The recent developments in proteomics particularly in the field of bioinformatics have given a new light to the diagnosis of specific diseases and also the formulation of the related cures. Genomics and changes associated with gene patterns have enabled the prediction of etiological factors leading to a disease being shed light on genetic mapping and prognosis on alterations of DNA in novel diseases. This has been done using techniques such as polymerase chain reaction, gene restriction, use of probe hybridization and also using primers. The study of protein function and structure has also been done in pathophysiology under a field known as proteomics since proteins are common biomarkers of a disease. The arrangement of their three-dimensional structure of proteins, genes and their rate of multiplication could be used to test the presence of diseases. Mutations causing emergence of novel diseases are also being studied particularly for infectious diseases. The adoption of proteomics and genomics has been used in virtual diagnosis of majority of endemic diseases due to their complexity and lack of cure. These diagnostic methods are at the cellular and molecular level since both DNA and proteins are major components of a cell. These two fields have created opportunities to find better prognosis and diagnosis methods for diseases through their ability to correlate. These methods have been used in the diagnosis of infectious diseases such as influenza and Severe Acute Respiratory Syndrome (SARS). Upon discovery of a new disease, careful analysis on the disease must be done before initiating screening, diagnosis and treatment methods to be used.
Background on Major Infectious Diseases
According to many epidemiological studies, the common infectious diseases are malaria and influenza. These are caused by plasmodium, a parasite, and influenza virus. These diseases are caused by pathogens, which are microorganisms and can be transmitted by air through sneezing, aerosolized droplets and coughing (in the case of influenza) and through skin bites (in the case of malaria) (Ryan & Ray 2004).
In the case presented, the patients with the new disease experienced head and muscle aches, general body weakness, lassitude, frequent fluctuations in body temperature and frequent coughing. The disease was epizootic like the H1N1 flu with the manifestation of low hemoglobin count among the patients but did not respond to treatment with H1N1 drugs. However, of concern was the speed at which incidences of the disease were reported which was very high. For any disease, understanding its molecular basics, methods of diagnosis and certain developments to prevent or cure it is crucial since it will increase preparedness once the disease strikes hence preventing social economic fallout in any society. The investigations of the outbreak showed an accelerated increase in lymphocytes due to an increase in immune responses but were ruled out to be malaria due to the presence of coughs thus there was a high possibility that a mutation on the influenza virus had occurred since the manifestations were similar to known types of influenza. On administering of the common medication for tuberculosis and other related diseases, the disease was found to persist thus resistance to such drugs. This ruled out the possibilities of other diseases narrowing our research to changes in the influenza virus changes. Other symptoms included fever and lack of appetite, which contributed to the experience of weight loss and lack of vigor.
The physical methods used to diagnose this novel kind of influenza include the checking of accelerated temperatures ranging between 100-104˚F, presence of tachycardia, nasal discharge, reddening and also watery eyes. Pulmonary findings at times are used such as rhonchi and clear lungs can also access the presence of the disease. Care however must be taken since most influenza virus strains are similar and misdiagnosis can occur thus physical symptoms would not be reliable diagnose the new disease (Nelson & Williams 2007; M’ikanatha & Lynfield 2007). Other more advanced methods have been used since research on disease trends has intensified.
Polymerase Chain Reaction Methods
Molecular diagnostic methods for amplification have been used to diagnose influenza but considering it is caused by a virus that replicate fast the use of accurate diagnostic procedures should be used. Further, the procedures require several basics in proteomic and genomics for any significant change to be observed. In this case, the basic principle is to detect specific DNA and RNA sequences via hybridization of a probe, detecting any complementary sequence on the cDNA of this bacterium. PCR techniques are also used to diagnose the disease and check for any drug resistance against any antipyretics and antivirals before initiating any treatment. This according to Ellis & Zambon (2002) allows accurate diagnosis of any novel disease using nucleic acids by the procedure of real time PCR. Such molecular techniques involving the amplification of nucleic acids have been done, but the problems of sensitivity, parameters used to cycle, the kind of samples and also the limited screening capability cannot be ignored.
Commercialized and direct amplification tests such as COBAS AMPLICOR to analyze any of the viruses of influenza using nucleic acid 16SrRNA genes have been done. This RNA is the one that is augmented and then identified using a probe that is a complex of the virus and another RNA sequence. Another of those methods is the chain reaction using ligase enzyme done using a minute particle of the enzyme and is immune assayed using a flourimetric analyzer. The BD test that uses both polarizations in flourimetry using the 16SrRNA co-amplified with IS6110 gene has also been very effective. These methods have been used to diagnose this disease and also check its mutations causing novel diseases that are resistance other normal medication. These tests have a major shortcoming in that they have not yet been approved though they are very effective. They use the principle of polymerase chain reaction wherein a section of either RNA that can hybridize with a given probe is taken and multiplied under a certain condition to produce many copies of the section that can be used for the intended analysis. This method is more preferred that culture and immune fluorescent method that use swabs thus require intact cells and are time consuming. They are also very specific and allow further diagnosis of the associated virus for the novel disease though they are expensive, require experts, specialized equipment and the major problem is that the viruses cannot be recovered on analysis.
