Laboratory diagnosis, Treatment and Prevention of Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) is a Gram-positive, lancet-shaped bacterium that causes various infections, such as pneumonia, meningitis, otitis media, sinusitis, and bacteremia. Laboratory diagnosis of pneumococcal infection is important for timely and appropriate treatment and prevention.

The main methods for laboratory diagnosis of Streptococcus pneumoniae are:

• Microscopy: This involves examining the specimen under a microscope after staining with Gram stain or other special stains. Gram staining of sputum, cerebrospinal fluid (CSF), or other body fluids can show lancet-shaped Gram-positive cocci in pairs or chains . This can provide a presumptive diagnosis of pneumococcal infection, but it is not specific enough to differentiate it from other streptococci or enterococci. A more specific test is the quellung reaction, which involves mixing fresh emulsified sputum with antiserum against pneumococcal capsular polysaccharide. This causes capsule swelling and increased refractility of the bacteria under the microscope . The quellung reaction can also be used to identify the serotype of pneumococcus by using specific antisera.
• Culture: This involves growing the bacteria on suitable media and observing their colony morphology and hemolytic pattern. Sputum, blood, CSF, or other specimens are plated on blood agar and incubated at 37°C in the presence of 5–10% carbon dioxide. After overnight incubation, gray colonies with alpha-hemolysis (partial hemolysis) are observed . These colonies can be further identified by performing biochemical tests, such as optochin sensitivity test, bile solubility test, and inulin fermentation test . Optochin sensitivity test involves placing a disk impregnated with optochin (ethylhydrocupreine dihydrochloride) on a blood agar plate inoculated with the isolate. A zone of inhibition around the disk indicates sensitivity to optochin and presumptive identification of pneumococcus . Bile solubility test involves adding a drop of bile or sodium deoxycholate to an isolated colony and observing for lysis. Pneumococci are lysed rapidly by bile due to the presence of an autolytic enzyme that breaks down their cell wall . Inulin fermentation test involves inoculating the isolate into a broth containing inulin (a polysaccharide) and observing for acid production. Pneumococci can ferment inulin and produce acid, unlike other streptococci .
• Animal inoculation: This involves injecting the specimen into mice and observing for signs of infection and death. Sputum or blood specimens containing few pneumococci can be isolated by intraperitoneal inoculation in mice. Pneumococci can be demonstrated in the peritoneal exudate and heart blood of the mice, which die 1–3 days after inoculation . This method is rarely used nowadays due to ethical and practical issues.
• Antigen detection: This involves detecting pneumococcal antigens in body fluids using immunological methods. Pneumococcal C polysaccharide is excreted in urine and can be detected using a commercially available immunoassay . This test can be useful for diagnosing pneumococcal pneumonia in adults, especially when sputum culture is negative or unavailable. The counterimmunoelectrophoresis (CIEP) is another test that can detect pneumococcal capsular polysaccharide antigen in CSF, blood, or urine for diagnosis of meningitis, bacteremia, or pneumonia . Latex agglutination test using latex particles coated with anti-CRP antibody can detect C-reactive protein (CRP), which is an acute phase protein produced by the liver in response to inflammation. CRP can be used as a prognostic marker in acute cases of pneumococcal pneumonia and other infectious diseases.
• Antibody detection: This involves measuring specific antibodies against pneumococcus in serum using serological methods. The indirect hemagglutination, indirect fluorescent antibody test, and enzyme-linked immunosorbent assay (ELISA) are some of the methods that can demonstrate specific pneumococcal antibodies in invasive pneumococcal diseases . These tests can be useful for epidemiological studies and evaluation of vaccine efficacy.
• Nucleic acid-based tests: These involve amplifying and detecting pneumococcal DNA or RNA in specimens using molecular methods. Nucleic acid probes and polymerase chain reaction (PCR) assays are some of the methods that can identify S. pneumoniae isolates in culture or directly from clinical specimens . These tests are more sensitive and specific than conventional methods and can also provide information on serotype, genotype, and antibiotic resistance.

