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Laboratory Diagnostics of the Diphtheriae and Pertussis

Diphtheria     Corynebacterium diphtheriae causes diphtheria. Usually the bacteria multiply on the surface of the mucous membranes of the throat where they cause inflammation. The inflammation may spread to the voice box (larynx) and may make your throat swell, narrowing your airway. Disease-causing strains of C. diphtheriae release a damaging substance (toxin), which can also involve the heart, brain and nerves. The bacteria may cause a thick, gray covering in your nose, throat or airway a marker of diphtheria that separates it from other respiratory illnesses. This covering is usually fuzzy gray or black and causes breathing difficulties and painful swallowing.
Risk factors for diphtheriae People who are at increased risk of infection by diphtheria include: children and adults who don't have up-to-date immunizations; people living in crowded or unsanitary conditions; undernourished people; people who have a compromised immune system.
Morphology, staining and culturing of Corynebacterium diphtheriae Diphtheria bacteria are Gram-positive, pleomorphic, often club-shaped rods. The individual cells tend to group in V, Y, or palisade arrangements. Neisser staining reveals the polar bodies (polyphosphates stored at one end of the rod). Löffler nutrient medium, which consists of coagulated serum and nutrient broth, is used for the primary cultures. Selective indicator mediums containing tellurite are used in selective culturing. K tellurite is used to inhibit the accompanying flora. The K tellurite is also reduced to tellurium, coloring the colonies a brownish black.
Extracellular toxin of Corynebacterium diphtheriae Diphtheria toxin consists of two functionally distinct fragments, A and B, whereby B stands for binding to receptors of target cells and A stands for toxic activity. Fragment A irreversibly blocks protein synthesis translation in the target cells, which then die. The toxin gene is always a prophage genome component.
Epidemiology of diphtheriae Infection sources include infected persons and carriers (rare).  
Transmitted of diphtheriae The disease is usually transmitted by droplet infection, or less frequently indirectly via contaminated objects.
Laboratory Diagnosis of diphtheriae Specimens obtained depend on the disease process and include a nose, throat, nasopharyngeal, and wound swab. Specimens are cultivating on cysteine-tellurite agar and Loeffler’s coagulated serum. In addition, differential diagnosis necessitates primary cultivation on blood and chocolate agar. Identification of gray-black colonies on cystein-tellurite agar, the typical Chinese letter, beaded, barred, or palisading arrangment of pleomorphic rods with accentuated metachromatic granules by methylene blue staining of colonies on Loeffler’s coagulated serum, constitute presumptive evidence for C. diphtheriae. Definitive identification of the organism is based upon the demonstration of exotoxin pruduction by a virulence test in guinea pigs or by a modified in vitro “Elek” test utilizing specific antitoxin.
Treatment of diphtheriae Specific antitoxin must be administered immediately. Inasmuch as the antitoxin is generated in horses, a skin test to ensure the absence of hypersensitivity to horse protein is essential prior to its use. Although antibiotics have no effect upon the toxemic disease process, penicillin or erythromycin is effective in killing organism and thus preventing further toxin production.
Prevention and Control of diphtheriae   1. Active immunization with formalin-inactivated toxoid. Vaccination is highly effective. It is first administered early in infancy along with tetanus toxoid and killed Bordetella pertusis (DTP) and must be followed by periodic toxoid boosters throughout childhood and adulthood. 2. The schick test.
Bordetella pertussis   B. pertussis organisms do not grow on common laboratory media; Bordet-Gengou medium, formulate with potatoes, glycerol, and added sheep blood, has been traditionally the medium of choice. These organisms are nonmotile and oxidize amino acids but do not ferment carbohydrates. The other Bordetella species are less fastidious and can grow on blood and Mac Conkey agars.
Pertussis toxin Pertussis toxin is a classic A-B toxin consisting of a toxic subunit (S1) and five binding subunits (S2 to S5). The S2 subunit binds to ciliated respiratory cells. The S3 subunit binds to receptors on phagocytic cells. Two S4 subunits are present in each toxin molecule.
Clinical Syndromes of Pertussis of Whooping Couph B. pertussis is a human disease with no other recognized animal or enviromental reservoir. The disease is still endemic worldwide and affects more then 60 million people annually. Infection is initiated by inhalation of infectious aerosol droplets and attachment and proliferation of the bacteria on ciliated epithelial cells. After a 7-10 day incubation period, the patient will experience the first of three stages. The catarrhal stage resembles a common cold, with serous rhinorrhea, sneezing, malaise, anorexia, and a low-grade fever. Patients in the catarrhal stage pose the highest risk to their contacts. After 1-2 weeks the paroxysmal stage begins, with classic whooping cough paroxysms. The paroxysms are characterized by a series of repetitive coughs fllowed by an inspiratory whoop. The paroxysms are frequently terminated with vomiting and exhaustion. After 2-4 weeks the disease inters the convalescent stage when the paroxysms diminish in number and severity, but secondary complications can occur.
Laboratory Diagnosis of Pertussis     Secretions collected on nasopharyngeal swabs consisting of calcium alginate or Dacron on wire handles are the best specimen. The swab must be inoculated immediately onto media at the patient’s bedside or transported in a moist, protective medium to the laboratory for cultivation. Direct fluorescent antibody stains for the organism are the fastest diagnostic tool. Although method is only 60% sensitive, it is very specific. Specimens are inoculated onto special media, such as Regan-Lowe agar and freshly made Bordet-Gengou agar. Cultures are incubated in a humidified atmosphere without added carbon dioxide for a minimum of 10 days. Once the characteristic colonies (colonies are shiny and have a characteristic metallic or pearly sheen) appear, they can be identified using fluorescent antibody stain. B. pertussis is oxidase positive and microscopic examination should reveal very tiny gram-negative coccobacilli.
Treatment of Pertussis   Treatment with macrolide (erythromycin, azithromycin) is effective in eradicating organisms and reducing length of infectious stage. Treatment does not allevviate symptoms.
Prevention and Control of Pertussis   Vaccination is the key to control; all susceptible populations should be immunized. Immunization of unvaccinated children older than 7 years of age or adults is not recommended at this time because of the decreased severity of disease among the age groups. Immunization with the current vaccine of completely killed cells of B. pertussis in a trivalent vaccin containing diphtheria and tetanus toxoids confers a high degree of protection.

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Date: 2016-01-14; view: 1113


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