Abstract

There is a lot of concern regarding the control of outburst of infectious diseases. One major reason behind this is the inefficiency of most detection methods. While most of the methods are based on old and labour-intensive technologies, others are lengthy and unreliable. Researchers from all across the world are continuously working on developing rapid and reliable diagnostic tests for early detection of such diseases. One such disease which affects human body is Rheumatic Heart Disease which is caused by Streptococcus pyogenes.  S. pyogenes which is classified as Group A streptococcus (GAS) colonizes the throat or skin and is primarily responsible for a number of suppurative infections and nonsuppurative sequelae. The current diagnostic methods that are employed for the treatment of RHD are based on biochemical test and molecular assay, but these methods are time-consuming and often expensive. With increasing efforts towards the control of the disease along with various intervention strategies, small success has been achieved for the treatment of rheumatic heart diseases in India. This review outlines the use of a DNA based biosensor to detect Streptococcus pyogenes which leads to RHD. The molecular recognition property of DNA and the mechanism of charge transport in it allow DNA to be widely employed for biochemical sensors. The initial step involves the development of electrochemical DNA based biosensor by using screen printed titanium dioxide nanoparticles based interdigitated electrode. Titanium dioxide electrode exhibits unique tendency because of the ease to derivatise its surface. Oxidized Ti is chosen as an electrode material on a SIO2 substrate. The electrode surface was chemically functionalized with (3-aminopropyl) triethoxysilane (APTES) to provide contact between the organic and inorganic surfaces of a single-stranded DNA probe and TiO2 nanoparticles while maintaining the sensing system’s physical characteristics. The capacity of target DNA of GAS bound with the carboxylated-probe DNA could be translated into electrical signals and confirmed by the increased conductivity in the current-to-voltage curves. Titanium dioxide nanoparticles interdigitated electrode constitutes high sensitivity, stability which provides specific sensing platform for a wide range of bimolecular interactive analysis. The electrochemical detection was carried out using cyclic Voltammetry, differential pulse Voltammetry, electrochemical impedance and resistance.

Keywords: RHD, ARF, GAS, biosensor, titanium dioxide, APTES

Introduction

Rheumatic heart diseases continue to be a common health problem in the developing world, causing morbidity and mortality among children and adults. In developing countries, rheumatic fever and RHD continue unabated, with RHD affecting an estimated 5 to 30 million worldwide [1]. Rheumatic heart disease is defined as impairment or damage to one or more heart valves that remain after an intervention of acute rheumatic fever (ARF) [2].

Rheumatic heart disease is an autoimmune disease in which S.pyogenes show mimicry with skeleton and muscle protein. RHD is caused by multiple encounters of ARF (acute rheumatic fever), which is responsible for the inflammation of the heart. RHD is the most significant abnormality caused by infection with group A Streptococcus pyogenes which is one of the most frequent pathogens known to provoke a wide variety of diseases in the human body such as acute glomerulonephritis, reactive arthritis, etc. The metabolism of S. pyogenes is fermentative, the organism is Catalase- negative, aerotolerant anaerobe which requires enriched medium containing blood in order to grow (sheep blood agar media)[3]. The word rheumatic means pain in muscles, joints that became painful when touched. Rheumatic fever is immunologically mediated periarteriolar connective tissue diseases caused by S.pyogenes infection. S.pyogenes can be recognized as an exogenous invader with an extremely complex surface consisting of chemically diverse components which accounts for its virulence. The antigenic components present on its surface include a variety of surface protein like  M protein, fimbrial proteins, fibronectin-binding proteins, (e.g. Protein F); capsular polysaccharide (C-substance), cell wall peptidoglycan and lipoteichoic acid (LTA) which helps the organism in the process of adhesion and also provide protection from phagocytosis[4].   It also enables the organism in order to resist ingestion and removal of phagocytic cells. RHD affects children; young people living in unhygienic conditions such as poor sanitation, poverty and overcrowding are the main factors in causing RHD. Streptococcus pyogenes belongs to the family of gram-positive bacteria, responsible for wide variety of diseases in humans including toxic shock syndrome, cellulitis, erysipelas, etc. The stimulation of innate immune system due to pharyngeal infection leads to exposure of T and B cell to S.pyogenes antigen.

The pathogenic mechanism of group A Streptococci has been investigated. It consists of a capsule composed of hyaluronic acid and exhibit β hemolytic on blood agar associated with complete lysis of red blood cells and can make colony up to 5mm in size. Group A Streptococcus (GAS) organisms are also known as flesh-eating bacteria and are usually spread by droplets or by a person touching items recently handled by an infected individual [5]. The stimulation of innate immune system due to pharyngeal infection leads to exposure of T and B cell to S.pyogenes antigen so there will be an urgent need for diagnosis of RHD  in order to vanish severe form of diseases.

