Laboratory Diagnostics of Streptococcal Infections
Streptococcus
The genus Streptococcus is a diverse collection of gram-positive cocci typically arranged in pairs or chains. Most species are facultative anaerobes, and some grow only in an atmosphere enhanced with carbon dioxide (capnophilic growth). Their nutritional requirements are complex, necessitating the use of blood or serum-enriched media for isolation. Carbohydrates are fermented, resulting in the production of lactic acid, and unlike Staphylococcus species, streptococci are catalase-negative.
Streptococci can be classified by several schemes:
1. Hemolytic properties on blood agar.
Alfa-hemolytic streptococci cause a chemical change in the hemoglobin of red cells in blood agar, resulting in the appearance of a green pigment that forms a ring around the colony. Beta-hemolytic streptococci cause gross lysis of red blood cells, resulting in a clear ring around the colony. Gamma-hemolytic is a term applied to streptococci that cause no color change or lysis of red blood cells.
2. Serologic (Lancefield) Groupings
Many species of streptococci have in their cell walls a polysaccharide known as the C-carbohydrates, which is convalently linked to the cell wall peptidoglycan. The lancefield scheme classifies primarily ß-hemolytic streptococci into groups A through U based on their C-carbohydrate. The clinically most important groups of ß-hemolytic streptococci are types A and B.
3. Biochemical (physiologic) Properties
Group A, B, -haemolytic streptococci
Streptococcus pyogenes is the most virulent member of this group of gram-positve cocci. This bacterium is an important cause of a variety of suppurative and nonsuppurative diseases.
Structure and Physiology
Isolates of S. pyogenes are gram-positive, nonmotile cocci occuring in short or long chains, and occasionally singly and in pairs. Freshly isolated strains of group A streotococci are encapsulated, but the capsules are lost rapidly during the stationary phase of in vitro cultivation.
Group A, like most streptococci, are less resistance to enviromental conditions than staphylococci, although they can survive on dry swabs for weeks. They are killed rapidly by physical and chemical agents. Structural features that are involved in the pathology or identification of the group A streptococci include:
A. Antigenic structure.
B. Extracellular products.
Antigenic structure of group A, ß-Streptococci
The outermost layer of the cell is the capsule (K-antigen), which is composed of hyaluronic acid, identical to that found in connective tissue. For this reason, the capsule is nonimmunogenic (again in contrast with S. pneumoniae).
C-carbonhydrates are cell wall polysaccharides whose antigenic diversity forms the basis for the classification of streptococci into 20 serogroups lettered from A to V.
Lipoteicoic acid (LTA) exposed on the cell surface definite the ability of group A streptococci to bind to epithelial cells in the mouth and on the skin.
M protein is a major antigen associated with virulent streptococci. In the abscence of M protein, the strains are not infectious. The M protein also prevents interaction with complement.
Protein F (fibronectin-binding protein) mediates attachment to fibronectin in the pharyngeal epithelium.
Extracellular products of group A, ß-Streptococci
Streptolysin S is nonimmunogenic cell-bound hemolysin capable of lysing erythrocytes, as well as leukocytes and platelets, following direct cell contact. Streptolysin O is inactivated reversibly by oxygen. Unlike streptolysin S, antibodies are readily formed against streptolysin O and are useful for documenting a recent infection (ASO test). In addition to its ability to lyses human erythrocytes, streptolysin O is also capable of killing leukocytes by lyses of their cytoplasmic granules with release of hydrolytic enzymes.
Pyrogenic (erythrogenic) exotoxins are proteins responsible for the rash of scarlet fever. There are three antigenically distinct types designated A, B, and C, which are produced by more than 95% of group A streptococcal strains.
DNAse is using for identifying immunologically distinct deoxyribonucleases (A through D). These enzymes are not cytolytic but are capable of depolymerizing free DNA present in pus. This reduces the viscosity of the abscess material and facilitates spread of the organisms.
Other enzymes such as hyaluronidase and streptokinase (fibrinolysin).
Diseases caused by Streptococci Group A (Streptococcus pyogenes).
Pharyngitis. Group A streptococcus is the major cause of bacterial pharyngitis, with group C and G occationally involved. This is primarily a disease of children between the ages of 5 to 15 years, but infants and adults are also susceptible. The pathogen is spread by person-to-person contact via respiratory droplets. Pharyngitis generally develops within 2 to 4 days after exposure to the pathogen, with an abrupt onset of sore throat, fever, malaise, and headache. The posterior pharynx can appear erythematous with an exudate, and cervical lymphadenopathy can be prominant.
