Module 5.1 Diseases and Their Serologic Tests - Bacteria PDF

IMMUNOLOGY AND SEROLOGY INTRODUCTION TO IMMUNOLOGY BASIC CONCEPTS OF IMMUNOLOGY IMMUNE DISORDERS DISEASES AND SEROLOGIC TESTS DISEASES AND SEROLOGIC TESTS BACTERIAL DISEASES VIRAL DISEASES PARASITIC DISEASES FUNGAL DISEASES...

IMMUNOLOGY AND SEROLOGY INTRODUCTION TO IMMUNOLOGY BASIC CONCEPTS OF IMMUNOLOGY IMMUNE DISORDERS DISEASES AND SEROLOGIC TESTS DISEASES AND SEROLOGIC TESTS BACTERIAL DISEASES VIRAL DISEASES PARASITIC DISEASES FUNGAL DISEASES DISEASES AND SEROLOGIC TESTS LEARNING OUTCOMES Enumerate efficiently the different bacterial, viral, parasitic and fungal diseases Enumerate efficiently the methods of serologic detection for each Relate properly the application of the different serological tests to clinical diagnosis Report correctly results in serology using standard format of reporting Explain correctly the basic pathophysiology of the different bacterial, viral, parasitic and fungal diseases DISEASES AND SEROLOGIC TESTS BACTERIAL DISEASES VIRAL DISEASES PARASITIC DISEASES FUNGAL DISEASES “THE FIRST STEP TOWARDS GETTING SOMEWHERE IS TO DECIDE YOU’RE NOT GOING TO STAY WHERE YOU ARE.” - J.P MORGAN  DISEASES AND SEROLOGIC TESTS VIRULENCE refers to the extent of damage, or pathology, caused by the organism.  DISEASES AND SEROLOGIC TESTS Bacterial Virulence Factors Virulence Factors: Bacterial properties or features that determine whether an organism is pathogenic and able to cause disease. may be classified as either structural components (e.g., endotoxin is a component of the cell walls of certain bacteria) extracellular substances produced by the bacteria, such as exotoxins.  DISEASES AND SEROLOGIC TESTS PLASMID Plasmids are self-replicating extrachromosomal DNA molecules that are located in the bacteria’s cytoplasm and contain a limited number of genes mobile genetic elements that can be transferred between bacteria through various mechanisms. Acquisition of exogenous DNA that codes for the production of virulence factors can convert an avirulent strain into a virulent strain.  DISEASES AND SEROLOGIC TESTS Structural Virulence Factors Functions Lipopolysaccharide (LPS) Found in cell wall of gram-negative bacteria Has three parts: outer, inner and Lipid A Lipid A: powerful stimulator of cytokine production that leads to a variety of systemic manifestations and potentially fatal endotoxic (gram-negative) shock. Flagella Whip-like structures Facilitate adherence as well as movement of the bacteria toward a host cell. Common pili involved in specific attachment F or Sex pili function in the attachment of bacteria to host cells. Capsules major structural feature that plays an important role in increasing an organism’s virulence block the attachment of antibodies, inhibit activation of complement, or act as a decoy when capsular material is released into the surrounding host environment. DISEASES AND SEROLOGIC TESTS Extracellular Virulence Factors produced by bacteria also Example are Invasins; contribute to an organism’s hyaluronidase virulence by breaking down collagenase primary or secondary defenses phospholipases of the body, damaging the host lecithinases tissues and cells, or facilitating coagulase the growth and spread of the kinases organism. How does the body fight off bacterial infections? First line of defense Structural barriers - Skin, mucosal surfaces Soluble components - Lysozyme, interleukins, prostaglandins, leukotrienes, and APRs Phagocytosis Formation of antibodies Cell-mediated immunity How do bacteria evade immune responses? A. Inhibiting chemotaxis B. Blocking adherence of phagocytes to bacteria C. Blocking digestion by toxin production D. Inhibiting binding of C3b E. Cleavage of IgA  How do bacteria evade immune responses? 1. Avoiding antibody A. Altering bacterial antigens due to mutations (some Streptococci) B. Down-regulation of MHC molecules C. IgA proteases (N. gonorrhoeae, H. influenzae, S. sanguis)  How do bacteria evade immune responses? 