Clinical Bacteriology - 10 Obligates and Spirochetes

CLINICAL BACTERIOLOGY SPIROCHETES OBLIGATE INTRACELLULAR AND NONCULTURABLE BACTERIAL AGENTS, CELL WALL DEFICIENT ORGANISMS, AND THE SPIROCHETES GENERAL CHARACTERISTICS • • OUTLINE • • o Laboratory Diagnosis for Introduction Rickettsiaceae Spirochetes o Differential Characteristics of o General Characteristics Rickettsiaceae and Related o Treponema Organisms o Treponema pallidium & • Chlamydia Classification of Syphillis o Chlamydia trachomatis o Treponema pertenue o Related Infections & Diseases o Other Treponema o Laboratory Diagnosis o Diseases Related to Syphilis o Chlamydophila psittaci o Borrelia o Chlamydophila pneumoniae o Leptospira o Differential Characteristics of o Other Spirochetes Chlamydia Species • Rickettsia o Transmission and Spread of • Mycoplasma o Mycoplasma pneumoniae Rickettsia o Ureaplasma urealyticum o Pathogenesis of Rickettsia o Genital mycoplasma o Orientia o Laboratory diagnosis o Organisms related to Rickettsiaceae • TREPONEMA CHARACTERISTICS • • • INTRODUCTION • • Classified as obligate intracellular bacteria that are either difficult to culture or unable to be cultured are organisms of the genera: Chlamydia, Chlamydophila, Rickettsia, Orientia, Anaplasma and Ehrlichia. These genera are classified as prokaryotes that differ from most other bacteria with respect to their very small size and obligate intracellular parasitism. The cell wall deficient bacteria : Mycoplasma and Ureaplasma can colonize or infect humans and are not of animal origin. Besides lacking a cell wall these agents with their small cell size and small genome size, require sterols for membrane function and growth. Mycoplasmas are part of the normal flora of humans and are found mainly in the oropharynx, upper respiratory tract, and genito urinary tract. Considered clinically significant are the following species: M. pnenumoniae, M. hominis and U. urealyticum. M. pneumoniae is a cause of community acquired pneumonia; infections caused by this agent are distributed worldwide. The genus Treponema, Leptospira and Borrelia contains pathogenic organisms for humans. The spirochetes are long, slender, helically curved, gram negative bacteria with unusual morphologic features of axial fibril and an outer sheath. These fibrils, or axial filaments are flagella like organelles that wrap around bacteria's cell wall, are enclosed within the outer sheath, and facilitate motility of the organisms. treponema are slender with tight coils, Borrelia are somewhat thicker with fewer and looser coils; and Leptospira resembles Borrelia except for their hooked ends. The major pathogen in the genus Treponema is T. pallidum which s the known agent of syphilis. Borrelia are the causative agents of tickborne and louseborne relapsing fever and tickborne Lyme disease in humans. The leptospires include both free-living and parasitic forms. Pathogenic Leptospira species are currently classified in seven species based on DNA relatedness, with L. interrogans sensu stricto being the main species associated with human leptospirosis. They are unicellular organisms. They are slender, helically coiled, flexible organisms, motile, resulting from the action of axial filaments which are also responsible for its spiral shape. They are facultative anaerobes and multiply by transverse fission • • • • The word Treponema is derived from a Greek word meaning “turning thread.” They are corkscrew like organisms. The spirals are so thin that they are not readily seen unless darkfield illumination or immunofluorescent stain is employed. They are not stained very well with aniline dyes, but they do reduce silver nitrate to metallic silver that is deposited on the surface so the treponemes can be seen in tissues (Levaditi Silver Impregnation). Pathogenic strains has never been cultured with certainty on artificial media, in fertile eggs, or in tissue culture but maintained in the testicular chancres of rabbits. Non– pathogenic strains (Reiter strain) can be cultured anaerobically in vitro and grows on a defined medium of amino