Serological and Molecular Detection of Bacterial Infections PDF

Serological and Molecular Detection of Bacterial Infections MLS 227 Chapter 20  Host–Microbe Relationships Symbiotic Host and microbes live together long term Indigenous microbiota Commensalistic No benefit or harm to either organism...

Serological and Molecular Detection of Bacterial Infections MLS 227 Chapter 20  Host–Microbe Relationships Symbiotic Host and microbes live together long term Indigenous microbiota Commensalistic No benefit or harm to either organism Mutualistic Both host and microbes benefit Parasitic Microbes cause harm to the host  Infectivity, Pathogenicity, and Virulence Infectivity Organism’s ability to establish an infection Pathogenicity Ability of an organism to cause disease May be increased by virulence factors Virulence Extent of pathology caused by an organism when it infects a host Structural Components of Bacteria Bacterial Virulence Factors and Pathogenicity Endotoxin The lipid A portion of Lipopolysaccharide in gram-negative cell walls Powerful stimulator of cytokine release Pili Adherence to host cells; resistance to phagocytosis Flagella Adherence to host cells; motility Capsule Blocks phagocytosis, antibody attachment, complement Exotoxins Potent toxic proteins released from living bacteria Neurotoxins, cytotoxins, enterotoxins  Immune Defenses Against Bacteria Innate defenses Intact skin and mucosal surfaces (barriers to entry) Antimicrobial defense peptides (e.g., lysozyme, defensins, ribonucleases) Complement proteins, cytokines, acute-phase reactants Adaptive defenses Antibody production Binding of C’, opsonization, neutralization of bacterial toxins Cell-mediated immunity CD4 T cells produce cytokines that induce inflammation. Cytotoxic T lymphocytes attack host cells that contain intracellular bacteria  Bacterial Evasion Mechanisms A. Inhibit chemotaxis. B. Block adherence of phagocytes. C. Resist digestion by phagocytic cells. D. Block action of complement. E. Degrade IgA molecules.  Laboratory Detection of Bacterial Infections Culture of the causative agent Grow on broth or solid media Major means of diagnosis, but may take time or may not be possible Microscopic examination Gram stain or special stains Detection of bacterial antigens Rapid testing by ELISA, LFA, or LA Molecular detection of bacterial DNA or RNA Can obtain results in a few hours with PCR Proteomics Analysis of proteins produced by specific bacteria Microscopic Examination of Bacteria Figure A (bottom left) shows gram-positive cocci identified with the gram stain (Staphylococcus aureus). special acid-fast staining Figure B (bottom right) is a Gram stain of E. for Mycobacterium coli showing Gram-negative rods. tuberculosis.  Serology Tests for Bacterial Infections Detect antibodies to bacterial antigens Uses: To detect and confirm infections for which other laboratory methods are not available To diagnose infections for which clinical symptoms are nonspecific Current infection indicated by presence of IgM, a high IgG titer, or a fourfold rise in antibody titer between acute and convalescent samples To determine a past exposure to an organism (IgM–, IgG+) To assess reactivation or reexposure Disadvantages: Delay between start of infection and production of antibodies Low antibody production by immunosuppressed patients  Group A Streptococci (GAS) Streptococcus pyogenes Gram-positive cocci arranged in pairs or chains Person-to-person transmission Major sites of infection are upper respiratory tract and skin. Beta hemolytic – streptolysin endotoxin  Clinical Manifestations of Acute GAS Infection Pharyngitis (“strep throat”) Pyoderma (impetigo) Scarlet fever Toxic shock syndrome Necrotizing fasciitis Treated with antibiotics  Group A Streptococcal Sequelae 1. Acute rheumatic fever Develops 1 to 3 weeks after pharyngitis or tonsillitis in 2% to 3% of infected individuals. Features: fever, joint pain, inflammation of the heart Most likely caused by immune responses to streptococcal antigens that cross-react with human heart tissue. 2. Poststreptococcal glomerulonephritis May follow GAS infection of the skin or pharynx. Damages glomeruli, producing hematuria, proteinuria, edema, hypertension, malaise, backache, abdominal discomfort, and impairment in renal function. Deposits of immune complexes containing streptococcal antigens in glomeruli Laboratory Diagnosis of Acute Group A Streptococcal Infections Culture on sheep blood agar Small translucent colonies surrounded by clear zone of beta hemolysis Rapid assays to detect group A Streptococcal antigens LFA (Lateral flow immunochromatographic assay)  Serological Detection of group A streptococcal Sequelae 1. Antistreptolysin O (ASO) Nephelometric methods currently used that measure light scatter produced by immune complexes containing streptolysin antigen. Titer elevated in 85% of patients with acute rheumatic fever. Does not increase in patients with skin infection. 2. Anti-DNase B Produced by both rheumatic fever and impetigo patients. Tested by EIA and nephelometric methods. 