Scarlet fever is a complication of streptococcal pharyngitis seen when the infecting strain is lysogenized by a temperate bacteriophage that stimulates production of erythrogenic toxin. Within 1 to 2 days after initial clinical symptoms of pharyngitis, a diffuse erythematous rash will initially appear on the upper chest and then spread to the extremities. The area around the mouth is generally spared, as are the palms and soles. The tongue will initially be covered with a yellowish-white coating that will later be shed revealing a red, raw surface (“strawberry tongue”).
Erysipelas. This disease can affect all age groups. İt is a disease of the skin and subcutaneous tissues usually occurring on the face or lower extremities and characterized by a fiery red, advancing erythema.
Puerperal sepsis. This infection is initiated during or following soon after the delivery of a newborn. It can occur due to exogenous transmission (for example, by nasal droplets from an infected carrier, or from contaminated instruments), or endogenously, from the patient’s vaginal flora. It is a disease of the uterine endometrium in which patients suffer from a purulent vaginal discharge.
Acute rheumatic fever occurs most commonly among young children during the fall and winter, and can occur only when preceded by pharyngitis caused by any of group A streptococcal serotypes. The disease can occur in 0.1-3% of untreated patients from 1 to 5 weeks after pharyngial onset. Although the mechanism of pathogenesis is not entirely understood, a plausible hypothesis is based upon the presence of cross-reactive epitopes among M proteins and target tissues, including human cardiac tissue, and the binding of anti-M protein antibody to the tissue epitopes. The major clinical manifestations are carditis, polyarthritis, and subcutaneous nodules.
Acute hemorrhagic glomerulonephritis occurs most commonly in children and can result when preceded by pharyngitis or skin disease caused by goup A streptococci. This disease can occur in less then 1 to 15% of untreated patients from 1 to 5 weeks after pharyngial or skin disease onset. Evidence supports the concept that renal damage is the result of immune complex diposition on the glomerular basement membrane. The major clinical manifestations are renal glomerular damage, hypertension, edema, proteinuria, and hematuria.
Other diseases are impetigo, cellulitis, lymphangitis.
Laboratory Diagnosis
The specimens obtained depend on the disease process and include nose and throat swabs, lesion material, pus, sputum, blood for culture and immunoserology, urine, and spinal fluid.
For isolation and identification of group A streptococci initial cultivation on blood agar or specialized selective agar are required. Overnight incubation anaerobically or under aerobic conditions in the presence of 10% carbon dioxide at 37ºC is optimal for isolation of the organism. The organism may be identified as beta-hemolytic, gram positive, catalese negative coccus, and is inhibited by bacitracin.
Immunoserologic test. Both the anti-streptolysin O (ASO) and anti-DNAse B assays are useful in diagnosis.
Antistreptolysin O titre
Antistreptolysin O titre (AS(L)O titre or AS(L)OT) - titre of (serum) antistreptolysin O antibodies is a blood test used to assist in the diagnosis of a streptococcal infection or indicate a past exposure to streptococci. The ASOT helps direct the antimicrobial treatment and is used to assist in the diagnosis of scarlet fever, rheumatic fever and post infectious glomerulonephritis.
Treatment
Adequate drainage, debridement, and antibiotic therapy are essential for the treatment of localized, suppurative skin lesions. Penicillin is the drug of choice for acute diseases. Penicillin has no effect upon established rheumatic heart disease and acute hemorrhagic glomerulonephritis. Penicillin resistant strains have not been reported. Erythromycin is the drug of choice for penicillin allergic patients.
Group B Streptococci
Group B streptococci (S. agalactiae) are harbored in the female genital tract and male urethra of 15-25% of humans and animals, as well as in the pharynx and genital tract. The organism is transmitted from an infected mother to her infant in utero or at birth. Group B streptococci are encapsulated. These organisms far outnumber E. coli K1 as the leading cause of neonatal meningitis during the first 4 months of life. The antiphagocytic properties of the capsular polysaccharide allow the organisms to survivei multiply, invade epithelial cells, and induce an acute inflammatory response. In adults, the organisms may produce pneumoniae, septicemia, prosthetic joint disease, or puerperal sepsis originating from the female genital tract.
