Chapter 19 Immunodeficiency Diseases PDF

10/28/22 Chapter 19 Immunodeficiency Diseases 1 Primary versus secondary immunodeficiencies Classification of primary immunodeficiencies Chapter Overview Immunologic defec...

10/28/22 Chapter 19 Immunodeficiency Diseases 1 Primary versus secondary immunodeficiencies Classification of primary immunodeficiencies Chapter Overview Immunologic defects in selected immunodeficiency diseases Laboratory testing for immunodeficiency 2 1  10/28/22 Disorders in which part of the immune system is missing or dysfunctional Decreased ability to defend against infections and more susceptible to developing cancer Primary immunodeficiencies (PIDs) Inherited X-linked, autosomal recessive, autosomal dominant Secondary immunodeficiencies Acquired as a result of other conditions, such as infection (e.g., HIV), autoimmune disease, malignancy, immunosuppressive therapy Immunodeficiencies 3 Immune System Components Affected by PIDs Humoral immunity Caused by defects in B cells or Th cells Patients susceptible to bacterial infections, especially sinusitis and otitis media Cell-mediated immunity Involves T-cell deficiencies Patients susceptible to intracellular pathogens (e.g., viruses, fungi, some bacteria) and cancer Innate immunity Neutrophils or macrophages (bacterial infections) Complement (bacterial infections and autoimmunity) 4 2  10/28/22 PID Examples and their Effects on WBC Function 5 Nine Categories of PIDs Combined immunodeficiencies Predominantly Combined antibody immunodeficiencies with associated or deficiencies syndromic features Congenital defects Diseases of immune of phagocyte Defects in innate dysregulation number, function, or immunity both Phenocopies of Autoinflammatory Complement primary disorders deficiencies immunodeficiencies 6 3  10/28/22 Predominantly Low levels of serum Igs: agammaglobulinemia Involve genetic defects in B-cell maturation or defective interactions between B and T cells Antibody May involve all Ig isotypes or one (sub)class Deficiencies Compare patient Ig levels with age-matched reference range 7 X-linked Bruton’s tyrosine kinase (Btk) deficiency Arrested maturation at pre-B-cell stage Lack of mature CD19+ B cells in blood and plasma cells in lymphoid tissues Treated with gamma globulins Common variable immunodeficiency Heterogeneous group of disorders Can be congenital or acquired; symptoms begin at 20 to 30 years of age Failure of B cells to mature into plasma cells as a result of B- cell or Th-cell defects Treated with gamma Predominantly Antibody globulins Deficiencies 8 4  10/28/22 Predominantly Antibody Deficiencies Transient hypogammaglobulinemia of infancy Delayed development in Ig production (especially IgG) Normal numbers of B cells but delayed Th maturation Selective IgA deficiency Low IgA levels, perhaps because of impaired differentiation to IgA plasma cells Patients may be asymptomatic or more susceptible to infections, allergies, and autoimmunity 9 IgG subclass deficiencies Caused by mutations in IgG heavy chain genes CD154 deficiency Decreased levels of IgG, IgA, and IgE as a result of defect in B-cell switching Predominantly Antibody Deficiencies 10 5  10/28/22 Combined Immunodeficiencies Involve Involve a T-cell deficiency Complex; affect cell-mediated Affect immunity and humoral immunity Possible treatment: bone marrow Tx transplant to reconstitute immune function 11 Severe Combined Immunodeficiency Disease (SCID) Group of very serious PIDs All involve a defect in normal T-cell development May affect B-cell and NK-cell development, depending on type X-linked recessive form caused by IL2RG gene mutation (T–B+NK+/– phenotype) Autosomal recessive forms associated with various defects (T–B+/–NK+/– phenotype) 12 6  10/28/22 Bubble Boy 13 Wiskott-Aldrich Syndrome Rare, X-linked recessive PID Characterized by: Immunodeficiency Eczema Thrombocytopenia Defective T-cell function Decreased IgM, normal IgA and IgG, increased IgE Defect in CD43, an integral membrane protein needed for signal transduction in lymphocytes 14 7  10/28/22 DiGeorge Anomaly Developmental abnormality in third and fourth pharyngeal pouches in embryo Most patients have deletion in chromosome 22 Results in: Underdevelopment of thymus and decreased T cells Hypoparathyroidism and hypocalcemia Cardiac abnormalities Mental retardation Abnormal facial features 15 Rare autosomal-recessive syndrome Ataxia: involuntary muscle movements Ataxia- Telangiectasia Telangiectasias: capillary swelling and red (AT) blotches on skin Low number of T cells and defective antibody responses Involves mutation in AT gene on chromosome 11, resulting in defective DNA repair and gene rearrangements in T and B cells 16 8  10/28/22 17 Defect in Phagocytic Cell Function: Chronic Granulomatous Disease (CGD) Group of disorders inherited as X-linked recessive or autosomal recessive Neutrophils unable to generate oxidative burst Genetic defect in a component of the NADPH oxidase system Decreased killing of catalase (+) organisms Laboratory detection Nitroblue tetrazolium (NBT) test Dihydrorhodamine (DHR) assay 18 9  10/28/22 19 Most are inherited as autosomal recessive Deficiencies in early C’ components Associated with a lupus-like syndrome Complement C2 deficiency most common C3 deficiency associated with recurrent infections Deficiencies with encapsulated bacteria Deficiencies in late C’ components Associated with Neisseria meningitides infections Deficiency in C1 esterase inhibitor Causes hereditary neuroangioedema 20 10  10/28/22 Patient history CBC and WBC differential Serum levels of IgG, IgM, IgA, and IgG subclasses Screening for Isohemagglutinins (natural ABO antibodies) Suspected Immunodeficiency Antibody response after vaccination Delayed hypersensitivity-type skin reactions for cell-mediated immunity CH50 assay for complement function DHR assay for neutrophil function 21 Confirmatory Tests for Immunodeficiency Immunophenotyping and flow cytometry to determine numbers of B cells, helper T cells, cytotoxic T cells, NK cells 22 11  10/28/22 Confirmatory Tests for Immunodeficiency 23 Genetic testing Mitogen assays for T- or B-cell proliferation Mitogen stimulates mitosis in all T cells or all B cells Confirmatory Tests T-cell mitogens include PHA and ConA for Other assays for T-cell activation Immunodeficiency QuantiFERON TB and T-Spot Measure IFN-gamma production by T cells activated by Mycobacterium tuberculosis Cylex ImmuKnow assay Measures ATP production from mitogen-activated T cells 24 12  10/28/22 Evaluation of Immunoglobulins Quantitative measurement by nephelometry or radial immunodiffusion (RID) Serum protein electrophoresis (SPE) Immunofixation electrophoresis (IFE) 25 Newborn Screening for TRECs TRECs = T-cell receptor excision circles Produced as a by-product of TCR gene rearrangement Quantitated by PCR Reflect number of T cells Mandated by many states Used to screen for T-cell deficiencies (e.g., SCID) 26 13  10/28/22 Summary Immunodeficiencies can be broadly classified as primary (inherited) or secondary (acquired due to HIV infection, etc.) PIDs can affect humoral immunity, cell-mediated immunity, or components of the innate defense system The International Union of Immunologic Societies has grouped PIDs into nine categories 27 Summary Antibody deficiencies are characterized by low levels of serum immunoglobulins; they can be caused by defects in B-cell maturation or defective interactions between B cells and T cells Antibody deficiencies include Btk deficiency, common variable immunodeficiency, transient hypogammaglobulinemia of infancy, selective IgA deficiency, IgG subclass deficiencies, and CD154 deficiency Combined immunodeficiencies are complex disorders that involve a T- cell defect that can affect cell-mediated and humoral immunity Combined immunodeficiencies include SCID, Wiskott- Aldrich syndrome, DiGeorge anomaly, and AT 28 14  10/28/22 Summary CGD is a group of disorders affecting the neutrophil oxidative burst; patients with CGD have decreased NBT and DHR test results Complement deficiencies are autosomal-recessive disorders that can affect the early or late components of the complement system Initial laboratory tests in patients suspected of having an immunodeficiency include a CBC and differential, serum Ig levels, isohemagglutinins and antibody responses after vaccination, delayed hypersensitivity-type skin reactions, CH50 assay, and DHR assay 29 