2. Blocking phagocytosis A. Inhibits chemotactic factors (N. gonorrhoeae) B. Substances in cell wall interferes adhesion with phagocytes (M protein: S. pyogenes) C. Block fusion of lysosomal granules with phagosomes (Salmonella, M. tuberculosis, M. leprae) D. Polysaccharide capsule (N. meningitidis, S. pneumoniae, Y. pestis, H. influenzae)  How do bacteria evade immune responses? 3. Inactivating complement cascade Bacterial capsules do not bind C3b Protein H (S. pyogenes) binds to C1 but cascade does not occur Sialic acid in cell walls degrade C3b (group B streptococci) DISEASES AND SEROLOGIC TESTS BACTERIAL INFECTIONS Group A Streptococci Helicobacter pylori Mycoplasma pneumoniae Rickettsial infections Spirochete infections DISEASES AND SEROLOGIC TESTS BACTERIAL INFECTIONS Group A Streptococci Helicobacter pylori Mycoplasma pneumoniae Rickettsial infections Spirochete infections  Group A Streptococci Streptococcus pyogenes ✓ Gram positive ✓ Beta hemolytic ✓ Catalase-negative ✓ Spherical, ovoid, or lancet-shaped cocci ✓ Seen in pairs or chains Lancefield groups ✓ 20 groups; based on serologically reactive carbohydrates (A-H, K-V)  Group A Streptococci VIRULENCE FACTORS M protein F protein Lipoteichoic Acid Major virulence factor Enhance the ability of S. For bacterial adherence to Inhibits phagocytosis pyogenes pili to attach to respiratory epithelium Limits deposition of C3 on the host cells particularly to bacterial surface, thereby fibronectin. diminishing complement activation. Strains lacking M antigen can’t cause infection Group A Streptococci  Group A Streptococci VIRULENCE FACTORS 3. Exoantigens or exotoxins Proteins excreted by the bacterial cells as they metabolize during the course of streptococcal infections. protein molecules that are released from living bacteria and are considered to be some of the most potent molecules known to harm living organisms. A. Enzymes B. Pyrogenic and erythrogenic toxins  Group A Streptococci VIRULENCE FACTORS Enzymes Streptokinase promotes fibrinolytic activity by converting plasminogen to plasmin Hyaluronidase may enhance the spread of the organism through connective tissue Pathogenic significance of the DNases and of NADase is unknown. Streptolysin O – oxygen-labile streptococcal hemolytic exotoxin  Group A Streptococci VIRULENCE FACTORS: EXOANTIGENS/EXOTOXINS Streptolysin O Streptolysin S Oxygen-labile streptococcal hemolytic Streptolysin S exotoxin Inactive in the oxidized form; can cause lysis Oxygen stable enzyme of WBC and RBC in its reduced state Produces surface hemolysis on BAP Produce subsurface hemolysis on BAP Molecular weight of 2,800 (non-antigenic) Synthesized only by growing streptococci Synthesized by growing and resting cells Cardiotoxic: induce the release from atria of acetylcholine, which poisons the ventricles MW of approximately 70,000 daltons (antigenic/immunogenic)  Group A Streptococci Clinical Manifestations of Infection Acute infections: Major sites of infection: Upper respiratory tract (pharyngitis) and skin (impetigo or streptococcal pyoderma) Other acute complications: Otitis media, scarlet fever, erysipelas, cellulites, puerperal sepsis, and sinusitis.  Group A Streptococci Clinical Manifestations of Infection Acute infections: Upper respiratory tract Pharyngitis - fever, chills, severe sore throat, headache, tonsillar exudates) Rhinorrhea (sometimes purulent) Scarlet Fever - result of pharyngeal infection; URTI with rash due to erythrogenic toxin Tests: Dicks test (susceptibility test), Schultz-Charlton reaction (diagnostic test)  Group A Streptococci Clinical Manifestations of Infection Acute Infections SKIN Impetigo (Streptococcal pyoderma): Superficial infection; lesions that itches, eventually crusts and heals Cellulitis : subcutaneous infection; warm, red, tenderareathat may beswollen Erysipelas : aka St. Anthony's Fire;skin is usually very redand swollen and thereis a well-defined border between normal and infectedskin.  