1. Lec.4 Laboratory diagnosis of strep pyogenes Laboratory diagnosis of strep pyogenes 1.Specimens: 2. Smears:. 3.Culture:. Colonies of S. pyogenes (GAS) can be indentified by: a.Typical colony morphology. b.Bacitracin test (give positive test). c.PYR test (Positive).

2. 4.Antigen detection tests These tests are 60-90% sensitive, 98-99% specific compared to the culture methods. 5.Serology A rise in the titer of antibodies to many group A streptococcal antigens can be estimated. Such antibodies as antisterptolysin O (ASO) mainly in respiratory disease. antisterptolysin O (ASO) mainly in respiratory disease. A high titer (ASO > 250 units) indicate recent or repeated infection and found mostly in rheumatic fever individuals. A high titer (ASO > 250 units) indicate recent or repeated infection and found mostly in rheumatic fever individuals. anti-DNAase and anti hyaluronidase mainly in skin infection anti-DNAase and anti hyaluronidase mainly in skin infection

3. Treatment 1.All S. pyogenes are susceptible to penicillin G. 2.Antimicrobial drugs have no effect on glomerulonephritis and rheumatic fever. So penicillin need to be taken for 10 days for pharyngitis. This extensive therapy is necessary because large number of Streptococci persist in the pharynx even after 3-4 days of treatment. 3.Erythromycin and cephalosporins are used

4. streptococcus pneumoniae (pneumococcus) Pneumococci are normal inhabitants of the upper respiratory tract of 5-40% of humans and cause pneumonia, sinusitis, otitis, bronchitis. Bacteremia, meningitis and other infections. Pneumococci are normal inhabitants of the upper respiratory tract of 5-40% of humans and cause pneumonia, sinusitis, otitis, bronchitis. Bacteremia, meningitis and other infections. Morphological characteristics Cultural characteristics Cultural characteristics  Aerobic and facultatively anaerobic, grow best with 5-10% CO 2.  Colonies on blood agar are small, round low convex and produce partial hemolysis ( α hemolysis ) of blood.  differentiated from α hemolytic Streptococci colonies by being sensitive to optochin test and bile solubility test positive (soluble in bile).

5. Antigenic structure 1.The pneumococcal cell wall has peptidoglycan and teichoic acid like other Streptococci. 2.The capsular polysaccharide is bound to the peptidoglycan and to the cell wall. 3.More than 90 serotypes of Pneumococcus are distinguished due to polysaccharide capsular antigen. 4.The type of Pneumococcus is determined by its reaction with type-specific antisera by capsule swelling test (Quellung reaction). 5.This reaction is useful for rapid identification and typing of the organisms in sputum, pus or in culture.

6. Pathogensis  Pneumococci produce disease through their ability to multiply in the tissue.  The virulence of the organism is a function of its capsule, which prevent or delays ingestion by phagocytes.  Predisposing factors to infection with Pneumococcus are: 1.Abnormalities in the respiratory tract a.viral or other infection that damage surface cells. b.abnormal accumulation of the mucus (allergy). c.bronchial obstruction 2.Alcohol or drug intoxication 3.Other mechanisms: malnutrition, general debilitation, sickle cell anemia and complement deficiency.  The onset of Pneumococcal pneumonia is usually sudden, with fever and pleural pain. The sputum characteristically bloody or rusty colored.

7. Laboratory diagnosis A.A Gram-stained smear of rusty red sputum shows typical organisms, many PMN nutrophils, and red cells. B.Fresh emulsified sputum mixed with antiserum cause capsule swelling (the Quellung reaction) for identification of Pneumococcus. C.Sputum (or other specimens) cultured on blood agar and incubated at 37°C in CO 2.

8. Epidemiology and control 1.Strep. Pneumoniae account for about 60% of all bacterial pneumonia. 2.Healthy carrier is more important in disseminating Pneumococci than the sick patients. 3.A polysaccharide vaccine containing 23 types licensed in united state used for elderly and immunosuppressed individuals. Treatment 1. Penicillin is the drug of choice, but 5-10% of Pneumococci are penicillin resistant. 2. Resistance to erythromycin and tetracycline occur. 3. High resistant strains are treated by Vancomycin and moderate resistant strains by third cephalosporins.

9. Streptococcus agalactiae (group B Streptococci). 1.Are β hemolytic and produce zone of hemolysis about1-2mm in diam. around the colony. 2.Considered part of the normal vaginal flora in 5-25% of women, and an important cause of neonatal sepsis, meningitis and lung infection. 3.Hydrolyze sodium hippurate and give a positive CAMP test (Christie, Alkins, Munch, Peterson). 4.Ampicillin is the drug of choice for treatment..

10. Enterococci (group D) 1.Are part of enteric flora, they react with group D antisera. Enterococcus faecalis is the most common species that cause enterococcal infection. 2.the most common cause of nosocomial infection mainly in ICU. 3.transmitted from patient to another primarily on the hands of hospital personnel. 4.The common sites of infection are urinary tract, wounds, biliary tract and blood, and may cause meningitis and bacteremia in neonates and endocarditis in adults. 5. nonhemolytic and occasionally α hemolytic, PYR positive, grow in the presence of bile, hydrolyze esculin and grow in 6.5% Nacl.

11. Antibiotic resistance A.Intrinsic resistance intrinsically resistant to cephalosporins, pencillinase resistant pencillins and monbactum, low-level resistance to aminoglycosides, intermediate resistant or susceptible to fluoroquinolones and less susceptible than Streptococci to penicillin and ampicillin. intrinsically resistant to cephalosporins, pencillinase resistant pencillins and monbactum, low-level resistance to aminoglycosides, intermediate resistant or susceptible to fluoroquinolones and less susceptible than Streptococci to penicillin and ampicillin. B. Resistance to aminoglycoside Combination of penicillin or vancomycin plus an aminoglycoside is used for severe enterococcal infections.Combination of penicillin or vancomycin plus an aminoglycoside is used for severe enterococcal infections. Enterococci have intrinsic low level resistance to aminoglycosides. Some enterococci have high level resistance to aminoglycosides and are not susceptible to the synergism.Enterococci have intrinsic low level resistance to aminoglycosides. Some enterococci have high level resistance to aminoglycosides and are not susceptible to the synergism. C.Vancomycin resistance Resistance to vancomycin have increased in frequency, and certain strains are not synergistically susceptible to vancomycin+ aminoglycosides. Resistance to vancomycin have increased in frequency, and certain strains are not synergistically susceptible to vancomycin+ aminoglycosides. D.β- lactamase production and resistance to β- lactams. Gen encoding for β- lactamase is the same gen as found in S. aureus. Gen encoding for β- lactamase is the same gen as found in S. aureus. E.Trimethoprim- sulfamethoxazole (TMP-SMZ) resistance.

12. Viridance streptococci 1.Include many species as S. mitis an S. mutans. 2.α hemolytic but may be non hemolytic. 3.Not inhibited by optochin and colonies are not soluble in bile (bile solubility test). 4.prevalent members of the normal flora of the upper respiratory tract. 5.associated with dental caries and they are the leading cause of subacute bacterial endocarditis in persons with abnormal valve due to congenital heart diseases and rheumatic lesions.