Most commonly known symptoms of RHD include joint pain, fever, heart inflammation, nervous system (jerky uncontrollable movements) etc. These appear about two weeks after the origin of an untreated strep throat infection that leads to an onset of RHD. Rapid traditional methods for diagnosis of S. pyogenes include culture test, rapid antigen detection test (RADT), Biochemical test, serological test, C Reactive protein  (CRP), Polymerase chain reaction (PCR)  Test etc. S.pyogenes is a gram-positive, or spore-forming prokaryote which does not constitute nuclear membrane, organelles in the cytoplasm except ribosome has its genetic material in the form of single continuous strands in the form of coils or loops. They are also categorized on the basis of biochemical reactions, hemolysis and colony morphology. The estimated mortality ratio caused by infection with S.pyogenes is 500,000 deaths yearly while approximately 5-15% of the normal individuals have the bacterium in their respiratory tract [6].

 

 

 

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Figure 1: Cross-sectional view of the base of heart showing Heart with Normal Mitral Valve and Heart with Rheumatic Mitral Stenosis

Serotyping and antigenic types

During 1928 Rebecca Lancefield described a method of Serotyping of S.pyogenes based on antigenic differences in their cell wall, also based on streptococcal M protein. The M protein exhibited the property of resistance with phagocytosis also known as bacterial vial determinants. The cell wall structure of Streptococcus pyogenes consists of repeating units of N-acetylglucosamine and N- acetylmuramic acid along with structural proteins R and T which constitute phagocytosis resistance. The capsule of S.pyogenes is composed of hyaluronic acid and exhibits nonantigenic properties.

Streptococcal genome

The group A Streptococcal genome is a singular circular chromosome, approximately 1.9 MB in size, where 10% of the genome consists of variable regions including prophage-like elements, the rest roughly 90% of the genome is called core- genome. The core genome encodes for various virulence factors such as M protein, streptolysin O,   and streptolysin S. The streptolysin O and streptolysin S are acute toxins as well the basis of beta-hemolytic property. They are the potent cell poison affecting many types of cell including neutrophils, platelets and subcellular organelle [7].

the biodaily s pyogenes
Figure 2: Circular map of Streptococcus pyogenes genome (Adapted from Nakagawa, et al. , 2003)

Biochemical tests for detection of S.Pyogenes:

Bacitracin Susceptibility: Bacitracin is a bactericidal drug useful in tropical preparation as a polypeptide antibiotic for the treatment of superficial skin infections or works as dermatological irritants but too toxic for systemic use and can lead to kidney damage if used internally. Bacitracin is a polypeptide antibiotic produced by Bacillus subtilis. This drug restrains the peptidoglycan synthesis of bacteria. The possible method for identification of group A Streptococci (GAS) is usually done by testing for sensitivity to Bacitracin.

CRP Measure: C-reactive protein (CRP) is a group of acute phase protein produced by the liver in response to inflammation.  It is also known as high-sensitivity C-reactive protein (hs-CRP) and ultra-sensitive C-reactive protein (us-CRP) due to their enhanced release in response to certain inflammatory cytokines. A high level of CRP in the blood increased the risk of cardiovascular disease of inflammation, whereas other markers for generation of increased risk of CRP is due to wide variety of conditions including trauma, burns, infections, such as pneumonia or tuberculosis etc.

Gram Staining: Gram staining or Gram stain is also called Gram’s stain method after Danish microbiologist Hans Christian gram (1853-1958). Gram staining is one of the most popular staining technique required for classifying bacterial species into two large groups as well as distinguish them on the basis of physical and chemical properties of their cell wall [10]. S. pyogenes is found to be Gram Positive coccus growing in pairs or as chains on gram staining.

Catalase Test: Catalase is a common enzyme found in nearly all aerobically respiring organisms exposed to oxygen (such as bacteria, plants, and animals). It catalyzes the decomposition of hydrogen peroxide into water and oxygen. It is a very important enzyme involved in the catabolic pathway and protecting the cell from oxidative damage by reactive oxygen species (ROS) [11].

Detection of Rheumatic heart diseases based on molecular assay

Polymerase chain reaction (PCR) is one of the most important techniques developed in 1983 by Kary Mullis used in molecular biology in order to amplify a single copy or a few copies of a segment of DNA across several orders of size, generating thousands to millions of copies of a particular DNA sequence. It is an easy, cheap, and reliable way to repeatedly replicate a focused segment of DNA, a concept which is applicable to numerous fields in modern biology and related science [12].  The objective of PCR is based on utilization of DNA polymerases to synthesize new strand of DNA complementary to given template strand because DNA polymerases can add a nucleotide only on to a pre-existing 3″OH group, it needs a primer to which it can add the first nucleotide. These requirements enable the researchers to mark the specific segment of template sequence that the researcher wants to amplify [13].