Group A Streptococci Severe infections Necrotizing fasciitis Toxic shock syndrome Can be also caused by Klebsiella, - life-threatening multisystem disease Clostridium, E. coli, S. aureus, that initiates as a skin or soft tissue Aeromonas hydrophila infection and may proceed to shock and Results from a skin infection that renal failure due to overproduction of invades the muscles of the extremities cytokines or the trunk. Commonly called a "flesh-eating infection“ Exotoxins deep in the fascia results in ischemia, tissue necrosis, and sepsis Most common cause is group A Streptococcus  Group A Streptococci Poststreptococcal disease Results from host’s response to infection Complications Organism itself may no longer be present Diagnosis through serologic testing / Sequelae Main damaging sequelae: Acute Rheumatic Heart Fever Poststreptococcal glomerulonephritis  Group A Streptococci Acute Rheumatic Heart Fever (RHF) Glomerulonephritis Develops as a sequela to pharyngitis or tonsillitis a condition characterized by damage to the glomeruli in in 2-3% of infected individuals; Rheumatic heart the kidneys. disease is damage to the heart valves caused by a Deposition of streptococcal- antistreptococcal immune bacterial (streptococcal) infection called complexes in the glomeruli and subsequent activation of rheumatic fever (CDC). complement Production of antibodies directed against M Kidney disease affecting the capillaries of the glomeruli, characterized by albuminuria, edema, and hypertension. protein that cross-reacts with myocardial tissue A heart or tissue cross-reactive antigen of S. pyogenes that shares immunologic epitopes with, but is distinct from, the M protein has been identified Group A Streptococci Diagnosis of acute streptococcal Laboratory infections Plated on Blood Agar Diagnosis: Presumptive ID: susceptibility to Culture bacitracin, positive PYR test, growth in trimethoprimsulfamethoxazole  Group A Streptococci Laboratory LATERAL FLOW IMMUNOCHROMATOGRAPHIC ASSAYS increasingly being used for the detection of bacterial, Diagnosis: viral, fungal, and parasitic antigens in clinical samples. largely replaced enzyme immunoassay (EIA) and LA Detection of assays to detect the antigens. Group A MOLECULAR METHODS: hybridization of specific rRNA Streptococcal sequences and real-time PCR, have also been developed as a means to rapidly detect Group A streptococcal Antigens infections.  Group A Streptococci The test is interpreted by the presence or absence of visually detectable pink-to- Lateral flow purple colored lines. A positive result is immunochromatographic indicated by production of both a sample assays (LFAs) line and a control line (shown on left), whereas a negative assay will produce only the control line (shown on right).  Group A Streptococci Detection of Streptococcal Antibodies Laboratory Used to identify rheumatic fever and glomerulonephritis The use of at least two tests for antibodies to different Diagnosis: exotoxins is recommended. Most diagnostically important antibodies: Detection of anti-streptolysin O (ASO)* anti-DNase B* Antibodies anti-NADase anti-hyaluronidase  Group A Streptococci Laboratory Detect antibodies to SLO, which is able to lyse red blood cells. Diagnosis: Presence of ASO indicates recent Anti- streptococcal infection in patients suspected of having acute rheumatic fever Streptolysin or poststreptococcal glomerulonephritis O following a throat infection.  Group A Streptococci Laboratory ASO Titer Diagnosis: Titers typically increase within 1 to 2 weeks after infections (240 Todd units – adults; 360 Todd units – children) Anti- Peak between 3 to 6 weeks following the initial symptoms Occurs in only about 85% of acute rheumatic fever Streptolysin O patients Usually do not increase in individuals with skin infections. Testing  Group A Streptococci Anti-Streptolysin O (ASO) Testing Laboratory ASO Neutralization (Tube) Test Test was based on the ability of Diagnosis: antibodies in the patient’s serum to neutralize the hemolytic Anti- activity of streptolysin O Streptolysin INTERPRETATION: O Positive (+) = no hemolysis Negative (-) = hemolysis  Group A Streptococci ASO Slide Test Laboratory Rapid latex agglutination ASO Polystrene latex particles are Diagnosis: coated with streptolysin O antigen ASO Slide Positive result is Test agglutination