acids, vitamins, salts, minerals and serum albumin. They remain viable in whole blood or plasma for at least 24 hours which is of potential importance in blood infusion. They are killed rapidly at 42oC and is used as a basis for syphilis therapy A life cycle has been postulated from T. pallidium consisting of granular stage, cyst–like spherical bodies and spirochetal form. There are two types of antibodies produced: reagin, an antibody like substance and antitreponemal antibody, a true antibody, both of which can be used for the serologic diagnosis of syphilis. Treponema pallidium & CLASSIFICATION OF SYPHILLIS ACQUIRED SYPHILLIS • Stages of Syphillis o Primary stage – dry lesion appear in genitalia known as hard chancre or hunterian chancre. It appears after 10 days to several months incubation period. ▪ Laboratory diagnosis • Dark field microscopy • Special stains such as Levaditi Silver Impregnation, Fontana Tribondeau (gold in color) • Serological test ▪ During this stage, RPR test is more sensitive than VDRL; similarly, the FTA–ABS becomes reactive before the MHA–TP. o Secondary stage – lesions are found all over the body known as condylomata lata. It appears 6–8 weeks after the appearance of initial chancre. They may also be involvement of CNS, eyes, bones and liver. Latent stage – shows no sign and the disease is recognized only through serological tests o Tertiary stage – takes place when the latent stage is not treated; involvement of the deep organs of the disease (cardiac syphilis, neurosyphilis) known as gummas. Other internal organs involved are bone & skin. ▪ Jerisch–Herxheimer reaction – reaction most commonly observed in the early stages of syphilis when treated after 2–12 hours with either heavy metals of penicillin. Commonly observed reactions are headache, malaise and a temperature above 38oC. It is due to the release of toxic products from dying or killed spirochetes. Non–specific or Non–treponemal antibody test o The organism is not utilized as an antigen but instead a cardiolipin– lecithin antigen to detect antibody like substance called reagin. o Complement Fixation Test ▪ Wasserman ▪ Kolmer o Flocculation Test ▪ Venereal Disease Research Laboratories (VDRL) – used heat inactivated serum which is added to antigen. ▪ Rapid Plasma Reagin (RPR) – used unheated serum which is added to RPR antigen; performed on a white, plastic coated card. Treponemal Antibody Test – organisms containing antigen detects specific treponemal antigen. o Treponema Pallidium Immobilization – uses live and motile treponemes + patient’s serum + complement; if immobilized, the patient’s serum contains specific treponemal antibodies. o Microagglutination Assay for Treponema pallidium antibodies (MHA–TPA) – based upon agglutination by specific antibodies in serum with lyophilized, formalinized, tanned sheep RBC sensitized with Treponema pallidium antigen. o Fluorescent Treponema Antibody Test o Fluorescent Treponema Antibody–Absorption (FTA–ABS) – the most specific test for syphilis; a modification of FTA which uses as antigen ▪ Fluorescent Antibody Test (FAT) – used to confirm the validity of (+) reagin test. It is also used to diagnose congenital syphilis and the late stages of syphilis. Antigen used is a lyophilized Nichol strain organism. ▪ FTA–ABS and MHA–TP – heat inactivation not required. o Reiter Protein Complement Fixation – antigen used is an extract from non – virulent treponemes (a Reiter strain); non – reactive in the late stages of syphilis. o • • Treponema pertenue • • • OTHER Treponema • • Transmission of the disease from syphilitic mother to the fetus through the placenta. The following may result: o Some of the fetus may die, others are still born alive o Other maybe born alive with symptoms like ▪ Interstitial keratitis ▪ Hutchinson’s teeth ▪ Saddle nose ▪ Periotitis ▪ CNS anomaly Treponema carateum o Causative agent carate or pinta which is characterized by hyperpigmented lesion o Affects only the skin, initially appears as red and blue lesions but later depigmented Treponema vincentii – together with Bacteroides