These are some of the main methods for laboratory diagnosis of Streptococcus pneumoniae infection. Depending on the type and source of specimen, one or more methods may be used to confirm the diagnosis and guide the treatment.

The type and quality of the specimen collected for laboratory diagnosis of Streptococcus pneumoniae are crucial for accurate and reliable results. The specimen should be obtained from the site of infection and transported to the laboratory as soon as possible. The specimen should also be collected before the initiation of antibiotic therapy, as antibiotics can inhibit the growth and detection of pneumococci.

Depending on the type of infection, different specimens can be collected for diagnosis of S. pneumoniae. Some of the common specimens and their collection methods are:

• Sputum: Sputum is the most common specimen for diagnosis of pneumococcal pneumonia. It should be collected by deep coughing or expectoration into a sterile container. The sputum should be purulent and free of saliva or oral contaminants. If the patient cannot produce sputum, induced sputum can be obtained by inhalation of hypertonic saline or bronchodilators.
• Blood: Blood is another common specimen for diagnosis of pneumococcal bacteremia or sepsis. It should be collected by venipuncture into sterile blood culture bottles containing appropriate media and anticoagulants. At least two sets of blood cultures should be obtained from different sites and at different times to increase the yield and avoid contamination.
• Endotracheal aspirate: Endotracheal aspirate is a specimen obtained from patients who are intubated or mechanically ventilated. It is collected by suctioning the lower respiratory tract through the endotracheal tube using a sterile catheter and syringe. The aspirate should be transferred to a sterile container and transported to the laboratory promptly.
• Bronchoalveolar lavage: Bronchoalveolar lavage is a specimen obtained from patients who have diffuse pulmonary infiltrates or who are immunocompromised. It is collected by bronchoscopy, which involves inserting a flexible tube with a camera and a washing device into the lungs. A sterile saline solution is instilled into a segment of the lung and then aspirated back along with cellular and microbial material. The lavage fluid should be transferred to a sterile container and transported to the laboratory promptly.
• Cerebrospinal fluid (CSF): CSF is the most important specimen for diagnosis of pneumococcal meningitis. It should be collected by lumbar puncture, which involves inserting a needle into the lower back and withdrawing fluid from the subarachnoid space. The CSF should be transferred to sterile tubes and transported to the laboratory promptly. The CSF should also be examined for cell count, glucose, protein, and gram stain.
• Pleural fluid: Pleural fluid is a specimen obtained from patients who have pleural effusion, which is an accumulation of fluid in the pleural space between the lungs and the chest wall. It should be collected by thoracentesis, which involves inserting a needle into the pleural space and withdrawing fluid using a syringe. The pleural fluid should be transferred to sterile tubes and transported to the laboratory promptly. The pleural fluid should also be examined for cell count, glucose, protein, pH, lactate dehydrogenase (LDH), and gram stain.
• Joint fluid: Joint fluid is a specimen obtained from patients who have septic arthritis, which is an infection of the joint space. It should be collected by arthrocentesis, which involves inserting a needle into the joint space and withdrawing fluid using a syringe. The joint fluid should be transferred to sterile tubes and transported to the laboratory promptly. The joint fluid should also be examined for cell count, glucose, protein, LDH, and gram stain.
• Abscess fluid: Abscess fluid is a specimen obtained from patients who have localized collections of pus in various organs or tissues. It should be collected by aspiration or drainage using a sterile needle or catheter. The abscess fluid should be transferred to sterile tubes and transported to the laboratory promptly. The abscess fluid should also be examined for cell count, glucose, protein, LDH, and gram stain.
• Bones and other biopsy material: Bones and other biopsy material are specimens obtained from patients who have osteomyelitis or other invasive pneumococcal infections involving solid tissues. They should be collected by surgical excision or biopsy using sterile instruments. The tissue specimens should be transferred to sterile containers with appropriate transport media and transported to the laboratory promptly.