Significant titer: >200 IU/mL  Group A Streptococci Streptozyme Test Slide agglutination test screening test for detection of antibodies to 5 streptococcal antigens Laboratory Streptolysin Streptokinase Diagnosis DNAse NADase Hyaluronidase  Group A Streptococci Laboratory Diagnosis Principle: NEUTRALIZATION If anti-DNase B antibodies are present, they will neutralize reagent DNase Highly specific for anti- B, preventing it from Interpretation: DNase produced in depolymerizing DNA. The Positive (+) = DNAse infection presence of DNase is B neutralized and Anti-DNAse Test Better than ASO test for measured by its effect on a unable to decolorize cases of DNA-methyl-green conjugate (green glomerulonephritis conjugate. This complex is color persists) green in its intact form; however, when hydrolyzed by DNase, the methyl green is reduced and becomes colorless DISEASES AND SEROLOGIC TESTS BACTERIAL INFECTIONS Group A Streptococci Helicobacter pylori Mycoplasma pneumoniae Rickettsial infections Spirochete infections  Helicobacter pylori Gram-negative, microaerophilic spiral bacterium Major cause of gastric and duodenal ulcers Associated with 90% of duodenal ulcers and nearly all gastric ulcers May lead to gastric carcinoma if untreated PEPTIC ULCER : an ulcer in the wall of the stomach or duodenum resulting from the digestive action of the MOT: oral–oral and fecal–oral routes gastric juice on the mucous membrane when the latter is rendered susceptible to its action  Helicobacter pylori CagA: Major Virulence Factor, highly immunogenic VacA: Vacuolating Factor, Toxin Virulence precursor. Factors Urease: providing a buffering zone around the bacteria that protects it from the effects of PEPTIC ULCER : an ulcer in the wall of the stomach or duodenum resulting the stomach acid. from the digestive action of the gastric juice on the mucous membrane when the latter is rendered susceptible to its action  Helicobacter pylori 1. Endoscopy and biopsy the most expensive and invasive methods for diagnosing an infection with H. pylori. Laboratory 2. CLOtest Diagnosis: detects urease activity in gastric mucosal biopsies During the endoscopy, a small biopsy is taken (1 to 3 Invasive mm). The specimen is placed in the test cassette, resealed following the manufacturer’s instructions, Procedures and sent to the laboratory. If urease is present, the yellow gel will turn a hot- pink color because of an increase in pH in the presence of urease. If urease is not present, the gel will remain yellow  Helicobacter pylori Urease testing Laboratory 1. Rapid urease - detected by placing biopsy specimens directly into urea broth Diagnosis: 2. Urea breath test Antigen noninvasive test Detection the patient ingests urea labeled with radioactive carbon ( 14C) or, in newer tests, a nonradioactive 13C urea is broken down by the Procedures urease enzyme of H. pylori, producing ammonia and bicarbonate. The bicarbonate is excreted in the breath and the labeled carbon dioxide is measured by detection of radioactivity for 14C.  Helicobacter pylori Rapid EIA Antigen Identify antigen in stool Detection samples Procedures Used to determine success of therapy  Helicobacter pylori Best for initial screening method Anti-H. Most methods detect IgG Antibody levels: Untreated individuals – remain elevated for years. pylori Treated individuals – decrease after about 6 months to about 50% detection Not as reliable as antigen testing to evaluate therapy Techniques: ELISA (method of choice), immunoblot, latex agglutination DISEASES AND SEROLOGIC TESTS BACTERIAL INFECTIONS Group A Streptococci Helicobacter pylori Mycoplasma pneumoniae Rickettsial infections Spirochete infections  Mycoplasma pneumoniae Belongs to a unique group of bacteria (Class Mollicutes , Genus Mycoplasma) because they have NO CELL WALL Pleomorphic Spherical or pear shaped to filamentous with branching Resistant to beta-lactams Lack of a reaction to Gram stain  Mycoplasma pneumoniae Colonizes the mucosa of the respiratory tract Causes primary atypical pneumonia/walking pneumonia Stevens–Johnson syndrome Raynaud syndrome aka Erythema Multiforme Major a transient vasospasm of the a condition in which the top digits in which the fingers turn layer of the skin dies and sheds white when exposed to the cold.  