melaninogenicus, produces a spirochetal disease known as Vincent’s angina (trench mouth) DISEASES RELATED TO SYPHILIS • • Bejel – an non–venereal form of syphilis which closely resembles yaws. It is now given a subspecies designation, T. pallidium subspecies endemicum. It is characterized by highly infections skin lesions. Rabbit syphilis – caused by Treponema cuniculi, a non– venereal infection of rabbits producing minor lesions on the genitalia. Borrelia CHARACTERISTICS • • • • They are highly flexible, spiral organisms which are loosely coiled and irregular in shape, motile with a corkscrew like motion in fresh blood. They are microaerophilic They can be visualized through aniline dyes They can cause relapsing fever, borreliosis or famine fever Borrelia recurrentis • • CONGENITAL SYPHILLIS • Causative agent of Yaws or Frambresia which is a disease of bone and skin. A non–venereal disease and transmitted to man through direct contact or through the aid of flies. o Primary lesion or Mother Yaws or Framboise – initial lesion which develops 3 – 4 weeks after exposure o Secondary lesion or Daughter Yaws – develops 6– 12 weeks after initial lesion o Tertiary lesion or Gummatous ulceration Crab yaws – infection of the feet which causes a crippling form of disease. Causative agent of Lousebourne or Epidemic Relapsing fever which is characterized by a short, febrile paroxysms lasing 2 or 3 days, alternating with afebrile periods of 3 or 4 days and resulting in 2 to 10 relapses, more commonly the lesser number, each attack terminates by crisis Transmitted to man by body louse, Pediculus humanosus corporis and Pediculus humanus capitis Borrelia turicatae or Borrelia parkeri or Borrelia duttoni • • Causative agent of Tickborne or Endemic Relapsing Fever which is similar to Louseborne except relapses are more frequent. Transmitted to man by ticks, Ornithodoros sp. Borrelia burgdorferi Leptospira interrogans • • • • • Named after Dr. Burgdorf. Causative agent of Lyme disease: Lyme arthritis, Banwarth’s syndrome, Erythema Chromicum Migrans (ECM) Transmitted to man by Ixodes dammimi and Ixodes pacificus Stages: o Stage I: A papule at the site of the tick bite an expanding annular ▪ Lesion (erythema chromicum migrans) o Stage II: Disseminated disease which is characterized by intermitted disease symptoms (fatigue, headache, mild stiff neck, bone and muscle aches, fever) and produce a syndrome similar to ▪ Infectious mononucleosis with conjunctivitis, pharyngitis, pneumonitis. ▪ Involvement of cardiovascular system and the CNS (meningoencephalus) o Stage III: Late disease is indicated by a persistent, chronic arthritis ▪ A patient may have one or all the stages and the infection may not become symptomatic until Stage II and III. OTHER Borelia • • • Borrelia anserine – causative agent of avian spirochetes Borrelia buccalis – a spirochete which occurs in every normal mouth Borrelia refringens – a spirochete found in normal genitalia that may be confused with T. pallidium. This organism is found in proximal tubules of kidneys of infected animals and is passed in human. So, human infection results from contact with animal urine or water which has been contaminated with urine like soil, natural water (flood), foodstuff. Leptospirosis develops after an incubation period of 10–12 days. • The signs and symptoms vary from a mild fever to severe illness including jaundice, kidney failure and meningitis. Human disease L. interrogans Clinical findings serovar Fort Bragg Fever autumnalis fever, rash over tibia Infectious jaundice canicola influenza–like illness, aseptic meningitis Marsh fever grippotyphosa fever, prostration aseptic meningitis Seven day fever hebdomadis fever, jaundice Weil’s disease icterohemorrhagiae jaundice, hemorrhages, aseptic meningitis Swinehard’s disease mitis aseptic meningitis LABORATORY DIAGNOSIS • • • • • LABORATORY DIAGNOSIS • • • • Light microscopy – Giemsa, Wright, Romanowsky, Leischman stained blood smears. The only spirochete found in the peripheral blood stained by direct stain in which the finding of stained spirochetes is sufficient