These are some of the common specimens and their collection methods for laboratory diagnosis of S. pneumoniae. Other specimens, such as urine, middle ear fluid, peritoneal fluid, pericardial fluid, etc., can also be collected depending on the clinical presentation and suspicion of pneumococcal infection.

Microscopy is a useful technique for the preliminary identification of S. pneumoniae from clinical specimens or culture isolates. It involves staining the bacteria with a dye and observing their morphology and arrangement under a microscope.

One of the most common microscopy techniques is Gram staining, which differentiates bacteria based on the structure of their cell wall. S. pneumoniae are Gram-positive cocci, meaning they have a thick peptidoglycan layer that retains the purple dye. They typically appear as lancet-shaped diplococci (pairs of oval-shaped cells) or chains of cocci . If observed by experienced microscopists, these features have high specificity for the presence of S. pneumoniae and should be reported as such.

Another microscopy technique is capsule staining, which detects the presence of a polysaccharide capsule around the bacterial cells. The capsule is an important virulence factor of S. pneumoniae that protects it from phagocytosis and complement-mediated lysis. Capsule staining can be done by mixing fresh emulsified sputum with antiserum against S. pneumoniae and observing the capsule swelling (the quellung reaction) under a microscope . This technique can also help to identify the serotype of S. pneumoniae based on the specificity of the antiserum.

Microscopy techniques are rapid and inexpensive methods for the identification of S. pneumoniae, but they have some limitations. They require skilled personnel, adequate specimen quality, and sufficient bacterial load to be reliable. They also cannot distinguish S. pneumoniae from other closely related streptococci that may share similar morphology and capsule production. Therefore, microscopy techniques should be confirmed by other methods such as culture, antigen detection, or nucleic acid-based tests.

Culture methods are widely used for the isolation and identification of Streptococcus pneumoniae from clinical specimens. Culture methods involve the following steps:

• Specimen collection and types: The most common specimens for culture are sputum, blood, cerebrospinal fluid (CSF), pleural fluid, joint fluid, abscess fluid, bones, and other biopsy material. The specimens should be collected aseptically and transported to the laboratory as soon as possible. Sputum specimens should be checked for quality by Gram stain to exclude saliva or oral flora contamination.
• Inoculation and incubation: The specimens are inoculated onto blood agar plates and incubated at 37°C in the presence of 5–10% carbon dioxide for 18 to 24 hours. Blood agar is an enriched medium that supports the growth of fastidious bacteria and also allows the detection of hemolytic properties .
• Colony morphology and hemolysis: Streptococcus pneumoniae typically produces gray, mucoid colonies with a depression in the center (umbilicated) on blood agar. The colonies are surrounded by a zone of alpha-hemolysis, which appears as a greenish discoloration of the agar due to partial lysis of red blood cells . Some strains may also show double-zone hemolysis, which consists of a narrow inner zone of beta-hemolysis (complete lysis) and a wider outer zone of alpha-hemolysis.

• Identification tests: Several phenotypic tests are used to confirm the identity of Streptococcus pneumoniae from culture. These include:

  • Catalase test: Streptococcus pneumoniae is catalase-negative, which means it does not produce bubbles when exposed to hydrogen peroxide. This test helps to differentiate it from other Gram-positive cocci that are catalase-positive, such as staphylococci .
  • Optochin sensitivity test: Streptococcus pneumoniae is sensitive to optochin (ethylhydrocupreine dihydrochloride), which inhibits its growth. A disk saturated with optochin is placed on a blood agar plate inoculated with the isolate. A zone of inhibited bacterial growth around the disk indicates a positive result .
  • Bile solubility test: Streptococcus pneumoniae produces an autolytic enzyme (amidase) that breaks down its cell wall when exposed to bile salts. A drop of bile is added to an isolated colony on a blood agar plate. The colony dissolves or becomes transparent if it is bile-soluble .
  • Inulin fermentation test: Streptococcus pneumoniae can ferment inulin, a polysaccharide, and produce acid. This test helps to differentiate it from other alpha-hemolytic streptococci that cannot ferment inulin. A broth medium containing inulin and a pH indicator is inoculated with the isolate. A color change from purple to yellow indicates a positive result .