They are more effective than smears of sputum done using microscopy and other radiographic X-rays. Its other advantages are that it uses a single specimen and that it is easy and fast, making it user friendly. Since it uses hybridization techniques the possibility of two strains of the virus that have undergone genetic mutation cannot be accurately recognized thus, to improve its efficiency, it should be done in conjunction with culture and microscopy to observe morphological changes. The common PCR techniques performed to diagnose any new strain of influenza must be either multiplexed PCR that tests more than one assay per analysis resulting to accurate results but must have extensive optimization to ensure no false competition of primers bringing false negatives occur. The incorporation of EIA in PCR that is not limited to size of the sample to be assayed has been done but this does not allow further analysis of sample and require thorough evaluation prior use. Of all these methods the newest and most effective is the real time PCR for diagnosis of this novel disease but fears that sometimes it is not feasible are there (Marechal et al. 2011).
Cellular Techniques of Diagnosis
With the knowledge that this disease is caused by a virus which is has peculiar characteristics such as reproducing in a host cell, the fact that it is smaller than other microorganisms and contains only RNA, there is a need to incorporate microscopy of higher magnification, centrifugation and cell line fixation techniques in order to diagnose this new strain of influenza. This will show alterations in size, shape and morphology of the nucleolus showing any alterations in the cell genome.
The use of aspiration biopsies using fine needles has been used to diagnose influenza since it can affect internal organs such as the lungs which are not easily reached. This is done by insertion of a needle to extract cells that are then stained and examined under a microscope. This has been shown to be less traumatic than surgical biopsies especially in children, but the problem of wrong diagnosis or false positive results is highly likely since this can also be done in Tuberculosis. According to Pearson and Patrick (2000) to improve the efficiency of this method its incorporation with fluoroscopy and bronchoscopy is advised.
Gold diagnostic methods are isolation and identification from cultures and serology of a highly purified sample protein of the viruses which have been used in the monitoring of viral changes as the disease progresses since they are more specific and sensitive to a particular strain of this virus can be used. Other cytology tests such as white cell count and leukocyte count have been done to ascertain presence of the disease. In influenza types, the mesothelial and endothelial cells of the respiratory system seem to change their morphology due to injuries caused by the pressure and friction during coughing. According to Das, Vaidyanathan, and Indudhara (1992), these cytological aspects can be incorporated with sonography, making the process more efficient and successful though can be misdiagnosed with TB.
Proteomics of diagnosing a new influenza disease
According to Baas et al. (2006), the use of proteomic fingerprinting together with mass spectrophotometry and profiling of serum biomarkers that are mainly proteins have been discovered and are efficient in the diagnosis of novel diseases. The process of fingerprinting has been done with the help of the super vector machine (SVM) and other immunoassay techniques using proteins such as Hemagglutinin and neuraminidase antigens found in any influenza strains. Depending on the peaks of these biomarkers displayed on a resultant graph, diagnosis can be deduced since the results are directly proportional to the degree of inflammation which is a common trait in all influenza diseases. Also according to Cole et al. (1998), some genome databases have managed to sequence and come up with the full-length sequences of these biomarkers of influenza in the process of gene profiling. This to a great deal has assisted diagnosis by comparison with sequences that are known. In a given sample, its three-dimensional structure can show any mutation that could lead to resistance thus it is an essential tool in analyzing the strains of this virus. However the difficulty is that most viruses are irregular thus their characterization is quite indefinite. These databases such as Gene bank, EMBL and NCBI have been very helpful in the discovery of novel diseases. Such databases provide information on the 3-D structures of proteins and experiments done concerning gel electrophoresis on the proteins. The use of electrophoresis has also been done to help in the diagnosis of previous influenza strains such as HINI, H5NI and others like type A and Type B. This involves separating viral proteins based on size and electrical charge of the proteins. In this case, the biomarkers from a patient suspected to be suffering from the novel strain are span in gel on being placed in an electric field of opposite charge then isolation occurs depending on size. These are compared with standard proteins of known strains to see if they are closely related. This has been incorporated using PCR to make it more efficient however this process requires a specialized gel with smaller pores considering that viruses are far much smaller for it to be successful. Gel electrophoresis is also used to determine the genetic difference between individuals and how susceptible they are to a given strain of flu using techniques such as Random Amplified Polymorphic DNA (RAPD) and Restriction Fragment Length Polymorphism (RFLP) (Lashkari et al. 1997). This has increased the use of comparative genomics in diagnosis of diseases. The use of Western blotting technique that identifies the proteins and separates them using SDS-PAGE has been done in the presence of TBST buffer as an effective method to discover proteins in a sample thus giving clues of a novel disease. These proteomic techniques are effective but are limited to proteins found in the databases and are too dynamic thus keep on changing. This method is thus limited for this study since influenza viruses are prone to mutations causing new flu diseases. Using DNA microarrays which are gene expression signatures (Greninger et al: 2009) that use oligo nucleotides of the DNA sequences for the biomarkers can help in the hybridization process and has also been done. It is expected to help in the diagnosis of influenza. This has been successful for various upper respiratory diseases since it offers comprehensive analysis.
