Infection with Streptococcus agalactiae (group B streptococci)

A study to identify the causative agent of streptococcal infection (Streptococcus pyogenes), during which the genetic material (DNA) of streptococcus in a sample of biomaterial is determined using the real-time polymerase chain reaction method.

Synonyms Russian

Streptococcus pyogenes, group A beta-hemolytic streptococcus, GABHS, group A beta-hemolytic streptococcus, β-HSA [real-time polymerase chain reaction, real-time PCR].

English synonyms

S. pyogenes, Group A streptococcus, GAS, β-hemolytic streptococcus group A, β-hemolytic streptococcus group A.

Research method

Real-time polymerase chain reaction.

What biomaterial can be used for research?

A swab from the pharynx (oropharynx), a swab from the nasopharynx, a urogenital scraping.

General information about the study

S. pyogenes is a Gram-positive, spherical bacterium that is the causative agent of acute pharyngitis and impetigo, as well as a spectrum of more severe destructive diseases such as pneumonia, sepsis and septic arthritis, toxic shock syndrome, cellulitis and necrotizing fasciitis.

Traditionally, to confirm the diagnosis of streptococcal infection, bacteriological seeding of biomaterial on special media is used. The biomaterial can be discharge from the tonsils or nasopharynx (if acute pharyngitis is suspected), or sputum (for pneumonia). As a rule, the result can be obtained only after a few days, which is associated with a delay in making an accurate diagnosis and prescribing specific treatment. However, it has been revealed that timely diagnosis and specific treatment not only reduce the duration of the disease and the intensity of symptoms, but also reduce the risk of developing complications such as peritonsillar abscess, otitis media, acute rheumatic fever and post-streptococcal glomerulonephritis (for acute pharyngitis), streptococcal sepsis (for pneumonia ), joint destruction and disability (with septic arthritis). Empiric therapy with penicillin antibiotics is based on the premise that S. pyogenes is the main bacterial causative agent of acute pharyngitis (5-15% of cases in adults and 20-30% in children). It should be noted that acute pharyngitis can be caused by other bacterial or viral agents, for which the appointment of such empirical therapy is not only not justified, but is also dangerous due to the development of resistant strains of microorganisms. Such diagnostic difficulties can be overcome using RT-PCR - polymerase chain reaction - this method allows you to get an accurate result in a shorter time.

The RT-PCR reaction uses specific primers for the S. pyogenes DNA fragment. This feature makes it possible to identify only pyogenic streptococcus, and not other streptococci related to it - representatives of the normal microflora of the pharynx (Streptococcus mutans, Streptococcus viridians). In terms of specificity, RT-PCR is not inferior to bacteriological culture and is significantly superior to other methods of identifying the pathogen (for example, a rapid test for determining the antigen).

Quite often, biomaterial is taken for research during the treatment of the disease. As a result, the sensitivity of diagnostic methods decreases and the likelihood of obtaining a false negative result is higher. In such a situation, the RT-PCR method has some advantages over other types of diagnostics. This is explained by the fact that to identify the pathogen in the reaction, the genetic material of the pathogen is used - both the genomic DNA of a living microorganism and DNA fragments of a bacterium that has undergone lysis. In this case, it becomes possible to identify not only living, actively reproducing microorganisms, but also bacteria killed as a result of treatment. Unlike RT-PCR, a prerequisite for other diagnostic methods (enzyme-linked immunosorbent assay or bacteriological culture) is the presence of living microorganisms. Therefore, if the biomaterial is given during treatment, the RT-PCR method should be preferred to other diagnostic methods.

The high sensitivity of RT-PCR allows this method to be used in the diagnosis of complications of acute pharyngitis: acute rheumatic fever and post-streptococcal glomerulonephritis. Complications of acute pharyngitis are autoimmune diseases in which S. pyogenes - associated pharyngitis is a causative factor in the pathological immune response, but at the time of diagnosis of these complications it has already resolved (on its own or as a result of treatment). Therefore, it is possible to identify the pathogen through bacteriological culture of tonsil and nasopharynx discharge in patients with complications of acute pharyngitis only in a small percentage of cases. On the other hand, proof of previous S. pyogenes infection is a necessary criterion for diagnosis, and also greatly facilitates the differential diagnosis of diseases. The RT-PCR method is characterized by high sensitivity and therefore can be used in the diagnosis of complications of acute pharyngitis.

The detection of S. pyogenes in so-called sterile environments is always a pathological sign. On the other hand, the detection of this microorganism in non-sterile environments (sputum, nasopharyngeal discharge) does not always indicate the presence of a disease. It was found that about 12-20% of school-age children and 2.4-3.7% of adults are asymptomatic carriers of S. pyogenes. In such cases, the interpretation of a positive test result should be assessed taking into account the bacterial load and in combination with some other clinical and laboratory signs. Typically, asymptomatic carriage is characterized by fewer bacteria compared to active infection. Therefore, the finding of a high bacterial load of S. pyogenes in a throat swab from a patient with fever, sore throat, and painful regional lymphadenitis confirms the diagnosis of acute streptococcal pharyngitis. And, conversely, the detection of a low bacterial load of this microorganism in a throat smear in the absence of complaints and clinical picture should be regarded as asymptomatic carriage. RT-PCR is a semi-quantitative method that allows an indirect assessment of bacterial load. Therefore, it turns out to be especially useful when examining “healthy carriers” of streptococcus. Asymptomatic carriage of S. pyogenes should be suspected in a patient when streptococcus is detected after an adequate course of antibiotic therapy, as well as in family members of a patient with frequent exacerbations of streptococcal pharyngitis.

