28 October 2016

Streptococcus pneumoniae (S. pneumoniae) is one of the most common bacterial respiratory pathogens worldwide and responsible for a variety of life threatening infectious diseases including pneumonia, meningitis and septicaemia 1. In the developing world, 25% of all preventable deaths in children under five years old are attributed to pneumococcal septicaemia, with over 1.2 million infant deaths per year. The true burden of invasive pneumococcal disease is thought to be highly underestimated, with only a small portion of presumptive cases confirmed by conventional techniques.

S.pneumoniae typically resides in the upper respiratory tract of healthy carriers. Although carriage is often asymptomatic, if S. pneumoniae gains access to other parts of the airway the inflammatory response results in disease. Children, the elderly, and immunocompromised patients are most at risk to developing pneumonia 2. Severe cases may result in bacteraemia, which is associated with increased morbidity and mortality. As with many infectious diseases, early detection can be crucial to patient outcomes and survival rates.

Laboratory techniques for identifying pneumococcal infections are dependent on traditional microbiological methods, many of which are laborious and limiting. The time constraints associated with identifying S. pneumoniae hinder diagnosis, treatment efficacy and ultimately patient outcomes.

The analytical methods currently employed to detect S. pneumoniae suffer from poor sensitivity and are not always conclusive in identifying species present. Many clinical laboratories identify S. pneumoniae by microscopic and colony morphology, or by phenotypic traits such as catalase negativity and α-haemolysis on blood agar 3. These tests exclude most, but not all viridians species, and the inadequacies present a major obstacle for accurate diagnosis. This was exemplified by a recent study highlighting the original misidentification of viridians species in samples isolated from HIV-seropositive patients 4.

Conventional culture methods are also employed. However, these are time consuming and can be compromised by spontaneous autolysis or antibiotic treatment. The detection of pneumococcal metabolites in urine, blood, breath and sputum samples represents an attractive alternative, being non-invasive. Though again it has a drawback, including positive results weeks after the onset of infection and inability to conclusively identify S. pneumoniae from viridians species 5.

Aptamers targeting pneumoniae

Klebsiella pneumoniae (K. pneumoniae) is a well-known source of community-acquired pneumonia in certain at-risk groups, causing community-acquired urinary tract infections with bacteraemia 7. K. pneumoniae is also a common cause of nosocomial infections, with the associated risks and costs; prolonged antibiotic treatments, extended hospital stays, financial burdens on healthcare providers.

Technical difficulty in prompt detection of K. pneumoniae makes it difficult to respond infection, and treatments are reactionary rather than pre-emptive. The resistance to antibiotics adds a further complication and swift treatment is paramount. An aptamer for K. pneumoniae has been developed with dissociation constant (Kd) of 5.377 nM and the developers outline its potential use in a aqueous environment compatible sensor 8.

Another example involving an aptamer demonstrated a simple and versatile colorimetric biosensor for specific detection of S. pneumoniae by combining the signal transducer and catalysed DNA hairpin assembly-based signal amplification. This strategy has been successfully applied to detect S. pneumoniae as low as 156 CFU ml−1, and shows good specificity against closely related streptococci and pathogenic bacteria 9. The developed method enables naked eye analysis of S. pneumoniae in clinical samples and demonstrates excellent assay versatility (Figure 1).

figure1

Figure 1:- DNA transducer-triggered signal switch for visual colorimetric bioanalysis 9.

(A) Specificity of the assay for DNA detection: complementary target DNA (T0), single-base mismatched DNA (T1), two-bases mismatched DNA (T2), non-complementary DNA (NT), and blank. Inset: The corresponding photographs of the colorimetric responses. (B) Selectivity of the assay for bacteria detection: S. pneumonia D39 (1), S. pneumonia 19F (2), S. pneumoniae R6 (3), S. pneumoniae 6B (4), S. pneumoniae TIGR4 (5), S. pneumoniae TIGR3 (6), S. pneumoniae 49619 (7), S. mitis (8), Streptococcus pyogenes (9), Haemophilus influenza (10), Staphylococcus aureus (11), Enterococcus faecalis (12), Klebsiella pneumoniae (13), E.coli (14), Pseudomonas aeruginosa (15), and blank (16). Inset: Agarose gel electrophoresis of different bacterial PCR products. The error bars were standard deviations of three repetitive measurements.

In both the above, aptamers are shown to be reagents capable of detecting S. pneumoniae sensitively and in a user-friendly way. However, the limited number of examples highlights the need to exploit the favourable properties of aptamers in point-of-care diagnostics further. Relatively small size, lack of immunogenicity, rock-solid reproducibility in oligonucleotide production and ease of chemical modification make aptamers ideal components in diagnostic devices 6. This is reflected by an ever-increasing interest for incorporating aptamers into diagnostics, working in conjunction with, or replacing, traditionally used antibodies.

To learn more about Aptamer Group’s diagnostic arm, please visit Aptadx.