Summary Confirmatory tests for immunodeficiencies include genetic testing, T- and B-cell mitogen tests, tests for T-cell activation, and evaluation of immunoglobulins by SPE and IFE TRECs are T-cell receptor excision circles that are produced during TCR gene rearrangement They reflect the number of mature T cells Newborn screening for TRECs is mandated by many states to detect T-cell deficiencies such as SCID 30 15  9/1/23 Chapter 17 Tumor Immunology Copyright © 2017 F.A. Davis Company 1 Chapter Overview Tumor biology Tumor antigens Clinically relevant tumor markers Laboratory tests for tumors Immune defenses against tumors Immunoediting and tumor escape Immunotherapy Copyright © 2017 F.A. Davis Company 2 1  9/1/23 Introduction to Tumor Biology Tumor (neoplasm) = an abnormal cell mass Benign or malignant Cancer = malignant tumor that can spread Caused by mutations in proto-oncogenes and tumor-suppressor genes Uncontrolled cell division and other characteristics Copyright © 2017 F.A. Davis Company 3 Neoplasm results from new growth of cells (neoplasia) that would not ordinarily induce the multiplication of cells, and thus, the growth of tissue Copyright © 2017 F.A. Davis Company 4 2  9/1/23 2 Kinds of Neoplasm 1. Benign neoplasm: slow growth, restricted anatomic location and does not cause usually cause death 2. Malignant neoplasm: slow or rapid growth, anaplasia, invasion of the body and metastases, can result in death. It is also termed as CANCER. Copyright © 2017 F.A. Davis Company 5 Tumor - swelling resulting from anaplasia which is due primarily to an increase in tissue mass Copyright © 2017 F.A. Davis Company 6 3  9/1/23 Evidence for Immune Response 1. Spontaneous regression 2. Long term indolence 3. Mononuclear response 4. Post surgical detection of malignant cells 5. Autologous tumors 6. Positive immediate and delayed hypersensitivity skin test Copyright © 2017 F.A. Davis Company 7 Characteristics of Cancer Cells Sustained proliferation Altered metabolism Resist death Evasion of immune Induce angiogenesis defenses Immortality Genomic instability Invasion and Accelerated growth in metastasis presence of inflammation Avoid suppressors of cell growth Copyright © 2017 F.A. Davis Company 8 4  9/1/23 TSA 1. Oncofetal antigens 2. Viral component proteins or enzymes Copyright © 2017 F.A. Davis Company 9 Oncofetal Antigen present during normal fetal devt, lost during differentiation of tissue and reappear during malignancy Copyright © 2017 F.A. Davis Company 10 5  9/1/23 alpha feto protein synthesize and secreted in the liver, found in the cord blood detected in hepatomas also seen in non malignant cases (cirrhosis / hepatitis) prognostic index or marker of chemotherapeutic success Copyright © 2017 F.A. Davis Company 11 Tumor-Specific Antigens (TSAs) Unique to the tumor Coded by mutated proto-oncogenes or tumor- suppressor genes Examples BCR/ABL gene rearrangement in CML Antigens coded for by cancer-causing viruses (e.g., EBV, HPV, HTLV-I) Copyright © 2017 F.A. Davis Company 12 6  9/1/23 Tumor-Specific Antigens (TSAs) Copyright © 2017 F.A. Davis Company 13 Tumor-Associated Antigens (TAAs) Expressed in tumor cells and normal cells Shared tumor-specific antigens On many tumors but not most normal tissues e.g., MAGE proteins on melanoma tumors Differentiation antigens On immature cells of a particular lineage e.g., CD10 (CALLA), CEA, AFP, PSA Overexpressed antigens Found in higher levels on malignant cells e.g., HER2, CA- 125, CA 19-9 Copyright © 2017 F.A. Davis Company 14 7  9/1/23 CEA detected in the gut, liver & pancreas of normal fetus during 2nd trimester of pregnancy adult: low levels in serum 70% of colon CA cases appear also in non malignant cases (cigarette smoking) prognostic marker Copyright © 2017 F.A. Davis Company 15 Clinically Relevant Tumor Markers Biologic substances found in increased amounts in blood, body fluids, or tissues of patients with a specific type of cancer Concentration in serum depends on amount of tumor proliferation and tumor size Elevated level indicates significant tumor mass Copyright © 2017 F.A. Davis Company 16 8  9/1/23 Clinical Uses of Tumor Markers Population screening To identify cancer in asymptomatic people (e.g., PSA) May detect cancer at an early stage but can get false- positive or false-negative results Diagnosis Identifies cancer in a particular patient (e.g., PSA) Patient prognosis Predicts clinical outcome and aids in selection of therapy (e.g., HER2 in breast cancer) Copyright © 2017 F.A. Davis Company 17 Clinical Uses of Tumor Markers Monitoring response of cancer patient to therapy (e.g., CA 125) Decreasing levels over time suggest effective treatment Increasing levels indicate ineffective treatment or tumor recurrence Copyright © 2017 F.A. Davis Company 18 9  9/1/23 Common Serum Tumor Markers Marker Cancer(s) Uses AFP Liver, testicular S, D, P, M CA 125 Ovarian S, D, P, M CA 19-9 Pancreatic D, P, M CEA Colorectal, breast P, M hCG Testicular, D, P, M trophoblastic PSA Prostate S, D, P, M Copyright © 2017 F.A. Davis Company 19 AFP 1. - primary hepatocellularcarcinoma 2. α HCG - Pituitary tumors 3. β2M - B cell neoplasia 4. Ca 15-3 - breast carcinoma 5. CA 19-9 - Pancreatic and gastric carcinoma 6. CA 72-4 - Gastric carcinoma 7. CA-125 - Ovarian carcinoma 8. β HCG - Choriocarcinoma 9. BJP - Multiple myeloma 10. Bombesin - Oat cell cancer Copyright © 2017 F.A. Davis Company 20 10  9/1/23 11. CA 549 - Breast cancer 12. CA M26 - Breast cancer 13. Calcitonin - Medullary carcinoma 14. c-erbB-2-ocoprotein -Breast carcinoma 15. Chromogranin A -Pheochromocytoma 16. CYFRA 21-1 - Squamous cell carcinoma of the lung 17. Desmesterol - Brain tumors 18. DHEA - Adrenal/pituitary cancer 19. DNA - Cervical carcinoma 20. EPO - Renal carcinoma Copyright © 2017 F.A. Davis Company 21 21. Ferritin - Acute myelocytic leukemia 22. Galactosyltransferase - Ovarian cancer 23. Galactosyltransferase isoenzyme II – Pancreatic cancer 24. Gastrin - Gastrinoma 25. Histaminase - Medullary thyroid cancer 26. HCG (intact mol) - Choriocarcinoma 27. Hyaluronic acid - Mesothelioma 28. IgA - Multiple myeloma 29. IGF-I - Pituitary cancer 30. IL-2 receptor - Leukemia Copyright © 2017 F.A. Davis Company 22 11  9/1/23 31. Igs - Multiple myeloma 32. Inhibin - Granulosa cell tumor 33. Insulin like growth factor - Non-islet cell tumor 34.Katakalcin - Medullary thyroid cell cancer 35. 17-Ketosteroids - Adrenal/pituitary cancer 36. LASA-P - Various carcinoma, Hodgkin’s L 37. Melanoma-assoc ag - Melanoma 38. Metanephrines - Pheochromocytoma 39. Neuron-specific enolase - small cell lung carcinoma 40. Pancreatic polypeptide - Endocrine tumor Copyright © 2017 F.A. Davis Company 23 41. P 21 protein - Breast cancer 42. Plasma catecholamines - Pheochromocytom 43. PNP - Leukemia 44. POA - Pancreatic cancer 45. PSA - Prostrate cancer 46. PS-2 protein - Breast cancer 47. SCC - Leukemias 48. TdT - Acute lymphocytic leukemia 49. Thyroglobulin - Thyroid cancer 50. TPA - non specific Copyright © 2017 F.A. Davis Company 24 12  9/1/23 51. TAG 72 - Gastric carcinoma 52. Urokinase inhibitor - Bladder tumors 53. VIP - Vipoma Copyright © 2017 F.A. Davis Company 25 Laboratory Tests for Tumors Immunohistochemistry Uses labeled antibodies to detect tumor antigens in tissue biopsies Immunoassays Measure levels of circulating tumor markers Molecular methods Detect genetic mutations associated with cancer e.g., PCR, FISH, microarray, DNA sequencing Proteomics Detects protein profile of a tumor population e.g., antibody arrays Copyright © 2017 F.A. Davis Company 26 13  9/1/23 Immunoassays for Serum Tumor Markers Sensitive, automated Factors to consider: Reagents may vary Cross-reactivity can produce false positives Antigen excess can produce false decrease (high-dose hook effect) Interference by heterophile, anti-animal, or autoantibodies Copyright © 2017 F.A. Davis Company 27 Immunoassays for Serum Tumor Markers Copyright © 2017 F.A. Davis Company 28 14  9/1/23 Immune Defenses Against Tumor Cells Immunosurveillance Immune system patrols the body for cancer cells and destroys them before they become clinically evident Innate defenses NK cells, macrophages Adaptive immune responses CTLs, dendritic cells, cytokines, antibodies Copyright © 2017 F.A. Davis Company 29 Immune Defenses Against Tumor Cells Copyright © 2017 F.A. Davis Company 30 15  9/1/23 Relationship Between Immune System and Cancer Copyright © 2017 F.A. Davis