What is the research used for?

For diagnostics:

• acute pharyngitis;
• acute rheumatic fever and post-streptococcal pharyngitis in patients with or without a history of an episode of acute pharyngitis;
• asymptomatic carriage of S. pyogenes in a patient with frequent relapses of pharyngitis;
• asymptomatic carriage of S. pyogenes in family members of a patient with frequent relapses of streptococcal pharyngitis;
• community-acquired pneumonia;
• septic arthritis.

When is the study scheduled?

• For symptoms of acute pharyngitis: pain when swallowing, swelling and erythema of the pharynx mucosa, purulent discharge from the surface of the tonsils, painful regional lymphadenitis;
• for symptoms of acute rheumatic fever: migratory polyarthritis or arthralgia, a feeling of interruptions in the heart, pain in the heart, unmotivated weakness, ring-shaped erythema and subcutaneous nodules, as well as neurological symptoms in the form of choreic hyperkinesis;
• for symptoms of post-streptococcal glomerulonephritis: edema (periorbital or generalized), gross hematuria, unmotivated weakness, proteinuria less than 3.5 g/day, arterial hypertension;
• when examining a patient with recurrent pharyngitis;
• when examining family members of a patient with recurrent pharyngitis;
• when examining family members of a patient with frequent relapses of acute streptococcal pharyngitis;
• in the presence of a risk factor for S. pyogenes (associated pneumonia) such as the H1N1 influenza virus;
• with symptoms of community-acquired pneumonia: sudden onset of illness, fever, shortness of breath, chest pain, cough with purulent sputum;
• in the presence of risk factors for septic arthritis: infancy and old age, immunosuppressive therapy, somatic diseases, hemodialysis;
• for symptoms of septic arthritis: pain in the joint at rest and with movement, erythema of the skin over the joint area, impaired mobility in the joint, increased body temperature and unmotivated weakness.

What do the results mean?

Reference values: negative.

Reasons for a positive result

In the discharge of the nasopharynx and tonsils:

• acute pharyngitis;
• acute rheumatic fever;
• poststreptococcal glomerulonephritis.

In sputum:

• pneumonia;
• acute bronchitis, smoker's bronchitis.

What can influence the result?

• The use of penicillin antibacterial drugs (amoxiclav, ampicillin), cephalosporins (cefixime, ceftibuten) and macrolides (azithromycin, clarithromycin) before taking the material for analysis can lead to a negative result.

Infection with Streptococcus agalactiae (group B streptococci)

Serogroup B streptococci (SGB) have long been known as causative agents of mastitis in cattle and small ruminants and are called Streptococcus agalactiae. Previously, until 1937 inclusive, this microorganism was considered as a commensal of the human body. Since the 60s, this species, as a human pathogen, has occasionally been isolated from a variety of nosological forms of infection: endocarditis, pyelonephritis, meningitis, pneumonia, abscess, peritonitis, obstetric sepsis, neonatal sepsis, pharyngitis, etc. During 1970–1980 GBS is becoming one of the common causative agents of purulent-septic infections in newborns and mothers in the USA and Western Europe. In the early 90s, the first publications about the role of this type of streptococcus in the pathology of pregnancy and in purulent-inflammatory diseases of newborns also appeared in our country. To date, there has been no official registration of diseases caused by this microorganism in the Russian Federation.

Streptococci of this species, according to modern nomenclature according to the Bergay key, are included in the genus Streptococcus of the family Sreptococcaeae, species S.agalactiae. The modern classification of streptococci is based on the division into serological groups according to the Lensfield classification. S.agalactiae are classified as serogroup B and are its only representatives. GBS is a heterogeneous population consisting of a number of serotypes and their combinations. Currently, 9 serotypes have been identified, designated by Roman numerals: I, II, III, IV, V, VI, VII, VIII. Type I is divided into subtypes Ia and Ib.

The pathogen's habitat is the human body, large and small livestock, the possibility of its transmission from animals to humans and vice versa has not been proven. Localization of GBS in humans is the gastrointestinal tract (large intestine), vagina, urethra, sometimes the microorganism can colonize the pharynx. Pathogenicity factors: ability for adhesion and invasion, resistance to phagocytosis, hemolysin, hyaluronidase, peptidase (impairs the chemotaxis of polymorphonuclear leukocytes to the site of GBS infection).

