Detecting Mycoplasma using Spectroscopy

Biological characteristics of Mycoplasma pneumoniae

Mycoplasma is the smallest and simplest self-limiting bacteria belonging to the class Mollicutes and family Mycoplasmataceae.

Mycoplasma

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They can be distinguished from bacteria through the lack of cell wall structure, which makes it susceptible to beta-lactam and anti-microbial agents and prevents them from being stained via gram staining.

They have a particularly small genome of roughly 0.58-2.20Mb. It is also an extracellular pathogen whereby its survival depends on the adherence to the respiratory epithelium.

This adhesion is primarily driven through interactive adhesion and accessory proteins.

Mycoplasma infection

The prokaryote Mycoplasma pneumonia (M. pneumonia) is a leading cause of respiratory disease in humans and is often acquired through respiratory secretions manifesting itself in non-specific respiratory tract symptoms, which could progress through to tracheobronchitis and atypical bronchopneumonia.

M. pneumoniae accounts for approximately 20% of all community-acquired pneumoniae and is the leading cause of pneumonia in both older children and younger adults.

However, due to the complexity of the pathogenesis which can involve extrapulmonary spread and chronic problems such as asthma and COPD the diagnostic strategies are somewhat complicated by the aspects of the disease.

Current mycoplasma detection

Serologic testing has been a long-standing foundation for the diagnosis of M. pneumoniae due to the considerable problems associated with direct culture.

Within the US, an enzyme-linked immunoassay (ELISA) is the most widely used commercial serologic test, which, under ideal conditions can yield similar sensitivities to PCR. Nonetheless, this is a time-consuming process as sufficient time is required following infection for an antibody response to develop and the availability of paired acute and convalescent-phase sera, therefore limiting its value which is important for point of care testing.

Similarly, PCR can exhibit a high degree of sensitivity yielding a positive detection sooner than serologic testing. Like an ELISA its diagnostic potential is limited due to several issues.

PCR as a diagnostic test is limited through its reliability, standardization and the costs associated with it. Due to the problem associated with current diagnostic standards, it often means there is a delay in the initiation of treatment, thus prolonging the morbidity and increasing the likelihood of continued transmission.

This, therefore, highlights a critical need for a new diagnostic platform with a high degree of sensitivity, specificity, and expediency.

Detection using spectroscopy

There is currently little research in the way of spectroscopy as a diagnostic tool in Mycoplasma infections.

However, vibrational spectroscopy is looking to be an attractive candidate due to its unique biochemical specificity in the identification of infectious agents.

Raman spectroscopy has several features fitted to the identification of biological samples inclusive of narrow bandwidths, good spatial resolution and its applicability in aqueous solutions.

However, it does possess an inherently low scattering cross-section preventing the more widespread application to biosensing. This has previously been overcome through enhancing the Raman spectra of an analyte by the proximity to a metal surface, otherwise known as Surface Enhanced Raman Spectroscopy (SERS), which demonstrated an increased level of spectral intensity without a loss of specificity.

This method of molecular fingerprinting has shown success with other micro-organisms. However, this has been limited by the reproducibility in the preparation of the SERS-active metal substrates.

A further critical element in vibrational spectroscopy as a diagnostic tool is the use of algorithms for feature selection, rather than the use of individual peaks.

Chemometric analysis reduces the dimensionality of the dataset, therefore, maximizing the variance among the spectral fingerprints providing a level of reproducibility and sensitivity of the spectroscopic method.

Hennigan et al. (2010) evaluated a silver nanorod assay as a biosensing platform for the detection and differentiation of M. pneuomoniae both in culture and within true clinical throat swab samples by SERS- dubbed now NA-SERS offering 95-100% specificity and 94-100% sensitivity.

Through this method, they identified a lower detection limit exceeding a typical PCR diagnostic assay. Furthermore, they were able to yield a clinically relevant result with >90% accuracy and sensitivity.

This suggests the biochemical specificity of Raman Spectroscopy, combined with the spectral enhancement of silver holds a great degree of promise for a rapid and sensitive detection platform.

Potential challenges in the future

There have been advances in the detection and characterization of Mycoplasma through PCR, serology, and culture, this has been augmented by knowledge of its genome. Through establishing the genome, it has given us a wide appreciation of its role as a human pathogen.

The establishment of NA-SERS has great promise for the future, however, there are still potential challenges for use in widespread detection.

For example, the biochemical complexity and the degree of variability in clinical specimens could confound the interpretation of spectral patterns.

Thus, a further, more comprehensive analysis of mycoplasma isolates is needed to assess the discriminatory capacity of the test. Furthermore, it is necessary to expand the testing spiked samples to include a broader population as background controls to assess alternative samples.

Sources

  • Kashyap S, Sarkar M. Mycoplasma pneumonia: Clinical features and management. Lung India. 2010;27(2):75–85. doi:10.4103/0970-2113.63611
  • Hennigan SL, Driskell JD, Dluhy RA, Zhao Y, Tripp RA, Waites KB, et al. (2010) Detection of Mycoplasma pneumoniae in Simulated and True Clinical Throat Swab Specimens by Nanorod Array-Surface-Enhanced Raman Spectroscopy. PLoS ONE 5(10): e13633. https://doi.org/10.1371/journal.pone.0013633
  • Shanmukh S, Jones L, Driskell J, Zhao Y, Dluhy R, et al. (2006) Rapid and sensitive detection of respiratory virus molecular signatures using a silver nanorod array SERS substrate. Nano Letters 6: 2630–2636.

Further Reading

Last Updated: Mar 13, 2020

Gemma North

Written by

Gemma North

Gemma has a BSc in Biological Sciences from the University of East Anglia and an MSc(Res) Translational Oncology from the University of Sheffield. Her master’s thesis explored drug redeployment in multiple myeloma specifically looking at the effects of niclosamide and valproate. The study has broader implications for myeloma patients where therapies are often aggressive and not always suitable due to the demographic of patients.

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