Culture methods are reliable and inexpensive for the identification of Streptococcus pneumoniae, but they have some limitations, such as:

• They require viable organisms and adequate specimen quality.
• They may take several days to provide results.
• They may be affected by prior antibiotic therapy or mixed infections.
• They may miss some non-culturable or atypical strains.

Therefore, culture methods may be supplemented by other methods, such as antigen detection, antibody detection, or nucleic acid-based tests, for rapid and accurate diagnosis of pneumococcal infections .

There are several tests that can be used to identify S. pneumoniae from culture isolates or clinical specimens. These tests are based on the biochemical, antigenic, or genetic characteristics of the bacterium. Some of the commonly used tests are:

• Optochin sensitivity test: S. pneumoniae is sensitive to optochin (ethylhydrocupreine dihydrochloride), a chemical that inhibits its growth. A disk saturated with optochin is placed on a blood agar plate inoculated with the isolate. A zone of inhibition around the disk indicates a positive result .
• Bile solubility test: S. pneumoniae produces an enzyme called amidase that breaks down its cell wall when exposed to bile salts. A drop of bile is added to a colony of the isolate on a blood agar plate. The colony dissolves or becomes transparent if it is S. pneumoniae .
• Inulin fermentation test: S. pneumoniae can ferment inulin, a polysaccharide, and produce acid. A broth medium containing inulin and a pH indicator is inoculated with the isolate. A color change from purple to yellow indicates a positive result .
• Antigen detection tests: S. pneumoniae produces a polysaccharide capsule that can be detected by immunological methods. The most common antigen detection test is the latex agglutination test, which uses latex particles coated with antibodies against the pneumococcal C polysaccharide antigen. The test can be performed on urine, cerebrospinal fluid (CSF), or other body fluids .
• Nucleic acid-based tests: S. pneumoniae can be identified by amplifying specific DNA sequences using polymerase chain reaction (PCR) or hybridization probes. These tests can be performed on culture isolates or clinical specimens, such as blood, CSF, or respiratory samples .

Sometimes, clinical specimens may contain very few pneumococci or may be contaminated with other bacteria. In such cases, animal inoculation can be used to isolate S. pneumoniae from the specimens. This method involves injecting the specimen into the peritoneal cavity of a mouse and observing the mouse for signs of infection and death. If pneumococci are present in the specimen, they will multiply in the mouse and cause peritonitis and septicemia. The mouse will usually die within 1 to 3 days after inoculation. The peritoneal exudate and heart blood of the mouse can then be collected and examined for the presence of pneumococci by microscopy and culture.

Animal inoculation is a sensitive and specific method for isolating S. pneumoniae from specimens such as sputum, blood, CSF, pleural fluid, and joint fluid. However, it is not routinely used in clinical laboratories because it is time-consuming, expensive, and requires special facilities and ethical approval. It is mainly used for research purposes or when other methods fail to identify the organism. Animal inoculation can also be used to test the virulence of pneumococcal strains by comparing their ability to cause disease and death in mice.

Antigen detection methods are based on the identification of pneumococcal capsular polysaccharide antigens (PCAs) in body fluids, such as urine, cerebrospinal fluid (CSF), blood, or sputum. These methods are rapid, simple, and useful for diagnosis of pneumococcal infections, especially when culture results are negative or unavailable.