3. Streptozyme test A rapid slide agglutination test that detects antibodies to five streptococcal products:  ASO  Anti-hyaluronidase (AHase)  Anti-streptokinase (ASKase)  Anti-nicotinamide-adenine dinucleotide (anti-NAD)  Anti-DNase B  Helicobacter pylori Gram-negative microaerophilic spiral bacterium Transmission likely by fecal-oral route Major cause of gastric and duodenal ulcers Can survive in acid environment of stomach because it produces urease, which provides a buffering zone around the bacteria. Treatment with antibiotics and anti-ulcer medications If untreated, can lead to gastric carcinoma or mucosa- associated lymphoid tumors.  Detection of Helicobacter pylori Infection Detect urease in stomach biopsy (CLOtest) Urea breath test H. pylori antigens H. pylori antibodies ELISA is method of choice. IgG in serum indicates an active infection. Titers decrease after successful treatment.  Mycoplasma pneumoniae Tiny bacteria that lack a cell wall Leading cause of respiratory infections Fever, headache, malaise, and cough “Atypical” or “walking” pneumonia Raynaud syndrome Causes Stevens-Johnson syndrome in minority of cases Spread by respiratory droplets Laboratory Diagnosis of M. pneumoniae Infection 1. Culture Produces mulberry colonies with a “fried egg” appearance on specialized media. Is the gold standard but rarely performed in clinical laboratories because the organism is difficult to grow. 2. Antibodies to M. pneumoniae Most useful diagnostic assay IgM antibodies = recent infection IgG antibodies = possible reinfection 3. Cold agglutinins Present in about 50% of patients with M. pneumoniae but not specific for the infection Cause RBC agglutination at 4°C; reversible at 37°C 4. Molecular methods Film array respiratory panel  Rickettsial Infections Obligate intracellular gram- negative bacteria 1. Spotted fever group (R. rickettsii) e.g., Rocky Mountain spotted fever 2. Typhus group (R.provazekii) e.g., epidemic typhus Organisms transmitted by arthropods (ticks, mites, lice, or fleas) through biting after feeding on an infected animal  Rocky Mountain Spotted Fever (RMSF) Caused by R. rickettsii Transmitted by three species of ticks Symptoms include headache, nausea, vomiting, diarrhea, skin rash; can rapidly progress to death Diagnosis Clinical presentation Serology by Indirect immunofluorescence Assay  Remember! Host–microbe relationships can be symbiotic, commensalistic, mutualistic, or parasitic. Bacterial virulence factors increase an organism’s ability to cause disease; these include some bacterial structural components (endotoxin, pili, flagella, capsule) and exotoxins. Endotoxin is an component that is released from the cell walls of dying gram-negative bacteria, which can cause massive cytokine production and death. Exotoxins are potent toxic proteins that are released from live bacteria.  Laboratory detection of bacterial infections can involve: Culture Staining and microscopic observation Rapid detection of bacterial antigens Molecular detection of bacterial nucleic acid Serological detection of antibodies to bacterial antigens Mass spectrometry for bacterial proteins Group A streptococci are gram-positive bacteria that most commonly cause acute infections of the upper respiratory tract and skin; some people who are untreated can develop one of two sequelae: acute rheumatic fever or glomerulonephritis. Laboratory diagnosis of acute streptococcal infections involves culture on sheep blood agar and rapid assays to detect streptococcal antigens.  Diagnosis of streptococcal sequelae requires serological methods to detect antibodies to streptococcal antigens, including ASO and anti-DNase B, because the bacteria are not likely to be present when symptoms appear. The streptozyme test is a rapid slide agglutination test that detects antibodies to five streptococcal products. Helicobacter pylori is a gram-negative, urease-producing bacterium that causes gastric and duodenal ulcers; untreated infections can progress to gastric carcinoma or MALT tumors. H. pylori infection can be diagnosed by urease detection in stomach biopsy tissue, the urea breath test, or an ELISA to detect H. pylori antibodies; serological tests and antigen tests of stool samples can be used to determine if the bacteria have been eliminated after treatment.  Mycoplasma pneumoniae are tiny bacteria that lack a cell wall; they are a major cause of respiratory infection often referred to as “walking pneumonia.” M. pneumoniae is difficult to grow in culture. The main methods of detection are serological assays for antibodies to the organism and PCR to detect DNA from the bacteria; cold agglutinins are produced in about one-half of patients. Rickettsia are obligate intracellular gram-negative bacteria that are transmitted by arthropods; two diseases caused by rickettsia are RMSF and typhus. Serological testing for antibodies to R. rickettsii by IFA is considered the gold standard for laboratory diagnosis of RMSF.