to establish a definitive diagnosis of relapsing fever. Animal inoculation using white mice wherein blood is taken and stained. Serologic test like VDRL, CF and use of Proteus OXK Culture using: o Kelly media A and chick embryo o Noguchi medium (with ascetic fluid) Leptospira OTHER SPIROCHETES • • • • • • • • • • They are tightly coiled spirochetes with very fine spirals. One end of the organism is often bent, forming a hook. They appear as minute cocci in chain as seen in the Darkfield Microscope. They are strictly aerobic, oxidase (+), catalase (+) and peroxidase (+). They grow best in protein rich semisolid medium (Fletcher’s) where they produce round colonies. They also grow on chorioallantoic membrane of embryonated egg. They can survive for weeks in water with alkaline pH. Spirillum minor – causes rat bite fever known as sodoku Spirillum volutans Magneto tacticum Azospirillum Rickettsia CHARACTERISTICS • Specimen used: blood, CSF, urine Darkfield microscopy – fluorescent antibody, silver impregnation/ staining with aniline dye. Animal inoculation test Serologic test Culture o Fletcher’s medium – contains frozen dried serum with hemoglobin o Ellinghausen’s medium – a semisolid medium o Tween 80 albumin, Stewart’s medium, Fletcher’s – contains rabbit serum as the added enrichment. • • • • • • • • The species of this genus have the simplest bacterial form and are considered transitional organisms between bacteria and viruses They are fastidious bacteria and obligate, intracellular parasites They have Gram-negative cell wall; are motile; and will not grow in culture media Multiply through binary fission Survive briefly outside of their host Do not undergo any intracellular developmental cycle Microscopy: small (0.5-2 um), pleomorphic, Gram-negative bacilli Culture: require living cells for growth Mode of acquisition: humans become infected following the bite of an arthropod vector Mode of prevention: avoid contact with the respective vectors • • • • • • • Accidental host: humans Agents of bioterrorism: R. prowazekii, R. rickettsia Rickettsia is divided into three groups” o Spotted fever group o Transitional group o Typhus group Bartonella • • • TRANSMISSION AND SPREAD OF Rickettsia • The organisms gain access into humans through skin abrasions or arthropod bites The organisms are disseminated homogeneously, thus causing vasculitis in the blood vessels The members of the typhus group reproduce in the cytoplasm cellular injury and death Rickettsia felis is maintained transovarially in cat flea, Ctenophalides felis • PATHOGENESIS OF Rickettsia • • Spotted fever group o Rocky mountain spotted fever (RMSF) is the most serious rickettsial infection o For RMSF, humans are the accidental hosts and ticks are the main vector and reservoir o The rashes develop on the palms of the hands and soles of the feet of RMSF patients o The boutonneuse fever exhibits rashes that are similar with that of RMSF but on th efface of the afflicted o Tache noires (black spots) are present in patient with boutonneuse fever o In ricketssialpox, the rashes are observed on the face and extremities Typhus group o Epidemic typhus is characterized by rashes on the face, palms, and soles of the feet of the sick o Rashes are not commonly observed in patients with endemic typhus o Inhalation of aerosol from dried infected flea feces is also a mode of transmission of Rickettsia typhi infection Coxiella burnetti • • • • • • • • • • • Orientia tsutsugamushi • • • • • • • • • • • • Anaplasma phagocytophilia • • • • • This genus belongs to the family Anaplasmataceae The species of the genus are Gram-negative coccobacilli that undergo an intracellular development cycle following the infection of circulating WBCs (replicate ion occurs in the leukocytes) Microscopy: presence of intravascular microcolony that resembles “mulberries” or a morula (identified through Wright-Giemsa staining) It has three developmental stages o Elementary body (infective form) o Initial bodies o Morulae Natural hosts: humans and animals (dogs & deer) Primary vector: Amblyomma americanum (lone