There are different types of antigen detection methods available, such as:

• Lateral flow immunochromatography tests (ICTs): These are commercially available kits that use monoclonal antibodies to detect PCAs in urine or CSF samples. The test result is indicated by a colored line on a test strip. The ICTs can detect 13 common serotypes of S. pneumoniae and have a high sensitivity and specificity for pneumococcal meningitis. However, they may have a lower sensitivity for pneumococcal pneumonia and may cross-react with some non-pneumococcal streptococci .

• Counterimmunoelectrophoresis (CIE): This is a laboratory technique that uses an electric field to separate antigens and antibodies in a gel matrix. The CIE can detect PCAs in CSF, blood, or sputum samples and can identify up to 23 serotypes of S. pneumoniae. However, the CIE is less sensitive and specific than ICTs and requires more time and expertise to perform .

• Latex agglutination test: This is a simple and inexpensive test that uses latex particles coated with anti-PCA antibodies to agglutinate PCAs in urine or CSF samples. The test result is indicated by visible clumping of the latex particles. The latex agglutination test can detect up to 25 serotypes of S. pneumoniae and has a moderate sensitivity and specificity for pneumococcal infections .

• Enzyme-linked immunosorbent assay (ELISA): This is a highly sensitive and specific test that uses an enzyme-labeled antibody to detect PCAs in urine or CSF samples. The test result is indicated by a color change in a substrate solution. The ELISA can detect up to 90 serotypes of S. pneumoniae and can quantify the amount of PCAs present in the sample .

Antigen detection methods are useful for diagnosis of pneumococcal infections, but they have some limitations, such as:

• They may not detect all serotypes of S. pneumoniae or may cross-react with other bacteria.
• They may not distinguish between current and past infections or between colonization and disease.
• They may be affected by prior antibiotic therapy or vaccination.
• They may not provide information on the antibiotic susceptibility of the organism.

Therefore, antigen detection methods should be interpreted in conjunction with clinical findings, culture results, and other laboratory tests .

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Antibody detection methods are used to demonstrate specific pneumococcal antibodies in invasive pneumococcal diseases, such as bacteremia, meningitis, and pneumonia. These methods can help to confirm the diagnosis of S. pneumoniae infection and to monitor the immune response to vaccination.

Some of the antibody detection methods are:

• The indirect hemagglutination (IHA) test, which measures the agglutination of sheep red blood cells coated with pneumococcal capsular polysaccharide antigens by serum antibodies.
• The indirect fluorescent antibody (IFA) test, which uses fluorescent-labeled anti-human immunoglobulin to detect serum antibodies bound to pneumococcal antigens on a slide.
• The enzyme-linked immunosorbent assay (ELISA), which uses enzyme-labeled anti-human immunoglobulin to detect serum antibodies bound to pneumococcal antigens coated on a microplate.

These methods require specific reagents for each pneumococcal serotype or serogroup and can be affected by cross-reactivity, non-specific binding, and previous exposure or vaccination. Therefore, they are not widely used for routine diagnosis of S. pneumoniae infection, but rather for research and surveillance purposes.

Nucleic acid–based tests are molecular diagnostic techniques that can detect specific DNA or RNA sequences of S. pneumoniae in clinical specimens or culture isolates. These tests offer potential advantages over conventional methods, such as high sensitivity, specificity, speed, and ability to detect nonviable organisms .

Some of the nucleic acid–based tests that have been used for identification of S. pneumoniae are:

• Nucleic acid probes: These are labeled DNA or RNA fragments that hybridize with complementary sequences of the target organism. Nucleic acid probes can be used to identify S. pneumoniae isolates in culture by dot blot or colony blot hybridization. Probes targeting the pneumolysin gene (ply) or the autolysin gene (lytA) have been shown to be specific and sensitive for S. pneumoniae .
• Polymerase chain reaction (PCR): This is a technique that amplifies a specific DNA fragment of the target organism using primers and a DNA polymerase enzyme. PCR can be used to detect S. pneumoniae DNA in clinical specimens, such as sputum, blood, CSF, or urine, or in culture isolates. PCR assays targeting the ply, lytA, cpsA (capsular polysaccharide synthesis gene), or spn9802 (a pneumococcal-specific sequence) genes have been developed and evaluated for S. pneumoniae identification . PCR can also be combined with other techniques, such as reverse line blot hybridization (RLB), real-time PCR, or multiplex PCR, to increase the specificity, sensitivity, or multiplicity of detection .
• Nucleic acid amplification tests (NAATs): These are techniques that use isothermal amplification of nucleic acids without the need for thermal cycling, such as loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), or transcription-mediated amplification (TMA). NAATs can be used to detect S. pneumoniae RNA or DNA in clinical specimens or culture isolates. NAATs targeting the lytA gene have been shown to be highly sensitive and specific for S. pneumoniae .