Mycoplasma pneumoniae LABORATORY DIAGNOSIS Detection of Antibodies to Culture Mycoplasma pneumoniae growth produces a IgM immunoglobulin is the “mulberry” colony with a most useful diagnostic test typical “fried egg” because it likely indicates a appearance. recent infection. ELISA methods have a specificity of more than 99% and a sensitivity of 98%  Mycoplasma pneumoniae 1. Detection of anti-M. pneumoniae Cold agglutinins IgM antibodies directed against the altered I antigens found on the surface of human RBCs in approximately 55% of patients IgM with anti-I specificity Titer peak at day 12-15, decreasing after day 20. The agglutinins are capable of clumping RBCs at 4°C. The reaction is reversible when the samples are warmed to 37°C. 2. Molecular Diagnosis of Mycoplasma Infections BioFire Diagnostics, Inc. (Salt Lake City, UT), now part of the BioMerieux corporation, received FDA approval in 2012 for its BioFire FilmArray Respiratory Panel. assay is able to detect 20 respiratory viruses and bacteria, including B. pertussis, C. pneumoniae, and M. pneumoniae DISEASES AND SEROLOGIC TESTS BACTERIAL INFECTIONS Group A Streptococci Helicobacter pylori Mycoplasma pneumoniae Rickettsial infections Spirochete infections Rickettsial infections Gram negative coccobacilli, obligate intracellular parasites Genus Rickettsia Spotted fever group (SFG) Spread of rash is from extremities to the trunk GIT complaints, arthralgias, conjunctivitis, stiff neck, DIC, etc Typhus group (TG) Rash appear as first on the trunk and later to the extremities  Rickettsial infections Rocky Mountain Spotted Fever Caused by Rickettsia rickettsii transmitted by American dog tick (Dermacentor variabilis), main pathophysiological event the Rocky Mountain wood tick (Dermacentor andersoni), and the brown dog tick (Rhipicephalus sanguineus) endothelial cell damage, which leads to increased vascular Pathogenesis: permeability, resulting in edema, hypovolemia, invade their target cells, the vascular endothelium. hypotension, and The organisms multiply by binary fission inside the endothelial hypoalbuminemia cells, are released, and infect adjacent cells. This leads to hundreds of contiguous infected cells, producing the lesions and skin rash associated with the infection.  Rickettsial infections Weil-Felix Test Rickettsiae & Proteus share certain antigens Serological P. vulgaris – OX-2 & OX-19 P. mirabilis – OX-K Diagnosis Insensitive and nonspecific! Should not be used except in developing countries in which no other method can be performed  Rickettsial infections Common Weil-Felix Reactions RICKETTSIAL SPECIES OX-19 OX-2 OX-K R. prowazekii ++ +/– – R. typhi ++ – – R. tsutsugamushi – – ++ R. ricketsii, R. conorii, R. australis, + + – R. sibirica R, akari, C. burnetii, R. quintana, Ehrlichia – – – Rickettsial infections Serodiagnosis is currently the method of choice for detecting rickettsial infections Current serological assays are organism-specific Indirect fluorescent assays (IFA)* Serological performed on two paired serum samples to demonstrate a significant (four-fold) rise in antibody titers Diagnosis Microimmunofluorescent assays (micro-IF)* Immunoperoxidase assays (IPA) ELISA Immunoblot assays (IBA) *Gold standard for detecting rickettsial antibodies Rickettsial infections Indirect fluorescent assays (IFA)  Rickettsial infections If the patient has a rash, molecular diagnosis using DNA from the skin lesions Molecular is of value. Several assays using real-time PCR have Diagnosis been described in the literature, but at the time of this writing, there are no FDA- approved assays. DISEASES AND SEROLOGIC TESTS BACTERIAL INFECTIONS Group A Streptococci Helicobacter pylori Mycoplasma pneumoniae Rickettsial infections Spirochete infections  Spirochete infections Spirochetes Many commensal and nonpathogenic species of spirochetes exist, and human disease is limited primarily to infection by members of three