star tick) Species: E. chaffeensis & E. ewingii Releated infection: Human monocytic ehrlichiosis (HME) LABORATORY DIAGNOSIS FOR RICKETTSIACEAE ORGANISMS RELATED TO RICKETTSIACEAE Causes human granulocytotrophic anaplasmosis Vector: Ixodes pacificus (Western black-legged tick) and Ixodes scapularis (deer tick) Reservoir: Peromyscus leucopus (white-footed mouse) Only species in the genus Coxiella Causative agent of Q (Query) fever which is a systemic infection of the lungs Extremely contagious and can be considered as a potential bioterrorism agent Can survive extracellularly because of its endospore-like body Can infect birds and rodents, which in turn excrete the organisms via their urine, feces, and birth products Not transmitted by arthropod vectors Does not multiply in routine bacteriologic culture media Modes of acquisition: inhalation of contaminated aerosols from dried animal feces and ingestion of contaminated unpasteurized milk Animal reservoir: cattle, goat, and sheep Ehrlichia Orientia Belongs to the family Rickettsiaceae Categorized as a separate genus fue to the absence of lipopolysaccharise and peptidoglycan, and the presence of 54- to 58-kDa major surface proteins Replicates in the cytoplasm of its host cell and is released through a process that involves “pinching off” the host cell Mode of acquisition: bite of an infected arthropod vector Vector: Leptotrombidium delicense (chigger) Accidental host: humans and rats The species of this genus are facultative, intracellular, and Gram-negative bacilli Can live within the RBC in their natural mammalian hosts Can be cultivated in CAP with 5% CO2 or in charcoal yeast extract agar Some species exhibit a “twitching motility” in wet mounts (B. bacilliformis, B. benselae) Species of Bartonella and their Associated Infections and Diseases o B. quintana - Trench fever (louse-borne disease) o B. benselae - Cat scratch disease o B. elizabethae - Infective endocarditis o B. bacilliformis - Oroya fever (chronic verruga peruana) and febrile acute hemolytic anemia) o B. clarridgeiae - Cat scratch disease (secondary agent) Specimens: biopsy of skin tissues, peripheral blood, and CSF Processing of specimens should be done in a biosafety level 3 laboratory • • • • Direct methods o Immunohistology (immunofluorescence or immunoenzyme stains) of skin biopsy material is a sensitive and specific test that is used to visualize Rickettsia in tissue sections o Giemsa stain is used for the detection of morulae during the febrile stage of ehrlichiosis Culture o Culture media: Yolk sacs of embryonated eggs and tissue cultures o Rickettsia, Ehlichia, and Anaplasma can be isolated from humans in an antibiotic-free, centrifugationenhanced shell vial cell culture o Columbia blood agar with 5% defibrinated blood is used for the cultivation of B. bacilliformis o Lung tissue cells are the preferred medium for C. burnetti Serological test o Only test that is performed for the diagnosis of rickettsial diseases o Used to confirm rickettsioses during convalescent stage o Antibodies to Rickettsia (except for R. rickettsii) cannot be detected until at least two weeks after infection o Rickettsioses are seldom diagnosed serologically during the acute stage of the illness due to the absence of an early antibody response o Indirect immunofluorescent antibody (IFA) assay ▪ The gold standard serologic test or reference method for rickettsioses and Q fever o Weil-Felix reaction ▪ Presumptive test for rickettsioses ▪ The agglutination of certain strains of Proteus vulgaris by serum from patients with rickettsial diseases is diagnostic ▪ Individuals with Q fever, ehrlichiosis, and rickettsialpox do not produce Weil-Felix antibody o Microimmunofluorescent dot test ▪ Excellent sensitivity for detecting antibodies to Rickettsia ▪ Used for the early diagnosis of RMSF after the onset of symptoms o Other serological tests: ▪ Latex agglutination ▪ Enzyme immunoassay ▪ Line blot Nucleic acid amplification (Polymerase chain reaction or PCR) o Cat scratch disease can be confirmed through PCR testing of lymph node aspirates and