Specimens for laboratory diagnosis depend on the disease process and include blood for culture, sputum, a cervical swab, and spinal fluid. These specimens are cultured on blood agar and incubated aerobically at 37ºC. Group B streptococci are beta-hemolytic, gram-positive, catalase negative cocci. They are only streptococci in which ability to hydrolyze hippurate and positive CAMP test.
Early therapy with penicillin plus an aminoglycoside is essential for the prevention of progressive, fatal disease. Heavily colonized mothers can be treated with penicillin intrapartum to prevent subsequent colonization of their newborns.
The viridans group of Streptococci
The viridans group often reffered to as “oral streptococci” do not contain C carbonhydrate but have been grouped based on rRNA cataloging and nucleic acid hybridization studies. These organisms are normal inhabitants of the oral, respiratory, and gastrointestinal mucosa of humans and animals. They are opportunistic pathogens and have generally been thought to be of low virulence. Viridans streptococci are, however, the major etiologic agents of bacterial endocarditis. Patients who develop streptococcal endocarditis usually possess a previously damaged heart valve (from previous rheumatic fever and other cause). Gingival disease or dental manipulations, including dental prophylaxis, are often predisposing factors in the development of endocarditis.
Viridans streptococci are able to adhere to epithelial and endothelial cells, and adherence is probably a key factor in their ability to cause disease. Streptococcus mutans has been definitively established as a major etiologic agent of dental caries in addition to being a cause of endocarditis. Extracellular sugars, called dextrans, serve as attachment mediators for tooth surfaces as well as heart valves. Specimens for laboratory diagnosis depend on the disease process and include blood for culture and urine. These specimens are cultured on blood agar and incubated aerobically at 37ºC. The species of the viridans group are alfa or gamma hemolytic, gram-positive, catalase negative cocci that are not inhibited by optochin. Although treatment with penicillin is effective, the occurence of penicillin resistant strains necessitate the use of penicillin plus an aminoglycoside.
Streptococcus pneumoniae
S. pneumoniae are gram-positive, nonmotile, encapsulated cocci. They are lancet-shaped, and their tendency to occur in pairs accounts for their earlier designation as Diplococcus pneumoniae. S. pneumoniae is the most common cause of pneumonia and otitis media, and is an important cause of meningitis and bacteremia. The risk of disease is highest among young children, older adults, smokers, and persons with certain chronic illnesses. Like other streptpcocci, S. pneumoniae is fastidious and is routinely cultured on blood agar and releases an α-hemolysin. This bacterium is an obligate parasite of humans and harbored in the nasopharynxs of 25-70% of the population. Pneumococci are very sensitive to enviromental, physical, and chemical agents. Antiseptic agents kill them rapidly.
The polysaccharide capsule is the sole basis for classification and the only known virulence factor. Distinct epitopes enable the recognition of more than 85 serotypes of pneumococci, 23 of which are responsible for greater than 85% of pneumococcal disease. The capsule inhibits phagocytosis and thus allows the organisms to establish themselves in host tissue, multiply, and produce disease.
Disease of
S. pneumoniae
S. pneumoniae is the most common cause of lobar and lobular (broncho) pneumoniae. This organism also is the most common cause of meningitis among adults and a major cause of otitis media and sinusitis among children. Most infections are caused by endogenous spread from the colonized nasopharynx or oropharynx to distal site (e.g., lungs, sinuses, ears, blood, and meninges). Colonization is highest in young children. Person-to-person spread through infectious droplets is rare.
Laboratory Diagnosis of Streptococcal pneumoniae
Specimens obtained depend on the disease process and include a nasopharyngeal swab, sputum (which may be rusty), blood for culture, spinal fluid, and pus. Gram stain of pus, sputum, and spinal fluid often shows gram-positive, lancet shaped, diplococci and numerous polymorphonuclear leukocytes.
Primary isolation and identification require initial cultivation on blood agar or in blood culture broth. Overnight incubation under aerobic conditions at 37ºC is optimal for isolation of the organism. The organism may be identified as α-hemolytic, gram-positive, catalase negative, coccus that is bile soluble and inhibited by optochin. Rapid identification of pneumococcal serotypes in spinal fluid can be accomplished by latex agglutination utilizing serotype-specific anticapsular antibody for the detection of capsular polysaccharide.
Treatment of Streptococcal pneumoniae
Although the penicillin is still the drug of choice, multiple-resistant strains are now appearing. Cephalosporins, erythromycin, chloramphenicol, vancomycin are used for patients allergic to penicillin or for treatment of penicillin-resistant strains.