GBS can cause severe infections in people. The most sensitive to them are newborns, pregnant women, diabetics, chronically ill people, children with dystrophy, the elderly, and people with reduced immunity. Spontaneous abortions, premature births, chorioamnionitis, pyelonephritis, urinary tract infections, endocarditis, sepsis, as well as complications after cesarean section, meningitis, mastitis and endometritis in postpartum women may be associated with GBS. In newborns, GBS mainly causes sepsis, meningitis and pneumonia. Transmission of the pathogen to a newborn can occur intranatally and postnatally. According to foreign sources, in recent years there has been a relative increase in the number of diseases caused by GBS in elderly people against the background of a slight decrease in the incidence among newborns. The observed phenomenon is associated, on the one hand, with the active prevention of this infection in maternity hospitals, and on the other, with an increase in the number of elderly patients with reduced immunity. In the latter, GBS usually causes infection of the skin and bones, sepsis without an obvious source of origin, urosepsis, pneumonia and peritonitis. Often the listed infections occur repeatedly in such patients.

An important role in the development of GBS infections in newborns is played by factors predisposing to the disease, the so-called “risk factors”, which can be divided into two groups:

• associated with the characteristics of childbirth or colonization of mothers with GBS;
• directly related to newborns.

The first group includes the presence of GBS on the cervical mucosa in pregnant women, the massiveness of colonization (isolation of the pathogen simultaneously from several examined loci), cases of GBS infections of children in the anamnesis of women in labor, premature birth, a long anhydrous period, a rise in temperature during childbirth, chorioamnionitis , bacteriuria caused by GBS.

The second group includes the fact and massive colonization of newborns with GBS, prematurity and low birth weight, birth by cesarean section, intrauterine instrumental examination of the fetus, lasting more than 12 hours.

Penicillin remains the most effective and common drug for treating streptococcal infections. Along with streptococci of serogroup A, GBS retain their sensitivity to this antibiotic, although in a small number of cases GBS strains show resistance to penicillin G. In addition to penicillin, streptococci have been shown to be highly sensitive to ampicillin, erythromycin, clindamycin, lincomycin, oxacillin and meroponem. The first three generations of cephalosprins, with the exception of cefoxitin and moxalactam, have a wider spectrum of activity compared to penicillin. High sensitivity of GBS to a number of relatively new drugs with a wide spectrum of activity was also noted: thienamycin, mezlocillin, azlocillin and piperacillin. GBS strains are often resistant to aminoglycoses, nalidixic acid, tetracycline, chloramphenicol, bacitracin, trimethoprim and metromnidazole. However, with the combined use of aminoglycosides with penicillin or ampicillin, experimental studies on laboratory animals revealed a synergistic effect of such combinations. In recent years, reports have appeared in foreign literature about the isolation of GBS strains resistant to beta-lactam antibiotics. The nature of this phenomenon is associated with the appearance in the cells of microorganisms of genes responsible for the synthesis of penicillin binding proteins (PBPs). According to a number of foreign researchers, the number of GBS strains resistant to erythromycin can range from 5.9 to 32.0% and the number of strains resistant to clindamycin from 6.9 to 14.3%. The use of therapeutic bacteriophages for the prevention and treatment of GBS infection is currently impossible due to the lack of active specific bacteriophages.

Indications for examination.

The birth of a child with a history of GBS infection in the mother, GBS bacteriuria during this pregnancy, the threat of premature birth, a rise in temperature during labor (≥ 38°C), an anhydrous period lasting more than 18 hours.

Material for research.

Blood, CSF, sputum, fetal fluid, joint fluid, fragments of affected tissue, oropharyngeal smears, smears/scrapings from the vaginal mucosa, rectal smears.

Etiological diagnosis includes

isolation of the pathogen and identification of its DNA.

Comparative characteristics of laboratory diagnostic methods.

Isolation of the pathogen in the diagnosis of GBS infections is the “gold standard” and represents an important step in a series of diagnostic measures. A standard culture using, for example, 5% blood agar is used. Timely isolation of the pathogen and determination of sensitivity to antibiotics largely determines the effectiveness of treatment and prevention of this disease.

If an infection is suspected, a variety of biological material is examined. In case of determination of GBS carriage in pregnant women, smears from the mucous membrane of the vagina and rectum are examined at 35–37 weeks. pregnancy. If it is not possible to sow immediately, it is possible to use transport media. The time required for isolation and identification of GBS usually ranges from 4 to 5 days.

Identification of a specific DNA fragment of S.agalactiae using the PCR method is carried out during the study of various biological materials. Scrapings of epithelial cells from the lateral walls of the vagina, perianal smears at 34–36 weeks of pregnancy are examined to detect colonization of the vagina and/or rectum with S.agalactiae; blood, CSF of a newborn - for the diagnosis of septic conditions; oropharyngeal smears - in newborns with clinical symptoms of pneumonia to confirm the diagnosis. The advantage of this study is that it allows testing to be completed within a few hours and thereby speeds up clinical decision-making for the prevention and treatment of the disease. The diagnostic sensitivity of the study is 81%, diagnostic specificity is 97.6%. Detection of S.agalactiae DNA by PCR can be performed in qualitative and quantitative format. The quantitative format of the test allows the study to be used to assess the degree of GBS colonization of a selected locus. However, detection of a specific DNA fragment of S.agalactiae by PCR does not allow identifying viable microorganisms and, accordingly, determining their sensitivity to antibiotics.