S. pneumoniae is difficult to diagnose in the laboratory. This is because of a multitude of factors playing their part in the diagnosis of non-bacteraemic pneumococcal pneumonia which accounts for the major burden of pneumococcal diseases. Some of these key factors are as follows:

• There is no gold-standard test for diagnosing non-bacteraemic pneumococcal pneumonia. A series of tests may be required for making a reliable diagnosis of pneumococcal infections²
   
• The different tests used have variable and limited capacities for the diagnosis of pneumococcal diseases. The diagnostic capacities of the most advanced tests, such as PCR, are also quite limited. Further, methods of sample collection, such as lung aspiration, may be considered as highly invasive and may not be permissible for all patients based on ethical grounds²
• ​​​​​The conventionally used culture-based methods of pneumococcal diagnosis are not utilised fully. Particularly, in our setting, most of the patients are treated empirically (before microbiological confirmation of the diagnosis). A major proportion of patients get treated by antibiotics before the need for a culture is realised. Because of the prior use of antibiotics, S. pneumoniae gets cleared from the patients’ samples. However, the disease may not be completely cured and may even worsen, leading to a critical situation. In such a scenario, it is not possible to estimate the true burden of pneumococcal diseases in the community¹
• The requirement of sheep or horse blood agar rather than the human blood. Many laboratories use human blood which is easily available rather than sheep or horse blood. Citrated sheep or horse blood is ideally required for pneumococcal diagnosis³
• S. pneumoniae is a very fragile bacterium. It contains within itself the enzymatic ability to disrupt and disintegrate the cells. The enzyme responsible for this is called autolysin (auto = self and lysis = destruction). This autolysis is a characteristic of pneumococci and hence, they remain very fragile outside the human body⁴
• Because of autolysis/prior use of antibiotics, the organism cannot grow in the culture. However, it is possible to identify dead bacteria by antigen detection tests or by PCR. These tests have come up in recent times. However, these tests are very expensive and demand a lot of technical expertise and are not routinely possible. Currently, PCR is not done routinely whereas the Binax Now is done at very few centres across the country^5-7
• Lack of technical expertise and equipped laboratories is an issue particularly relevant to s. pneumoniae isolation. Some of the good conventional methods of culture-based diagnosis (e.g. Quellung reaction) need good expertise and are rarely carried out. The problems are compounded by the lack of easy availability of appropriate culture media or test kits and the costs associated with newer molecular methods like PCR. On the physician’s end, the problems are compounded by practices such as starting empirical antibiotic therapy without realising the need of microbiological diagnosis, the lack of understanding the importance of 2 sets of cultures, the lack of appropriate sample collection and transport, etc⁸

Culture

This is the most frequently used method for pneumococcal diagnosis. The ability of diagnosis using this method is very limited as it requires living bacteria to grow which may not be possible in cases of autolysis, prior antibiotic use, etc. Transport and processing delays, and use of appropriate culture medium (e.g. sheep blood instead of human blood) are other challenges. Because of all these challenges, the isolation of pneumococci from the sputum culture is currently possible in <30% of the actual cases. Antibiotic sensitivity and serotyping can be done from cultured isolates by using further methods and processes. Serotyping can be done by latex agglutination. However, serotyping is not done routinely in clinical practice and is carried out only for research purposes. Results (particularly for the sputum culture) may be falsely positive because it may be the normal flora of the respiratory tract rather than the causative organism of the infection.^1,3

Microscopic Examination of the Sputum

Urinary Antigen Detection Test

Polymerase Chain Reaction