genera: Treponema Borrelia Leptospira Diagnosis often relies upon the demonstration of a patient’s serologic response to the offending agent. Direct detection by microbiological culture, microscopy, and genomic amplification Spirochete infections Spirochetes Gram-negative Long, slender, helically coiled Has axial filaments or periplasmic flagella Microaerophilic bacteria Corkscrew motility Two major spirochete diseases: Syphilis Lyme disease  Spirochete infections Spirochetes Clinical course: Proliferation of the organisms at the site of inoculation (localized skin infection) Spirochetemia with systemic dissemination to numerous organs Persistence of small numbers of microbes at various, often immune, “privileged” sites (latent infection). Cardiac and neurological involvement if disease remains untreated.  Spirochete infections: Syphilis Syphilis A commonly acquired spirochete disease typically spread through sexual transmission and is caused by Treponema pallidum subspecies pallidum aka Great pox or Evil pox, French disease, Italian disease, Spanish disease Other organisms: T. pallidum subspecies pertenue – yaws T. pallidum subspecies endemicum - nonvenereal endemic syphilis T. carateum - pinta.  Spirochete infections: Syphilis Syphilis Treponema pallidum subspecies pallidum 6–20 μm in length Thin, regular spiral organism Close-coiled with 10–13 coils Man is the only natural reservoir Very hard to stain, best observed by DFM Multiplies by transverse fission, occurs every 30 hours  Spirochete infections: Syphilis Syphilis Mode of transmissions 1. Sexual contact (30-50%) Sexual transmission is the primary mode of dissemination; this occurs through contact of abraded skin or mucous membranes with an open lesion. 2. Mother to fetus (placental) 3. Much less frequent MOT: Nonsexual contact with lesion Transfusion of fresh blood products Not recovered in blood stored at 4°C for >48 hours Accidental needle stick when handling laboratory specimens Spirochete infections: Syphilis Stages of Syphilis Primary Secondary Latent Tertiary  Spirochete infections : Syphilis Stages of Syphilis PRIMARY Hard chancre Hunterian chancre Initial lesion Appear at site of entrance after 10- CHANCRE 90 days (ave. 21 days) of infection Single, firm, painless, non-itchy skin Persists for 1-6 weeks ulceration with a clean base and sharp borders between 0.3 & 3.0 cm in size Evolves from a macule to a papule, and finally to an erosion or ulcer  Spirochete infections : Syphilis Stages of Syphilis SECONDARY This stage is usually observed about 1 to 2 months after the primary chancre disappears. 25% of untreated individual will progress to this stage Condylomata lata 6-8 weeks after the appearance of the 1° chancre Mucous membranes CONDYLOMATA LATA Bloodstream dissemination Rashes that become maculopapular or postural Generalized rash on trunk and extremities and form flat, whitish, wart- like lesions  Spirochete infections : Syphilis Stages of Syphilis LATENT Divided into: Early latent (1 year) Characteristics: Absence of clinical symptoms Patients are non-infectious May last for years or for the rest of the patient’s life Lab tests: Serologic tests  Spirochete infections : Syphilis Stages of Syphilis TERTIARY Lesions (Gummas) seen 3-10 years after 1° stage if left untreated Gummas can be found in the CNS (Neurosyphillis) Incomplete paralysis (paresis) General paralysis (tabes dorsalis) Cardiovascularsystem (aortic aneurysm) Eyes(blindness) GUMMAS Soft, tumor-like balls of Labs: Sero tests only inflammation which may vary considerably in size.  Spirochete infections : Syphilis TERTIARY Three major manifestations: 1. Gummatous syphilis Gummas most often found on bones, skin or subcutaneous tissue If in eyes, can cause blindness 2. Cardiovascular disease Aortic aneurysm, thickening of the valve leaflets causing aortic GUMMAS regurgitation, or narrowing of the ostia, producing angina pectoris. Soft, tumor-like balls of 3. Neurosyphilis inflammation which may vary considerably in size.  