Company 31 Immunotherapy Also known as biologic response modifier therapy Uses ability of the immune system to destroy tumor cells Active immunotherapy Stimulates patient’s immune system to respond to tumor antigens Nonspecific stimulation: Coley’s toxins, BCG Cancer vaccines: preventative (HPV, HBV) or therapeutic (TSA-specific, Provenge) Copyright © 2017 F.A. Davis Company 32 16  9/1/23 Passive Immunotherapy Administration of soluble components of the immune system Cytokines to nonspecifically boost the immune response and increase white blood cell production e.g., GM-CSF, IL-2, IFN-a Copyright © 2017 F.A. Davis Company 33 Passive Immunotherapy Monoclonal antibodies that target specific tumor antigens: Surface antigens Receptors Angiogenesis factors Molecules that block T-cell activation Antibody–drug conjugates Copyright © 2017 F.A. Davis Company 34 17  9/1/23 Adoptive Immunotherapy Transfer of cells of immune system TILs to melanoma patients Genetically engineered T cells Copyright © 2017 F.A. Davis Company 35 Summary Development of a malignant tumor (cancer) results from exposure to environmental factors that induce mutations in proto-oncogenes and tumor-suppressor genes Tumor cells possess altered properties, such as resistance to apoptosis, unregulated proliferation, invasion of nearby and distant tissues, genomic instability, and resistance to immune defenses Immune cells recognize altered antigens on the tumor cell surface; these antigens can be tumor specific or tumor associated Copyright © 2017 F.A. Davis Company 36 18  9/1/23 Summary The immune defenses that destroy tumor cells are the same mechanisms that attack pathogens Innate defenses involve NK cells and macrophages Adaptive defenses against tumors involve cytotoxic T cells, dendritic cells, and antibodies Through immunosurveillance, the immune system can recognize and destroy tumor cells before they become clinically evident Copyright © 2017 F.A. Davis Company 37 Summary Tumor cells can undergo genetic changes that allow them to change their phenotype to become less immunogenic (immunoediting); they can progress from a state of equilibrium to being able to escape immune defenses Some tumor antigens circulate in the serum and can be used in cancer screening, diagnosis, prognosis, and patient monitoring; examples include AFP, CA 19-9, CA 125, CEA, hCG and PSA Copyright © 2017 F.A. Davis Company 38 19  9/1/23 Summary Serum tumor markers are most often detected by sensitive, automated immunoassays; however, false positives can be caused by cross-reactivity or interference by heterophile antibodies, and false decreases can result from antigen excess Immunotherapy harnesses the ability of the immune system to destroy tumors Immunotherapy can be active (e.g., cancer vaccines), passive (e.g., cytokines and monoclonal antibodies), or adoptive (e.g., infusion of TILs). Copyright © 2017 F.A. Davis Company 39 20  11/26/22 SEROLOGICAL AND MOLECULAR DETECTION OF BACTERIAL INFECTIONS 1 CHAPTER OVERVIEW Host–microbe relationships Bacterial structure and virulence factors Immune defenses against bacteria and escape Laboratory detection of bacterial infections Group A streptococci Clinical manifestations and laboratory Helicobacter pylori detection of selected bacterial infections Mycoplasma pneumoniae Rickettsial infections 2 1  11/26/22 1. Symbiotic – Host and microbes live together long term – Indigenous microbiota 2. Commensalistic HOST– – No benefit or harm to MICROBE either organism 3. Mutualistic RELATIONSHIPS – Both host and microbes benefit 4. Parasitic – Microbes cause harm to the host 3 INFECTIVITY, PATHOGENICITY, AND VIRULENCE Organism’s ability to establish an Infectivity infection Pathogenicity Ability disease of an organism to cause Extent of pathology caused by an Virulence organism when it infects a host 4 2  11/26/22 S T R U C T U R A L C O M P O N E N T S O F B A C T E R I A 5 Endotoxin The lipid A portion of LPS in gram-negative cell walls Powerful stimulator of cytokine release Pili BACTERIAL Adherence to host cells; resistance to phagocytosis VIRULENCE Flagella FACTORS Capsule Adherence to host cells; motility Blocks phagocytosis, antibody attachment, C’ Exotoxins Potent toxic