Blood-based experiments demonstrated that immunological assays could be used to diagnose novel strains of influenza since for the longest time the use of fluids such as sputum and urine are being used though they are not specific to the particular strains of the disease unless combined with molecular techniques. This is on the basis that once the body is infested with new pathogens, antibodies are raised to defend the body against those microbes, and particularly lymphocytes that are increased in the blood. The use of flow cytometry to detect interferon-γ on incubation of sputum and other fluids that has been purified to obtain protein derivatives and show the activity of CD4 cells that produce the interferon has been done. It is purported in patients with influenza strains; the CD4 count should be low to achieve immune deficiency. It has also been used to type any strain of the virus thus a very effective method to discover this foreign disease as according to Greninger et al. (2009) based on microspheres. Using light scattering via fluorochromes, proteins formed in immune reactions will be displayed. Use of ELISPOT to detect spots formed by cells that produce interferon which is in response to immune reactions has also been applied. A similar technique that uses the principles of ELISPOT is the dot blot hybridization technique which can also be used as according to Fouchier et al. (2000). This enzyme-linked assay of immunosorbents has also been used to detect cytokines and antibodies raised against a given antigen due to the presence of reverse transcriptase enzyme in the virus The use of antibodies and antigens linked reactions such as ELISA has been explored in order to detect influenza, but it is very cumbersome and could even lead to mixtures of samples if screening was to be done although it is effective for small populations (Pai et al. 2003). It is time consuming and not accurate in typing of strains of a novel disease unless it is incorporated with molecular techniques. Use of immune assays combined with fluorescence and enzymology has helped generate good deductions of novel influenza strain screening, but this method is very expensive and requires to be done by experts to be effective.
Biotechnological Efforts to Diagnose
Biotechnology is also used to help in diagnosing novel diseases with the help of nanotechnology that entails the behavior and responses of particles of the size of 10 -9 which are collectively called nano-particles. These are more efficient than microscopy that can only observe up to10 -6 particles. It has also introduced the use of single nucleotide polymorphisms (SNPs), which have been used to check the patterns of diseases by using the knowledge of genomics; alterations at certain regulatory regions of the proteins sequences of this virus to increase the ease of detection. This has however been promising for diagnosis of new influenza strains and research is currently ongoing. Use of variable nucleotides tandem repeats (VNTR) have been done to ensure that using the sequence of samples from patients would show the biomarkers of the causative virus. This is expected to be successful in typing the different strains of influenza. These techniques are easy since they only involve PCR using specific primers but should be done by experts so that the proper deductions can be made thus increasing their cost.
The analysis of the pathology of novel disease is important since it offers clues on the characteristics and type of disease, risks associated with the disease, and also plans on how to formulate treatment for the disease. With the current increase in new diseases and mutations of existing diseases as in the case of influenza, there is a need to study the pathology of the disease so as to diagnose it. This has however been promoted by the emergence of molecular techniques which analyze diseases at the genetic level using nucleic acids and protein biomarkers. This has been effective in formulating treatment and has further been promoted by the new trends of proteomics and genomics, which both fall under a new field of bioinformatics. In the field of genomics, the use of full-length DNA and RNA and consequential sequencing has helped study the trends of the influenza virus marking its mutation which are the result of emergence of new pandemics such as the avian flu pandemic that killed millions. It has helped the incorporation of biotechnology using microsatellites and SNPs to ensure patterns making and fingerprinting in RNA of viruses have been done. In proteomics, new methods such as SDS PAGE and Western blotting among others have been introduced to ensure effective screening apart from microscopy which is too time-honored. Their incorporation with technologies such as real-time PCR and microarrays with flourimetry and immunological assays has resulted to efficient and cost-effective diagnosis of novel diseases as in the case if influenza strains by helping in their typing. This is thus foreseen to help in development of better cure for this disease that is cost effective and does not have side effects such as resistance, ensuring high life expectancy and cure for such patients. This is expected to bring a new revolution to patho physiology and pathology of diseases in the contemporary society.
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