Nucleic acid–based tests are useful tools for the rapid and accurate identification of S. pneumoniae in clinical and laboratory settings. However, some limitations and challenges remain, such as the need for standardization, validation, quality control, cost-effectiveness, and interpretation of results .

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, especially in children under five years of age and elderly people. The most common pneumococcal diseases are pneumonia, meningitis, otitis media, sinusitis, and bacteremia. Prevention of these diseases can be achieved by the following measures:

• Vaccination: There are two types of vaccines available for protection against pneumococcal infections: pneumococcal polysaccharide vaccine (PPV) and pneumococcal conjugate vaccine (PCV). PPV contains purified capsular polysaccharides from 23 serotypes of S. pneumoniae and is recommended for adults aged 65 years or older and people with certain chronic medical conditions. PCV contains polysaccharides conjugated to a protein carrier and induces a stronger immune response in young children. PCV covers 10 or 13 serotypes of S. pneumoniae and is recommended for infants and children under five years of age. Both vaccines are safe and effective in preventing invasive pneumococcal disease and reducing mortality. However, they do not prevent colonization or transmission of the bacteria and do not cover all serotypes that cause disease. Therefore, surveillance and monitoring of pneumococcal epidemiology and resistance patterns are essential to guide vaccine policies and strategies.

• Antibiotic stewardship: The emergence and spread of antibiotic-resistant pneumococci pose a serious threat to public health and limit the treatment options for pneumococcal infections. Antibiotic stewardship is a set of coordinated interventions to improve the appropriate use of antibiotics and reduce the development and transmission of antibiotic resistance. Antibiotic stewardship programs include education, guidelines, feedback, audit, restriction, and de-escalation of antibiotic therapy. These programs aim to optimize the selection, dose, route, duration, and timing of antibiotic administration based on the patient`s clinical condition, microbiological results, and local resistance patterns. Antibiotic stewardship can improve patient outcomes, reduce adverse effects, save costs, and preserve the effectiveness of antibiotics for future generations.

• Infection control: Infection control measures are important to prevent the spread of pneumococci in healthcare settings and in the community. These measures include hand hygiene, respiratory hygiene, cough etiquette, isolation precautions, environmental cleaning, disinfection, sterilization, and waste management. Infection control also involves the identification and management of outbreaks, contact tracing, screening, prophylaxis, and reporting of cases and clusters of pneumococcal disease. Infection control can reduce the risk of nosocomial infections, cross-transmission, and outbreaks caused by pneumococci.

• Health promotion: Health promotion is the process of enabling people to increase control over their health and its determinants. Health promotion strategies include advocacy, policy development, legislation, regulation, communication, education, empowerment, community participation, and social mobilization. Health promotion can address the social and environmental factors that influence the occurrence and severity of pneumococcal disease, such as poverty, malnutrition, overcrowding, indoor air pollution, smoking, alcohol consumption, HIV infection, immunosuppression, chronic diseases, and co-infections. Health promotion can also enhance the awareness, knowledge, attitudes, beliefs, behaviors, skills, and practices of individuals and communities regarding pneumococcal prevention.

By implementing these measures in an integrated and comprehensive manner, pneumococcal disease can be prevented effectively and efficiently.

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