proteins released from living bacteria Neurotoxins, cytotoxins, enterotoxins 6 3  11/26/22 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 Recognition of PAMPs by PRRs such as TLRs 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 7 BACTERIAL EVASION MECHANISMS 8 4  11/26/22 Culture of the causative agent Grow on broth or solid media Major means of diagnosis, but may take time or may not be possible LABORATORY Microscopic examination DETECTION OF Gram stain or special stains BACTERIAL Detection of bacterial antigens INFECTIONS Rapid testing by ELISA, LFA, or LA Molecular detection of bacterial DNA or RNA Can obtain results in a few hours with PCR 9 L A B O R AT O R Y DETECTION OF BACTERIAL INFECTIONS 10 5  11/26/22 Serology Detects 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 LABORATORY nonspecific DETECTION OF Current infection indicated by presence of IgM, a high IgG titer, or a fourfold rise in antibody titer between acute and BACTERIAL convalescent samples INFECTIONS 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 11 GROUP A STREPTOCOCCI Gram-positive cocci arranged in pairs or chains Person-to-person transmission 12 6  11/26/22 CLINICAL Pharyngitis (“strep throat”) MANIFESTATIONS Pyoderma (impetigo) OF ACUTE GROUP Scarlet fever A Toxic shock syndrome STREPTOCOCCAL Necrotizing fasciitis INFECTION Treated with antibiotics 13 C L I N I C A L M A N I F E S TAT I O N S OF ACUTE GROUP A STREPTOCOCCAL INFECTION 14 7  11/26/22 GROUP A STREPTOCOCCAL SEQUELAE Acute rheumatic fever Develops 1 to 3 weeks after pharyngitis or tonsillitis in 2% to 3% of infected individuals Symptoms: fever, joint pain, inflammation of the heart Most likely caused by immune responses to streptococcal antigens that cross-react with human heart tissue Poststreptococcal glomerulonephritis May follow strep 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 15 LABORATORY DIAGNOSIS OF ACUTE GROUP A STREPTOCOCCAL INFECTIONS Culture on sheep blood agar Rapid assays to detect group A streptococcal antigens Small translucent colonies surrounded by clear LFA zone of beta hemolysis 16 8  11/26/22 L ABORATORY DIAGNOSIS OF ACUTE GROUP A STREPTOCOCCAL INFECTIONS 17 S E R O LO G I C D ET E C T I O N OF GROUP A STREPTOCOCCAL SEQUELAE 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 Anti-DNase B Produced by both rheumatic fever and impetigo patients Tested by EIA and nephelometric methods 18 9  11/26/22 Streptozyme test Detects antibodies to five SEROLOGIC streptococcal products: ASO DETECTION OF Anti-hyaluronidase GROUP A (AHase) STREPTOCOCCAL Anti-streptokinase (ASKase) SEQUELAE Anti-nicotinamide- adenine dinucleotide (anti-NAD) Anti-DNase B 19 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 of production of 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 20 10  11/26/22 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 21 Mycoplasma pneumoniae Tiny bacteria that lack a cell wall Leading cause of respiratory infections Fever, headache, malaise, and cough “Walking pneumonia” Raynaud syndrome Causes Stevens-Johnson syndrome in minority of cases Spread by respiratory droplets 22 11  11/26/22 LABORATORY DIAGNOSIS OF M PNEUMONIAE INFECTION Culture Produces mulberry colonies with a “fried egg” appearance on specialized media Is gold standard but rarely performed in clinical laboratories because organism is difficult to grow Antibodies to M pneumoniae Most useful diagnostic assay IgM antibodies = recent infection IgG antibodies = possible reinfection 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 Molecular methods Film array respiratory panel 23 FEBR IL E A G G L U TINATION TESTS 24 12  11/26/22 1. Widal Test for Typhoid Fever CLASSICAL 2. Weil-Felix for Typhus Fever METHODS 3. Test for Brucella antigen 4. Test for Francisella tularensis 25 WIDAL TEST Antigens for S. typhii 1. O Somatic 2. H Flagellar Antigens for S. paratyphii 1. A 2. B (both are somatic) 26 13  11/26/22 WIDAL TEST Significant Titer: 1:80 Showing at least +2 27 INTERPRETATION O ANTIGEN H ANTIGEN Active or current infection Past infection Recent immunizations Convalescent Exposure 28 14  11/26/22 A OR B O AND A OR B Paratyphoid fever Mixed infection 29 TYPHIDOT TEST consisting of a dot ELISA kit that detects IgM and IgG antibodies against the outer membrane protein (OMP) of the Salmonella typhi. The typhidot test becomes positive within 2–3 days of infection and separately identifies IgM and IgG antibodies. 