biopsies o PCR is a diagnostic tool for ehrlichiosis DIFFERENTIAL CHARACTERISTICS OF RICKETTSIACEAE AND RELATED ORGANISMS Organism Spotted fever group Rickettsia conorri Infection/Disease Boutonneuse fever or Mediterranean spotted fever Rickettsia rickettsii Rocky mountain spotted fever (RMSF) Vector/Mode of Transmission Ticks (Rhipicephalus sanguineus) Wood ticks (Dermacentor andersoni) Organism Transitional group Rickettsia akari Rickettsia felis Typhus group Rickettsia prowazekii Rickettsia typhi Infection/Disease Rickettsialpox Flea-borne spotted fever Anaplasma phagocytophila Mouse mite (Liponyssoides sanguineus) Flea bite or feces Epidemic typhus/Brill- Body louse (Pediculus Zinsser disease bumanus corporis) Squirrel flea (Orchopeas howardi) Squirrel louse (neohematopinus sciuropteri) Endemic murine Rat flea (Xenopsylla typhus cheopis) Scrub typhus group Orientia Scrub typhus tsutsugamuchi Anaplasmataceae Ehrlichia chaffeensis Vector/Mode of Transmission Dog ticks (Dermacentor variabilis) Brown dog ticks (Rhipicephalus sanguineus, Amblyomma cajennense) Human monocytic ehrlichiosis Chigger (Leptotrombidium delicense) bite Lone star tick (Amblyomma americanum) Deer tick (Ixodes scapularis and Ixodes pacificus) Inhalation of aerosol from infected animals Feces of body louse (Pediculus humanus corporis) Kitten scratch or bite Coxiella burnetti Human granulocytotropic anaplasmosis Q fever Bartonella quintana Trench fever Bartonella henselae Cat scratch disease Bacillary angiomatosis Oroya fever and Sandfly (Lutzomyia) verruga peruana bite Bartonella bacilliformis CHLAMYDIA • • • • • • • The species of this genus are non-motile, small (0.21.5um), and have a Gram-negative cell wall Obligate, intracellular organisms that require living cells for growth Do not possess cytochromes and cannot synthesize their own ATP Called “energy parasites” because they depend on the eukaryotic cell of their hosts for metabolism, growth, and reproduction Replicate by binary fission in the cytoplasm of infected cells Species: Chlamydia trachomatis, Chlamydophila psittaci, and Chlamydophila pneumoniae Chlamydia has two morphologic forms: o Reticulate body (RB) - replicative and non-infectious form ▪ The intracellular and metabolically active form of Chlamydia ▪ Directs the reproduction of host cells to their own metabolic needs so they multiply by binary fission o Elementary body (EB) - infectious form ▪ The extracellular form of Chlamydia and is spherical in shape ▪ Resembles Gram-negative bacilli with a rigid cell wall ▪ Infects the host cell by inducing active phagocytosis ▪ Its two components are the major outer membrane protein (MOMP) and the lipopolysaccharide antigen Chlamydia trachomatis • • • • • • • • One of the major sexually transmitted pathogens One of the principal causes of pelvic inflammatory disease (PID) and ocular trachoma Can travel through the birth canal where infants can be infected during birth Associated with infertility and ectopic pregnancy Natural host: humans Unique feature: 10 stable plasmids Biovars: Lymphogranuloma venereum (LGV), mouse, and pneumonitis trachoma Serovars of C. trachomatis based on the MOMP antigenic differences: o Serotypes A, B, Ba, C - endemic trachoma o Serotypes L1, L2, L2a, L2b, L3 - LGV o Serotypes D to K, Da, Ia, Ja - PID, urethritis, cervicitis, epididymitis, inclusion conjunctivitis • • • • RELATED INFECTIONS AND DISEASES • • • • • Chlamydia infection is transmitted sexually, as well as during birth, from the mother to the infant The intra peritoneal spread may cause peritonitis or perihepatitis (Fitz-Hugh-Curtis syndrome) Trachoma o A chronic inflammation of the conjunctiva that leads to blindness o Can cause distortion of the eyelids (eyelashes become misdirected and turned-in) o Modes of transmission: contact with contaminated objects, hand-to-hand contact with carriers, and through vectors (flies) Lymphogranuloma venereum (LGV) o A sexually transmitted disease which has multisystem involvement o A small, painless ulcer or papule appears initially and then nodules (buboes) develop after several