Spirochete infections : Syphilis Congenital Syphilis Placental passage of T. pallidum from 18th week gestation Although the disease can be transmitted at any stage of pregnancy, typically the fetus is most affected during the second or third trimester Can cause late abortion, stillbirth, neonatal death, neonatal disease, or latent infection Treatment of mother prevents infection of fetus (esp. During the 1st four months) Penicillin can also cross the placenta  Spirochete infections : Syphilis Congenital Syphilis Presentation: Diffuse maculopapular desquamatous rash Hemolytic anemia, Hepatosplenomegaly and Jaundice Abnormal cartilage and bone involvement Mental retardation Stigmata: scars or deformities resulting from early or late lesions that have healed  Spirochete infections : Syphilis Diseases Related to Syphilis Disease Description Causative agent Lesions Chronic skin & bone disease T. pallidum 1. Yaws of the tropics subspecies Fambresia pertenue Ulcerative skin disease of 2. Pinta Central & South America T. carateum Pintid T. pallidum Non venereal endemic 3. Bejel subspecies --- syphilis endemicum Natural venereal infection Minor lesions 4. Rabbit of the rabbits T. cuniculi @ genitalia Syphilis  Spirochete infections : Syphilis Diseases Related to Syphilis T. pallidum subspecies pertenue Causative agent of YAWS (aka frambesia tropica, pian, parangi, paru & buba) Chronic skin & bone disease of the tropics Most prevalent of the nonvenereal treponematoses MOT: direct contact with skin lesions Frambesia: granulomatous or wart-like lesion, with a granular surface similar to a raspberry Approximately 10% develop late yaws, which shows irreversible, destructive lesions of bone, cartilage, soft tissue, and the skin  Spirochete infections : Syphilis Diseases Related to Syphilis Treponema carateum Causative agent of PINTA (aka carate, mal de pinto, azul) Lesions: Pintid Commonly occur in hands, feet & scalp Scaly psoriasiform plaques Skin appears to be the only organ affected in this disease  Spirochete infections : Syphilis Diseases Related to Syphilis T. pallidum subspecies endemicum Causative agent of BEJEL (non-venereal endemic syphilis) MOT: direct contact, sharing of eating or drinking utensils Lesions: Primary- oral cavity Secondary- oral mucosa Tertiary- skin/bones/nasopharynx Late stage - tissue destruction of the skin, bones, and cartilage  Spirochete infections : Syphilis Diseases Related to Syphilis Treponema cuniculi Causes Rabbit syphilis (venereal spirochetosis or vent disease) Natural infection of rabbits Produces minor lesions of genitalia  Spirochete infections : Syphilis Laboratory Diagnosis DIRECT by visualization (microscopy) of the organisms in material from lesions Darkfield Microscopy Fluorescence Microscopy Immunohistochemical Microscopy INDIRECT by immunologic methods Nontreponemal test Treponemal test  Spirochete infections : Syphilis Laboratory Diagnosis: Direct DARKFIELD MICROSCOPY (DFM) T. pallidum is a thin, tightly wound, rigid, spiral organism exhibiting little flexibility and does not move from place to place. Has a sensitivity of 80% in diagnosing syphilis, but non-specific DFM not recommended for oral lesions (Commensal treponemes in the mouth) Three Treponema spp. are normal inhabitants of the genital region: T. phagedensis, T. refringens, T. minutum  Spirochete infections : Syphilis Laboratory Diagnosis: Direct FLUORESCENCE MICROSCOPY Sensitive and highly specific alternative to DFM Can be performed by: Direct method - using a fluorescent-labeled anti-T. pallidum Indirect method - using anti-T. pallidum and a second labeled anti- immunoglobulin antibody. Live specimens are not required Tissue fluid dried and fixed to the slide Direct fluorescent antibody [DFA-TP]  Spirochete infections : Syphilis Non-treponemal Tests Treponemal Tests Detects non-treponemal antibodies or Reagin Detects treponemal antibodies (Abs to Reacts with lipid Ags (cardiolipin) treponemal Ags) Used to screen for disease, and to monitor the Reacts with T. pallidum and closely related course of disease after treatment strains Transient false positives occur in diseases such Used to confirm a positive non-treponemal as