30 15  11/26/22 The test is based on the presence of specific IgM and IgG antibodies to a specific 50Kd OMP antigen, which is impregnated on nitrocellulose strips. IgM shows recent infection where as IgG signifies remote infection. and time consuming. 31 The most important limitation of this test is that it is not quantitative and result is only positive or negative. Whereas a detailed Widal test can tell the titer of specific antibodies. However, both tests lack sensitivity and specificity. The Widal test is losing its value as it is labor intensive 32 16  11/26/22 COMPARATIVE EVALUATION DURING 1 ST WEEK OF DISEASE 33 WEIL-FELIX TEST Utilized Proteus antigens Significant titer: 1:160 showing +1 34 17  11/26/22 RICKETTSIAL INFECTIONS Obligate intracellular gram-negative bacteria Spotted fever group e.g., Rocky Mountain spotted fever Typhus group e.g., epidemic typhus Organisms transmitted by arthropods (ticks, mites, lice, or fleas) through biting after feeding on an infected animal 35 COMMON RICKETTSIAL INFECTIONS Diseases Organisms Vector Reservoir RMSF R rickettsia Tick borne Wild rodents, dogs Erlichiosis E canis Unknown Rickettsial pox R akari Mite borne Wild rodents Scrub typhus O tsutsugamushi Wild rodents Epidemic typhus R prowazeki Louse borne Humans, flying squirrels Trench fever R quintana Humans Murine typhus R typhii None Wild rodents Q fever C burnetti Cattle, sheep dogs 36 36 18  11/26/22 ROCKY MOUNTAIN SPOTTED FEVER (RMSF) Caused by R rickettsii Transmitted by three species of ticks Symptoms include headache, nausea, vomiting, diarrhea, skin rash; death Diagnosis Clinical presentation Serology by IFA 37 WEIL-FELIX Poor sensitivity and specificity IFA is the gold standard Useful in investigating outbreaks or epidemics 38 19  11/26/22 WEIL FELIX REACTIONS TO DIFFERENT PROTEUS ANTIGENS DISEASES OX19 OX2 OXK RMSF + + - Erlichiosis - - - Rickettsial pox - - - Scrub typhus - - + Epidemic typhus + + - Trench fever - - - Murine typhus + +/- - Q fever - - - 39 39 I.F.A. 40 20  11/26/22 SUMMARY 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 41 SUMMARY Host defenses against bacteria include: Innate defenses such as barriers provided by skin and mucous membranes, anti-microbial peptides, C’, cytokines, APRs, and PRR recognition Adaptive defenses involving humoral and cell-mediated immune responses Laboratory detection of bacterial infections can involve: Culture Staining and microscopic observation Rapid detection of bacterial antigens Molecular detection of bacterial nucleic acid Serologic detection of antibodies to bacterial antigens 42 21  11/26/22 SUMMARY 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 serologic 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. 43 SUMMARY 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; serologic 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 infections such as “walking pneumonia” 44 22  11/26/22 SUMMARY M pneumoniae is difficult to grow in culture. The main methods of detection are serologic 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. Serologic testing for antibodies to R rickettsii by IFA is considered the gold standard for laboratory diagnosis of RMSF 45 23  11/26/22 Chapter 21 Spirochete Diseases Copyright © 2017 F.A. Davis Company 1 Chapter Overview Syphilis Causative organism and transmission Clinical manifestations Laboratory diagnosis Lyme disease Causative organism and transmission Clinical manifestations Laboratory diagnosis Relapsing fever Causative organism and transmission Clinical manifestations Laboratory diagnosis Copyright © 2017 F.A. Davis Company 2 1  11/26/22 Syphilis A sexually transmitted disease caused by the spirochete bacterium Treponema pallidum Rapidly destroyed by heat, cold, and drying Direct contact with open lesion needed Transmission to fetus during pregnancy Bloodborne transmission rare Copyright © 2017 F.A. Davis Company 3 Clinical Manifestations of Syphilis Primary stage Development of chancre