weeks Inclusion conjunctivitis o Characterized by an abundant eyer discharge and a swollen conjunctiva o Affects infants who acquire it through aspiration or ocular exposure during birth LABORATORY DIAGNOSIS • • Specimens: urethra and cervical secretions, conjunctiva discharge, nasopharynx and rectal swabs; and materials aspirated from fallopian tubes and epididymis Use of Dacron or rayon-tipepd swabs is preferred • Culture (living tissue cell culture) o Culture media: buffalo green monkey kidney cells, HeLa 229 cells, Hep-2 cells, McCoy cells, and cycloheximide-treated McCoy cells o Procedure prior to culture: centrifugation of the specimen onto the cell monolayer o Shell vial technique facilitates the adherence of elementary o After 48-72 hours of incubation, the monolayers are stained with iodine or immunofluorescent stain to identify the presence of inclusion bodies Cytologic examination o Cell scrapings from the conjunctiva of newborns or persons with ocular trachoma are used as specimens o A direct fluorescent antibody (DFA) staining method that utilizes the fluorescein-isothiocyanate-conjugated monoclonal antibodies is used to identify the organisms in infected cells Antigen detection and nucleic acid amplification o Nucleic acid amplification is the most sensitive method for detecting C. trachomatis o More reliable in symptomatic patients o Lipopolysaccharide (LPS) antigen is the major antigen that is detected in genus-specific serologic tests for chlamydial infection o Specimens: endocervical and urethral swabs Serodiagnosis o The extractable LPS or elementary body with ketodeoxyoctonate is the primary antigen that can be identified in genus-specific tests o A negative test result can exclude chlamydial infection o Complement fixation ▪ Identifies family-reactive antibodies ▪ Used to diagnose LGV ▪ Genus-specific antigens can be detected by using this method ▪ (+) result; ≥ 1:64 titer o Microimmunofluorescence (Micro-IF) assay ▪ Used for the type-specific antibodies of C. trachomatis ▪ The preferred method for identification of antibodies to C. trachomatis ▪ Can be used for the diagnosis of LGC, trachoma, inclusion conjunctivitis, and chlamydial neonatal infection ▪ (+) result: IgM titer of 1:32 Immunoassay o Enzyme immunoassay (EIA) is the most commonly used rapid antigen assay o EIA detects either the outer membrane LPS chlamydial antigen or the MOMP antigen Chlamydophila psittaci • • • • • • Causative agent of psittacosis or ornithosis Endemic pathogen of bird species such as parrots, parakeets, chickens, and ducks Modes of acquisition: inhalation of infected aerosols from dried bird excreta or handling of infected birds Commonly used test: complement fixation w/ a titer of ≥ 1:32 Sensitive method: direct microimmunofluorescence Precautionary measures: only laboratories with biosafety level 3 facilities can perform the isolation and cultivation of the specimens Chlamydophila pneumoniae • • • • • • • Mycoplasma pneumoniae Formerly known as the Chlamydia pneumoniae strain TWAR Human pathogen that is transmitted through aerosol droplets One of the major causes of infectious respiratory diseases Associated with pneumonia, bronchitis, pharyngitis, and sinusitis Specimens for isolation: nasopharyngeal aspirates, sputum, and throat swabs Culture media: HeLa cells or HEp-2 cell lines Preferred method: microimmunofluorescent assay DIFFERENTIAL CHARACTERISTICS OF CHLAMYDIA SPECIES Properties Host range Elementary body Inclusion morphology and inclusion bodies Stains used Glycogencontaining inclusions Susceptibility to sulfonamides Diseases Number of serovars C. trachomatis Humans Round C. psittaci Birds Round Round, vacuolar Variable, dense Halbertstaeder- Levinthal-ColeProwazek bodies Lillie bodies Lugol’s iodine stain • • • • • • • • • • • • C. pneumoniae Human Pear-shaped • Round Giemsa stain Present Macchiavello stain and Giemsa stain Absent Susceptible Resistant Resistant Trachoma, LGV, and inclusion conjunctivitis 20 Psittacosis Pneumonia, pharyngitis 10 10 • Absent MYCOPLASMA • • • • Belong to the class Mollicutes