medicine, Room 1035, CB 7600, Chapel Hill, NC 27516, USA
Survival has improved in patients with cystic fibrosis (CF), in part because of aggressive antimicrobial management. Two multidrug-resistant environmental bacteria, the Burkholderia cepacia group and nontuberculous mycobacteria, have emerged. Improving genomic and proteomic technologies are allowing better identification of bacteria and fungi found in the CF lung and detection of viral agents that may be associated with pulmonary exacerbations. Anaerobic bacteria and Streptococcus angionsus group organisms may play a role in chronic CF lung infections. The diversity of organisms declines perhaps as a result of aggressive antimicrobial therapy, and an apex predator, Pseudomonas aeruginosa, may emerge in many patients with CF.
Keywords
Infection
Cystic fibrosis
Diagnostic microbiology
Update
Key points
• Cystic fibrosis is the most important genetic disease in Caucasians. Patients with this disease die prematurely primarily as a result of chronic lung infection. Staphylococcus aureus and mucoid Pseudomonas aeruginosa continue to be the key pulmonary pathogens.
• Survival has improved in patients with CF in part because of aggressive antimicrobial management. An unintended consequence of this therapy has been the emergence of 2 multidrug-resistant environmental bacteria: the Burkholderia cepacia group and nontuberculous mycobacteria.
• Burkholderia cenocepacia, a species within the Burkholderia cepacia complex, is associated with high mortality and is a contraindication for lung transplantation. The key nontuberculous mycobacterial pathogen, Mycobacterium abscessus, is not so virulent and is not a lung transplantation contraindication. Both present an infection control challenge, because they can be spread from person to person.
• Improving genomic and proteomic technologies are allowing better identification of bacteria and fungi found in the CF lung and to detect viral agents that may be associated with pulmonary exacerbations. Chronic rhinovirus infections are of particular interest.
• Microbiome studies have identified 2 groups of bacteria that may play a role in chronic CF lung infections: anaerobic bacteria and Streptococcus angionsus group organisms. Microbiome studies also show that as the diversity of organisms decline, perhaps as a result of aggressive antimicrobial therapy, an apex predator, etc., Pseudomonas aeruginosa, may emerge in many patients with CF.
Introduction, epidemiology, and clinical presentation
Cystic fibrosis (CF) is the most common autosomal-recessive genetic disease that occurs in non-Hispanic Caucasians populations, although other racial groups may have this disease as well.1 Affected individuals have mutation in the CF transmembrane conductance gene (CFTR), a membrane protein involved in sodium and chloride transport in epithelial cells.2 The resulting dysregulation in electrolyte transport leads to depletion in airway surface liquid on bronchial epithelial cell surfaces. As a result, patients with CF have thick, dry, tenacious mucus, which impairs mucociliary clearance of particulates, especially bacteria and fungal conidia, from the airways. This environment is ideal for the growth of a limited number of organisms, primarily those that thrive in natural environments such as water. This thickened mucus provides an ideal niche for the establishment of chronic infection. It is this chronic infection that results in the premature death that in seen in CF.3
More than 1800 CFTR mutations have been associated with CF.4 The most common mutation is F508del, which is found in ∼85% of people in the United States; approximately 47% are homozygous for this mutated gene.4 Further carrier rate for mutated CFTR genes is estimated to range from 1/25 for non-Hispanic Caucasians to 1/61 for African Americans to 1/94 for Asian Americans.1 CF is seen most frequently in North America, Northern Europe, Australia, New Zealand, Brazil, and Argentina. It is estimated that 1 in 3500 live births result in clinical disease.4
Currently life expectancy in US patients with CF is approximately 38 years, significantly less than that of the general population.4 Cardiopulmonary failure secondary to chronic lung disease is responsible for 85% of premature deaths in CF. The airways of patients with CF become infected in infancy. This situation begins periods of chronic infection and lung inflammation with accompanying cough, which is a lifelong reality in patients with CF. A hallmark of chronic infection and airway inflammation is periods of pulmonary exacerbations. Pulmonary exacerbations are characterized by worsening symptoms, including increased cough and sputum production, hemoptysis, shortness of breath, increased respiratory rate, loss of appetite, weight loss, increased neutrophil counts, and declining pulmonary function.5 The events that trigger these pulmonary exacerbations are not clearly understood, although viral infections and perhaps changes in the microbiome may be important.6,7 Exacerbations are characterized by the recruitment of neutrophils, cytokine release, and high level of neutrophil-derived elastases in the bronchi and bronchioles, causing significant lung disease.8 Antimicrobial therapy has been shown to be effective in treating exacerbations symptomatically.9 However, over time, lung function deteriorates and becomes so low, that it is no longer compatible with life (Fig. 1). Only lung transplantation can successfully reverse this disease course.8
Fig. 1 Lung function by age group, 2011. FEV1, forced expiratory volume in 1 second.
(From Cystic Fibrosis Foundation. Cystic Fibrosis Foundation patient registry 2011 annual data report. 2012.) Microbiology, diagnostic considerations, and susceptibility testing of agents of chronic infection
Over the past 4 decades, our understanding of the complex nature of chronic lung infections has greatly expanded. Over the past 3 decades, there has been more than a doubling in life expectancy in the population with CF.4 Three factors have been central to this improvement:
a. More effective antimicrobial therapy and treatment strategies, with early eradication of Pseudomonas aeruginosa being a key strategy
b. Improvement in airway clearance techniques
c. Improvements in infection control techniques to prevent the spread of organisms highly virulent to patients with CF, especially Burkholderia cenocepacia2,9
With the use of broader-spectrum antimicrobials, we are seeing a plethora of emerging highly resistant bacteria and fungi in the CF airways. Our understanding of the role of these organisms in chronic infection and inflammation is poorly delineated (Box 1). Over the past decades, new technologies (Fig. 2) have been developed and applied to this understanding. These technologies include nucleic acid amplification techniques (NAATs) for direct organism detection, including multiplex NAAT for viruses; the use of DNA sequence analysis for organism identification; molecularly based epidemiologic techniques, including pulsed field gel electrophoresis (PFGE), multilocus sequence type, whole genome sequencing; and matrix-assisted laser desorption ionization–time of flight mass spectroscopy (MALDI-TOF MS).
Box 1 Pathogenic potential of commonly recovered organisms from chronic CF airway infections or pulmonary exacerbations
• Known
Pseudomonas aeruginosa
Staphylococcus aureus
Methicillin resistant
Small colony variant
Burkholderia multivorans
Burkholderia cenocepacia
Burkholderia dolosa
Aspergillus spp
Scedosporium spp
Mycobacterium abscessus
Influenza virus
Respiratory syncytial virus
• Possible/likely
Haemophilus influenzae
Mycobacterium avium complex
Anaerobic bacteria especially Prevotella...