Secondary stage Generalized lymphadenopathy, malaise, fever, pharyngitis, rash Latent stage Asymptomatic Tertiary stage Gummatous, cardiovascular, neurosyphilis Copyright © 2017 F.A. Davis Company 4 2  11/26/22 Clinical Manifestations of Syphilis Treatment Effectively treated with antibiotics (e.g., penicillin) when detected in the early stages Congenital syphilis Transmission of treponemes to the fetus occurs when pregnant woman has early-stage or latent syphilis Causes death in 10% of cases Live-born infants may be asymptomatic at birth but develop symptoms later (runny nose, skin rash, generalized lymphadenopathy, hepatosplenomegaly, jaundice, anemia, bone abnormalities, neurosyphilis) Copyright © 2017 F.A. Davis Company 5 Laboratory Diagnosis of Syphilis Direct detection Demonstration of treponemes in active lesions Dark-field microscopy Fluorescent antibody staining Serologic tests Nontreponemal Treponemal Copyright © 2017 F.A. Davis Company 6 3  11/26/22 Nontreponemal Tests Detect antibody against cardiolipin (reagin), a lipid released from membranes of cells damaged as a result of the infection Venereal Disease Research Laboratory (VDRL) test Rapid plasma reagin (RPR) test Look for flocculation Screen: Test undiluted patient serum Titer: Test twofold dilutions of patient serum Copyright © 2017 F.A. Davis Company 7 VDRL Test Patient serum mixed on slide with cardiolipin- lecithin-cholesterol antigen suspension Rotated for 4 minutes at 180 rpm Viewed under light microscope for flocculation Results compared to controls Reactive = medium to large clumps Weakly reactive = small clumps Nonreactive = no clumps or slight roughness Copyright © 2017 F.A. Davis Company 8 4  11/26/22 RPR Test Patient serum mixed on card with charcoal particles coated with cardiolipin antigen Rotate 8 minutes, 100 rpm Observe for macroscopic flocculation Copyright © 2017 F.A. Davis Company 9 RPR Test Copyright © 2017 F.A. Davis Company 10 5  11/26/22 Treponemal Tests Detect antibody to T pallidum Fluorescent treponemal absorption (FTA-ABS) T pallidum particle agglutination (TP-PA) Automated immunoassays Enzyme-linked immunosorbent assay (ELISA) Chemiluminescent immunoassays (CLIA) Multiplex flow immunoassays (MFI) Copyright © 2017 F.A. Davis Company 11 FTA-ABS Test An indirect immunofluorescence test for antibody to T pallidum Procedure Patient serum is incubated with sorbent (an extract of nonpathogenic treponemes) to remove cross- reacting antibodies Absorbed patient serum is incubated with a microscope slide fixed with T pallidum Following a wash, anti-human Ig conjugated with fluorescein is added After a second incubation and wash, slides are examined under a fluorescent microscope Copyright © 2017 F.A. Davis Company 12 6  11/26/22 IFA Copyright © 2017 F.A. Davis Company 13 Positive Reaction Copyright © 2017 F.A. Davis Company 14 7  11/26/22 TP-PA Test Patient serum and controls diluted and incubated with unsensitized gel particles or gel particles sensitized with T pallidum antigen (+) test = agglutination (smooth mat covering surface of well) (–) test = no agglutination (button) Copyright © 2017 F.A. Davis Company 15 Automated Immunoassays ELISA CLIA MFI Copyright © 2017 F.A. Davis Company 16 8  11/26/22 Other Tests Treponema pallidum Immobilization test (TPI) Principle: The Antibody produced against Treponema pallidum plus complement can immobilize the 5 treponemes Reagent Antigen: Live actively motile Treponema pallidum organisms (extracted from the lesions of infected Rabbits) (+): >50% Immobilizaed Treponemes Copyright © 2017 F.A. Davis Company 17 Other Tests HATTS (Hemagglutination Treponemal Test for Syphilis) Principle: Hemagglutination Reagent: Glutaraldehyde stabilized turkey RBC coated with Treponemal Antigen MHA-TP (Microhemagglutination Treponema pallidum test) Principle: Hemagglutination Reagent: Tanned Formalin Sheep RBC coated with Treponemal Antigen Copyright © 2017 F.A. Davis Company 18 9  11/26/22 Other Tests Treponema pallidum Immobilization Test (TPI) Most specific Syphilis standard test Principle: the Antibody produced against T. pallidum + Complement can immobilize the live Treponemes Reagent: Live actively motile 20-49 – weakly reactive, doubtful