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S. pneumoniae is difficult to diagnose in the laboratory.1 Some salient points about the tests used for the diagnosis of pneumococcal infections are as follows.

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 country5-7
  • 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

S. pneumoniae is a difficult organism to diagnose in the laboratory.1 Some salient points about the tests used for the diagnosis of pneumococcal infections are as follows.​​​​​​​​​​​​​​


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
The usefulness of sputum tests is debatable because of the possibility of contamination by normal bacteria in the upper respiratory tract. Specimens should be obtained by deep cough and be grossly purulent. Ideally, the specimen should be obtained before treatment with antimicrobials and be transported to the laboratory immediately for prompt processing. In the absence of a culture, adequate sputum samples showing a predominance of pneumococci under the microscope may be considered for a presumptive diagnosis. A good sputum sample should contain <10 epithelial cells (normal airway cells) and >25 polymorphs (white blood cells) per low power field of the microscope.8
Urinary Antigen Detection Test
It is based on the concept of identification of the pneumococcal capsular antigen in the urine. In pneumococcal infections, the pneumococcal capsular antigens generally escape into the patient’s body fluids and are excreted in the urine. The urinary antigen detection test performed on the urine samples of such patients can detect pneumococcal infections. It is a good test if used properly, but is expensive and is used in few centres only. Apart from urinary antigen detection, this test can also be used for pneumococcal disease detection from the CSF, blood, BAL and pleural fluid. The results of this test are not affected by prior antibiotic use (since it can identify dead bacteria). In children, the test is of lower value because children generally carry pneumococci in their nasopharynx even without any disease (normal nasopharyngeal carriage). Hence, the test may be positive even when there is no infection. One of the drawbacks of this test is that the results remain positive for a long period even after the infection is resolved. This may be a problem during diagnosis if the patient is prone to frequently recurring pneumococcal infections.5
Polymerase Chain Reaction
There are 2 types of methods, conventional PCR and real-time PCR. Conventional PCR is not practiced at most places because it has been replaced by real-time PCR. Real-time PCR (a new type of PCR) can give results in a short span of time (within a few hours) as compared to conventional PCR. PCR is not done routinely in our country because it is very expensive and requires a lot of technical expertise which is not readily available. However it has some advantages, including detection of organisms even in antibiotic pre-treated cases and good diagnostic capacity. This test can be done directly on patient’s samples. Limited serotyping is possible by PCR as well.​​9,10


ACIP, Advisory Committee on Immunization Practices; BAL, bronchoalveolar lavage; CSF, cerebrospinal fluid; PCR, polymerase chain reaction; PCV13, 13-valent pneumococcal conjugate vaccine; PPSV23, 23-valent pneumococcal polysaccharide vaccine.



Werno AM, Murdoch DR. Medical microbiology: laboratory diagnosis of invasive pneumococcal disease. Clin Infect Dis. 2008;46(6):926-932.O’Brien KL, Wolfson LJ, Watt JP, et al; Hib and Pneumococcal Global Burden of Disease Study Team. Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates. Lancet. 2009;374:893-902.Arbique JC, Poyart C, Trieu-Cuot P, et al. Accuracy of phenotypic and genotypic testing for identification of Streptococcus pneumoniae and description of Streptococcus pseudopneumoniae sp. nov. J Clin Microbiol. 2004;42(10):4686-4696.Petti CA, Woods CW, Reller LB. Streptococcus pneumoniae antigen test using positive blood culture bottles as an alternative method to diagnose pneumococcal bacteremia. J Clin Microbiol. 2005;43(5):2510-2512.Charkaluk ML, Kalach N, Mvogo H, et al. Assessment of a rapid urinary antigen detection by an immunochromatographic test for diagnosis of pneumococcal infection in children. Diagn Microbiol Infect Dis. 2006;55(2):89-94.Dowell SF, Garman RL, Liu G, Levine OS, Yang YH. Evaluation of Binax NOW, an assay for the detection of pneumococcal antigen in urine samples, performed among pediatric patients. Clin Infect Dis. 2001;32(5):824-825.Hamer DH, Egas J, Estrella B, MacLeod WB, Griffiths JK, Sempértegui F. Assessment of the Binax NOW Streptococcus pneumoniae urinary antigen test in children with nasopharyngeal pneumococcal carriage. Clin Infect Dis. 2002;34(7):1025-1028.Musher DM, Montoya R, Wanahita A. Diagnostic value of microscopic examination of gram-stained sputum and sputum cultures in patients with bacteremic pneumococcal pneumonia. Clin Infect Dis. 2004;39(2):165-169.Falguera M, López A, Nogue´s A, Porcel JA, Rubio-Caballero M. Evaluation of the polymerase chain reaction method for detection of Streptococcus pneumoniae DNA in pleural fluid samples. Chest. 2002;122(6):2212-2216.Moore CE, Seng duang phachanh A, Thaojaikong T, et al. Enhanced determination of Streptococcus pneumoniae serotypes associated with invasive disease in Laos by using a real-time polymerase chain reaction serotyping assay with cerebrospinal fluid. Am J Trop Med Hyg. 2010;83:451-457.


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