which means “soft” Smallest free-living organisms capable of growing on artificial media Part of the microbial flora of humans and found mainly in the oropharynx, URT, and genitourinary tract Common parasites of the genital tract and their transmission is related to sexual activity Pleomorphic organisms that do not possess a cell wall; slow grower; fastidious, facultatively anaerobic; replicate by binary fission Have a small cell size (0.3-0.8 umZ) and small genome size Require sterols (cholesterol) for membrane function and growth Lack the enzymatic pathway for purine and pyrimidine synthesis - require complex media Species: M. pneumoniae, M. hominis, M. fermetans, M. pirum, M. penetrans, and U. ureayticum Eaton agent Etiologic agent primary atypical pneumonia/walking pneumonia Pleuropneumonia-like organism (PPLO); with gliding motility Does not occur as a natural commensal; extremely fastidious Has the ability to attach to respiratory mucosal cells (a surface parasite); escape phagocytosis, and modulate the immune system - initiation of disease Produced hydrogen peroxide and ammonia as waster products, causing ciliostasis and sloughing of superficial cell P1 protein - responsible for attachment and interacts with neuraminic acid-containing glycoproteins The interaction of this organism with the antigenic determinant of human erythrocytes, which contains the necessary 2,3-sialylated poly-N-acetylgalactosamine sequences, may alter the I antigen, converting it into a non-self antigen that stimulates production of cold agglutins Pathogenesis o Can occur singly or as outbreaks in closed populations such as families and military recruit camps; also causes tracheabronchitis o Infects children and adults and is spread by respiratory droplets produced by coughing o Intracellular protein antigens exposed during its phagocytosis and subsequent degradation stimulate T-lymphocyte reactions, which determine the severity of disease Ureaplasma urealyticum • • T-strain of mycoplasma Has the ability to metabolize urea GENITAL MYCOPLASMA • • • • • • U. urealyticum and M. hominis Has been recovered from genital tract of healthy adult Colonization among infants occur during passage through an infected birth canal May be recovered from the genital tract, nose and throat infants Cause invasive disease in immunosuppressed patients (agammaglobulinemia), prostatitis, bacterial vaginosis, and non-gonococcal urethritis M. hominis - cause postabortal and postpartum fever LABORATORY DIAGNOSIS • • Specimens o M. pneumoniae - throat swab, serum, bronchoalveolar lavage, sputum, lung tissue o Genital mycoplasma - urethral, vaginal or endocervical swab; blood, urine, prostatic secretions, seme No direct method for identification • • • Culture o Culture media: beef or soybean protein with serum, fresh yeast extract; biphasic SP-4, PPLO broth or agar with yeast extract and horse serum and Modified New York City Medium, E. agar, Shepard’s, Edward Hayflick agar o Mycoplasma and ureaplasma grow slowly than most other bacteria o pH requirement: pH 5.5-6/5 (ureaplasma) and pH 6-8 (Mycoplasma) o Mycoplasmsa - “fried egg colonies” on plated medium o U. urealyticum - “dark brownish clumps” on A7 or A8 agar medium o M. hominis - colonies can be stained with the Dienes or acridine orange; (-) urease o M. pneumoniae - requires biphasic culture system; sealed container up to 3 weeks in 5-10% CO2 o Glucose (dextrose) is incorporated into media selective for M. pneumoniae o Urea and/or arginine can be incorporated into media to detect U urealyticum and M. hominis o Definitive identification of M. pneumoniae: is by overlaying suspicious colonies with 0.5% guinea pig RBC in PO4-buffered saline Serodiagnosis o ELISA - most widely used method o Detection of specific IgM in a single serum sample is diagnostic of acute infection Rapid indentification - Manganous Chloride Urea Test (U. urealyticum) o Reaction is observed under a dissecting microscope o (+) dark brown precipitate of manganese oxide around the colonies o U. urealyticum utilizes manganous chloride in the presence of urea o