hepatitis, infectious mononucleosis, varicella, screening test or to confirm infection in the late herpes, measles, malaria, and tuberculosis, as latent which has a negative nontreponemal test well as during pregnancy, SLE, leprosy, Ex: TPI, TPPA, FTA-ABS, etc intravenous drug use, autoimmune arthritis, advanced age, and advanced malignancy. Ex: VDRL, RPR, USR, etc  Spirochete infections Treponemal antigens Cardiolipin aka Diphosphatidyl glycerol or Wassermann Ag Normal constituent - phospholipid found in liver & cardiac muscle a lipid material released from damaged host cells A “hapten” - must be bound to microbial cell to be immunogenic Microbial cell = foreign carrier Cardiolipin = immunologic determinant Spirochete infections : Syphilis Reagin the antibody against substances released by cells when they are damaged by T. pallidum (cardiolipin and lecithin) aka Anti-cardiolipin or Wasserman Antibody  Spirochete infections : Syphilis Treponemal antibodies Infected individuals produce both specific and nonspecific Abs Specific Abs - directed against T. pallidum Nonspecific Abs - directed against the protein Ag group common to pathogenic spirochetes Specific Abs in early or untreated early latent syphilis are largely IgM Largest elevation of IgG level are seen in the 2° stage Nonspecific IgA Abs increase significantly during the course of untreated syphilis  Spirochete infections NONTREPONEMAL TESTS Detects non-treponemal antibodies or Reagin Reacts with lipid antigens (cardiolipin) Used to screen for disease, and to monitor the course of disease after treatment Examples: Venereal Disease Research Laboratory (VDRL) Rapid Plasma Reagin (RPR) Toluidine Red Unheated Serum Test (TRUST)  Spirochete infections : Syphilis NONTREPONEMAL TESTS Venereal Disease Research Laboratory (VDRL) Specimen: Serum and CSF (very specific for the diagnosis of neurosyphilis) VDRL is the only serologic test approved for testing CSF VDRL Reagents Cardiolipin (0.03%) – main reacting component Cholesterol (0.9%)- enhance reacting surface of cardiolipin Lecithin - removes anti-complement property of cardiolipin  Spirochete infections : Syphilis NONTREPONEMAL TESTS Rapid Plasma Reagin (RPR) Specimen: Serum Reagent: modified VDRL reagent Cardiolipin, Cholesterol, Lecithin Charcoal - for macroscopic evaluation Choline Chloride - chemical inactivation of serum EDTA - prevents oxidation of lipids Thimerosal - preservative  Spirochete infections : Syphilis Laboratory Diagnosis: Indirect VDRL RPR Complement Yes (heating the serum at 56 degree Celsius No heating needed since the reagent used able Inactivation for 30minutes) to inactivate complement. Result Interpretation Put on a rotator for 4 minutes at 180 RPM Put on a rotator for 8 minutes at 100 RPM Microscopically Macroscopically (charcoal is added) Tests must be performed at room The antigen is similar to the VDRL antigen, temperature within the range of 23°C to with the addition of EDTA, thimerosal, and 29°C (73°F to 85°F) choline chloride, which stabilize the antigen and inactivate complement so that serum does not have to be heat-inactivated before use. Spirochete infections : Syphilis Laboratory Diagnosis: Indirect INTERPRETATION No clumping or slight Small clumps Medium to Large roughness  Spirochete infections : Syphilis TREPONEMAL TESTS Detects treponemal Abs (Abs to treponemal Ags) Uses T. pallidum cells as antigen source reacts with T. pallidum and closely related strains Used to confirm a positive non-treponemal screening test or to confirm infection in the late latent which has a negative non-treponemal test Examples: T. pallidum Immobilization Test (TPI) Fluorescent Treponemal Antibody Absorption Test (FTA-ABS) Microhemagglutination T. pallidum tests  Spirochete infections : Syphilis TREPONEMAL TESTS Treponema pallidum Immobilization (TPI) Test Interpretation Treponemal test of reference Standard test from w/c other tests are evaluated Positive >50% immobilized Used to confirm syphilis or to rule out false positive results Doubtful 20-50% immobilized Test of choice for spinal fluid, for neurosyphilis Negative