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Journal articles made easy: Detecting strep throat using mass spectrometry

Description

This article looks at detecting strep throat bacterium using touch spray mass spectrometry. It will help you understand the research the journal article is based on, and how to read and understand journal articles. The research article was originally published in our Analyst journal. 
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Strep throat spotted in seconds

Detection of strep throat bacterium directly from medical swabs by touch spray–mass spectrometry

 

Click here for the full article in the journal Analyst 

Click here for the article in Chemistry World

 

Authors: Alan K. Jarmusch, Valentina Pirro, Kevin S. Kerian, and R. Graham Cooks

 

Why is this study important?

Strep throat is a common illness that is diagnosed millions of times annually in the United States alone.1,2 One bacterium, Streptococcus pyogenes  or group A streptococcus (GAS), is responsible for the vast majority of cases. Diagnosis is crucial in children, elderly patients, and in regions in which rheumatic and scarlet fever are prevalent because life-threatening complications are possible. Clinical symptoms like sore throat and fever are not exclusive to S. pyogenes or strep throat making it hard to diagnose.1 Rapid strep tests are currently used for point-of-care diagnosis (ie in a doctor’s office), as shown in Figure 1A. The patient’s throat is swabbed and the swab inserted into the test receptacle, which functions similarly to a common pregnancy test. The test provides a binary (yes/no) answer on the presence/absence of S. pyogenes. Although the rapid test is easy to use and provides a straightforward outcome, the results are not reliable. Accordingly, alternative but simple methods to detect the bacteria responsible for infection (with increased confidence) are desirable, with the intention of improving patient care.

 

 

Figure 1

 

Figure 1. Strep throat diagnosis: (A) schematic of the standard procedure and (B) TS-MS procedure described in Jarmusch et al. Analyst (2014) 139: 4785-4789.3

 

Mass spectrometry (MS) is a powerful analytical technique capable of detecting molecules, such as lipids, in complex matrices. Lipids provide cellular structure and contribute to cell function.4 Lipid profiles, unique patterns of lipids and their amount, are characteristic of cell type and are specific enough to allow discrimination between different bacterial species.5,Therefore, lipid profiling by MS represents a possible alternative strategy for S. pyogenes detection, illustrated in Figure 1B.

 

Further information

 

What is the objective?

The objective was to develop a MS procedure for the detection of S. pyogenes, the primary pathogen of strep throat. The authors intended to adapt an ambient ionization method that would allow for direct detection of S.pyogenes from the throat swabs. Ambient ionization allows the generation of ions under atmospheric conditions (pressure, temperature, etc) and requires minimal sample preparation.7 Touch spray (TS) was used because sampling and ionization occur from the same substrate.8 In addition to this main objective, the authors' aim was also to create a method capable of differentiating multiple bacteria using a single method.

 

What was the overall plan?

  • Test medical swabs using touch spray-mass spectrometry

  • Detect S. pyogenes from culture

  • Compare the spectra from two related bacteria

  • Detect S. pyogenes in simulated clinical samples

 

What was the procedure?

Test medical swabs using touch spray-mass spectrometry (TS-MS)

TS-MS using swabs is performed by positioning the swab in front of the mass spectrometer (Figure 1B). Solvent is applied to the swab and then a high voltage is applied, generating a strong electric field that results in the emission of analyte-containing charged droplets. The initial step in the development of a TS experiment with swabs was to test many (and various) swabs in order to find those most suitable for the generation of ions for MS. Medical swabs are commonly used for sampling orifices and body surfaces with their specific construction based on application. Most medical swabs are comprised of a paper or plastic handle and a rounded adsorbent tip (for example cotton). The swab that performed best was designed for sampling the oral cavity, specifically the pharynx, and was made of rayon with an aluminium wire handle. This worked best (based on signal intensity and lowest background) for three major reasons: (1) the metallic handle of the swab allowed voltage to be easily applied and transferred to the swab, (2) the swab is a porous polymer from which ions are readily generated,9 and (3) the shape of the swab; rounded but coming to a point, which increased the electric field to allow ionization. A second step in the method development was to optimize spray stability and signal intensity. A vertical orientation of the swabs relative to the mass spectrometer was found to provide the best reproducibly. Different solvents for lipid extraction were tested to obtain reproducible lipid information. A single addiction of methanol (~40 µL) onto the swab tip was the final condition chosen.