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Figure 3: Use of PCR for the Molecular assay for detection of rheumatic heart diseases. (Retrieved from CSIR-IGIB)

Historically a throat culture was used to confirm GAS infections. The drawback of throat culture is their cost and appropriate follow-up treatment. However, imprecise the clinical diagnosis of streptococcal infection may be some of its features help to differentiate it from much more common viral throat infection.GAS diagnostic methods are time-consuming, expensive, nonspecific that leads to other limitations. On the basis of molecular recognition property of DNA, it is widely employed for biochemical sensors.  The initial step involves the development of electrochemical DNA based biosensor by using screen printed titanium dioxide based interdigitated electrode [14].

Biosensor:

Professor Leland C Clark Jr, the father of biosensor concept published the definitive paper in 1956 on the oxygen electrode. Based on his concept, biosensors were defined as analytical devices capable of providing both a qualitative result and quantitative result by employing a biological material to specifically interact with an analyte. The interaction produces some detectable physical changes that can be measured and converted into electrical signal by transducer [15].

Finally, the electrical signal is amplified, interpreted and displayed as analyte concentration in solution. An analyte is a compound whose concentration is detected by the biosensor. The biosensor works on a principle of producing either discrete or continuous digital electronic signals which are equal to a single analyte or related group of analytes or it involves interactions of biological elements to produce qualitative and quantitative result [16].

Classification of biosensor on the basis of the transducer:

  • Electrochemical biosensor: The biological signal can be used to process the electrical signal in order to generate current or conductivity between the two electrodes for the analysis of biochemical process for biotechnological purposes.
  • Amperometric biosensor: It involves the generation of current when the current is applied between two electrodes. Amperometric biosensor allows an electrochemical reaction to start at the electrode surface and giving rise to the current. The Amperometric biosensor was developed by Updike and hicks with the first use of enzyme electrode for diagnosis of glucose in diabetes.
  • Optical biosensor: The working of these biosensors is based on measurement of light absorbed or emitted due to the biochemical reaction. The most commonly used optical biosensors are surface Plasmon resonance (SPR)-based biosensors including SPR imaging and localized SPR.
  • Calorimetric biosensor: This type of biosensor has their application during biochemical reactions which involves the change in enthalpy, can be analyzed by calorimetric biosensor [17].

Sensors continue to make a significant impact in everyday life. There has been a strong demand for producing highly selective, responsive, and cost-effective sensors. The heart of the electrochemical system is Potentiostat. A Potentiostat is a device which will apply the potential across a pair of electrode and simultaneously measure the current which flows through the solution of an analyte. The unique requirements for each of the Voltammetry techniques are described under individual techniques; we are describing three electrode systems.

Reference electrode-carbon

Counter electrode -silver

Working electrode –titanium

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Figure 4: Screen printed electrode from dropsens.

Screen printed titanium composite electrode for early detection of Streptococcus pyogenes

Titanium dioxide electrode (TiO2) exhibits unique tendency because of the ease to derivatise its surface. Oxidized Ti is chosen as an electrode material on a SiO2 substrate. The electrode surface was chemically functionalized with (3-aminopropyl) triethoxysilane (APTES) to provide contact between the organic and inorganic surfaces of a single-stranded DNA probe and TiO2 nanoparticles while maintaining the sensing system’s physical characteristics. The complement of the target DNA of GAS to the carboxylated DNA probe could be translated into electrical signals and confirmed by the increased in the current [18].

Titanium dioxide nanoparticles interdigitated electrode constitutes high sensitivity, stability which provides specific sensing platform for a wide range of biomolecular interactive analysis. Efforts are being made to develop electrochemical sensor of high sensitivity and the low detection limit for quick and early detection of a pathogen from patient samples [19].

Cyclic Voltammetry has become an important and widely used electroanalytical technique in many areas of chemistry. It is widely employed for redox processes, in order to analyze the mechanism of reaction intermediates, and for obtaining stability of reaction products. Similarly, in differential pulse Voltammetry, the current is measured twice during the lifetime of each drop, and the difference in current is plotted [20].

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Figure 5: Schematic representation of immobilization of probe on screen-printed electrode

Conclusion

Biosensors can be used for quick detection of RHD as there is no confirmatory diagnostic test available for quick detection of RHD. All the available methods are either expensive or have some limitations. Therefore DNA sensors prove to be more specific, sensitive and cost-effective as compared to other current assays available. The traditional microbial diagnosis of Streptococcus pyogenes relied on a throat culture that appeared as β-hemolytic colonies on 5% sheep blood agar. The principle of DNA biosensor is that it is based on nucleic acid recognition processes which are rapidly being developed towards the goal of rapid and inexpensive testing of genetic and infectious diseases. It is advantageous to standardize this method for quick diagnosis of streptococcal infections in patients. In conclusion, this method is described here as a useful tool to improve non- culture diagnosis and case ascertainment in fulminating fatal cases of streptococcal infections.

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Authored By:

Karishma Sharma

M.Tech-Biotech

Research Scholar

CSIR-IGIB