 

Detect S. pyogenes from culture

After the swab and experimental conditions were selected, the next step was to test if the bacterium responsible for strep throat, S. pyogenes, could be detected from the swab. The authors cultured S. pyogenes on blood agar and then sampled about 20 micrograms of material from a single colony, a group of individual organisms living in close proximity, using the rayon tip of the medical swab. The swab was then put near the mass spectrometer and held vertically with a ring-stand and clamp. Touch spray ionization was performed, as described above, resulting in the spectrum shown in Figure 2. Negative ions generated from the swab appear as peaks in the spectrum. The peaks at approximately m/z 700-800 are identified as lipids that originate from the bacteria.

 

 

Figure 2

Figure 2. Negative ionization mode TS-MS spectra of a single colony of S. pyogenes sampled from culture. 

 

 

Compare the spectra from two related bacteria

There are many different types of bacteria that can cause strep throat, or more broadly bacterial infection of the pharynx. The authors aimed to develop a method that was able to differentiate multiple causes (differential diagnosis) in a single test, not currently possible in commercial rapid strep tests. The ability to differentiate two similar species in the same genus was tested in vitro. TS-MS spectra resulting from S. pyogenes and Streptococcus agalactiae, group B streptococcus (GBS), are displayed in Figure 3. The lipid profiles of the two Streptococcus spp. are visibly different in the m/z 600-800 region. It turns out that these patterns are characteristic of the bacteria, so detailed MS interpretation is not needed: pattern recognition allows diagnosis. The ability of TS-MS to reproducibly detect differences in the lipid profile of bacteria possessing similar culturing features (for example beta-hemolysis or the breakdown of red blood cells resulting in transparency) is notable, while the ability to differentiate Streptococcus spp. is highly clinically relevant. The visual differentiation of S. agalactiae serves to illustrate the capability for differential diagnosis from a single testing methodology. It also simultaneously presents another possible and independent application of this methodology, as rapid GBS detection, which is sought-after in neonatal care.10

 

Figure 3

Figure 3. Negative mode TS-MS spectra of S. pyogenes (A) and S. agalactiae (B) sampled using a single colony from culture. Annotated peaks are tentatively identified as bacterial lipids but the spectral pattern rather than individual components is used to recognize the microorganism.

 

Detect S. pyogenes in simulated clinical samples

The detection of S. pyogenes lipids was tested with a simulated clinical sample. Human oral fluid, saliva and cheek epithelial cells were used to test the method. The swab was dipped into ~1mL of human oral fluid, absorbing an estimated 40 microliters, and then subsequently used to sample a single colony of S. pyogenes from culture - simulating a clinical throat swab. Touch spray ionization was performed resulting in the detection of multiple ions attributed to human and bacterial components. The lipid region (m/z 700-900), Figure 4, contained ions corresponding to bacterial lipids. Bacterial lipids m/z 719.5 and 773.6 were similar in relative abundance to those seen in in vitro experiments whereas m/z 745.5 and 747.6 differed in pattern. The ions m/z 719.5 and 773.6 were only detected when S. pyogenes was present.

 

 

Figure 4

Figure 4. Negative ionization mode TS-MS spectrum displaying phospholipid ions corresponding to bacterial and human components of a throat swab.

 

 

What are the conclusions?

This study details the initial development of a new non-invasive diagnostic technique, directed towards a specific clinical condition (strep throat). It addresses the problem of diagnosis of bacterial infections at the point-of-care. TS-MS performed using medical swabs allowed the rapid analysis of S. pyogenes, and it required only seconds to obtain the test data. Detection of bacterial lipids originating from S. pyogenes was performed in vitro and from simulated throat swabs using a single colony. In vitro experimentation provided visual differentiation of S. pyogenes and S. agalactiae.

 

 

What are the next steps?

A clinical trial is required to test this method and this is now being planned. The future development of swab design and materials should facilitate MS analysis, improving performance including reproducibility, background reduction and improved signal levels. Additionally, extensions of the method to other microorganisms and the ability to detect bacterial and human lipids concurrently in clinical samples might provide additional data pertinent to disease. Since MS allows detection of small molecules, such as illicit drugs and pharmaceuticals, with extremely high sensitivity and specificity, the application of TS-MS experiments with medical swabs for oral fluid analysis is envisioned to be of great benefit for therapeutic drug monitoring, drug testing, etc. Indeed, switching from drug detection in blood to oral fluid is the current trend in several clinical and forensic applications.11,12

ADDITIONAL INFORMATION

Journal articles made easy are journal articles from a range of Royal Society of Chemistry journals that have been re-written into a standard, accessible format. They contain links to the associated Chemistry World article, ChemSpider entries, related journal articles, books and Learn Chemistry resources such as videos of techniques, and resources on theory and activities. They should facilitate students understanding of scientific journal articles and how to extract and interpret the information in them.