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agglutination, precipitation, complement fixation and virus neutralization tests) have proved useful in infectious disease diagnosis, but they suffer from several drawbacks such as poor performance and lack of standardization. The development and application of molecular diagnostic techniques has initiated a revolution in the diagnosis and monitoring of infectious diseases. Clinical microbiology laboratories increasingly rely on molecular diagnostic techniques. The various formats of nucleic acid amplification are the most frequently used molecular tests in the diagnosis of infectious diseases. In many clinical settings, polymerase chain reaction (PCR) is clearly the method of choice due to its exquisite sensitivity and specificity . Advances in molecular biology over the past 10 years have opened new avenues for microbial identification and characterization. When methods for microbial genome analysis became available, a new frontier in microbial identification and characterization was opened[2, 3]. Molecular screening programs for infectious diseases are developed to detect symptomatic and asymptomatic disease in individuals and groups. Persons at high risk, such as immunocompromised patients or those attending family planning or obstetrical clinics, are screened for CMV and Chlamydia infection respectively. Likewise, all blood donors are screened for blood-borne pathogens. One of the most highly touted benefits of molecular testing for infectious diseases is the promise of earlier detection of certain pathogens. The rapid detection of M. tuberculosis directly in clinical specimens by PCR or other amplification-based methods is quite likely to be cost-effective in the management of tuberculosis. Other examples of infectious disease that are amenable to molecular diagnosis and for which management can be improved by this technology include HSV encephalitis, Helicobacter pylori infection, and neuroborreliosis caused by Borrelia burgdorferi. For HSV encephalitis, detection of HSV in cerebrospinal fluid (CSF) can direct specific therapy and eliminate other tests including brain biopsy. Likewise, detection of H. pylori in gastric fluid can direct therapy and obviate the need for endoscopy and biopsy. PCR detection of B. burgdorferi in CSF is helpful in differentiating neuroborreliosis from other chronic neurologic conditions and chronic fatigue syndrome[1, 4, 6, 7]. Literature Review Molecular diagnostic techniques and tools have proven readily adaptable for use in the clinical diagnostic laboratory and promise to be extremely useful in diagnosis, therapy, and epidemiologic investigations and infection control. Molecular methods such as genetyping, nucleic acid amplification techniques, microarray analysis and nucleic acid hybridization, have allowed identification of different subtypes of pathogens and have greatly reduce the time taken in diagnosis over conventional microbiologic testing in many ways . Automation and high density oligonucleotide probe arrays (DNA chips) also proved to be of great clinical significance in infectious disease diagnosis. Commercial kits for the molecular detection and identification of infectious pathogens have provided a degree of standardization and ease of use that has facilitated the introduction of molecular diagnostics into the clinical microbiology laboratory (Table 1). The use of nucleic acid probes for identifying cultured organisms and for direct detection of organisms in clinical samples has greatly improve the clinical diagnostic process. Although these probe tests are still widely used, amplification-based methods are increasingly employed for diagnosis, identification and quantitation of pathogens, and characterization of antimicrobial-drug resistance genes. Nonetheless, molecular strain typing, or genotyping, has proven useful in guiding therapeutic decisions for certain viral pathogens and for epidemiologic investigation and infection control.
2 Table 1: Commercially Molecular diagnostic test kits for infectious disease  Test method Companies Chlamydia trachomatis PCR, LCR,TMA, Hybrid- Roche Abbott, Gen-Probe, detection capture Digene Neisseria gonorrhoeae LCR, PACE2, Hybrid- Abbott, Digene, Murex capture Mycobacterium PCR, TMA Roche, Gen-Probe tuberculosis HPV screening Hybrid-capture Digene CMV Hybrid-capture, NASBA digene HIV quantitation PCR Roche T. vaginalis Hybridisation Becton-Dickinson
The hybrid capture systems detect human papilloma virus (HPV) in cervical scrapings, herpes simplex virus (HSV) in vesicle material, and cytomegalovirus (CMV) in blood and other fluids. All these tests have demonstrated sensitivity exceeding that of culture or immunologic methods for detecting the respective pathogens but are less sensitive than PCR or other target amplificationbased methods. Therefore among the most widely employed molecular diagnostics techniques nowadays we have: Nucleic Acid Amplification Techniques: Nucleic acid amplification techniques increase sensitivity dramatically while still retaining a high specificity. Invented by Cetus scientist Kary Mullis in 1983 [2, 3], Nucleic acid amplification provides the ability to selectively amplify specific targets present in low concentrations to detectable levels; thus, amplification-based methods such as polymerase chain reaction (PCR), Isothermal amplification, Ligase chain reaction (LCR) Nucleic acid sequence base amplification (NASBA) Transcription mediated amplification (TMA) and Strand displacement amplification (SDA) offer superior performance, in terms of sensitivity, over the direct (non-amplified) probebased tests. PCR (Roche Molecular Systems, Branchburg, New Jersey) was the first such technique to be developed and because of its flexibility and ease of performance remains the most widely used molecular diagnostic technique in both research and clinical laboratories. Because the target DNA or RNA may be present in very small amounts in clinical specimens, various signal amplification and target amplification techniques (e.g. PCR) have been used to detect infectious agents in clinical diagnostic laboratories. The basic technique of PCR includes repeated cycles of amplifying selected nucleic acid sequences[1, 2]. Each cycle consists of three steps: denaturation, annealing, and polymerization. During denaturion, the 2 strands of the helix of the target genetic material are unwound and separated by heating at 90° to 95°C. During annealing, or hybridization, oligonucleotide primers bind to their complementary bases on the single-stranded DNA. This step requires a much cooler temperature, 55°C. Finally, during polymerization (at 75°C), the polymerase reads the template strand and quickly matches it with the appropriate nucleotides, resulting in 2 new helixes consisting of part of the original strand and the complementary strand that was just assembled. The process is repeated 30 to 40 times, resulting in millions of identical DNA copies[1-3]. PCR is increasingly applied in the diagnosis of viruses, bacteriae, parasites, and fungi. Qualitative PCR is widely used for a range of infections including herpesviruses (HSV, VZV, CMV, EBV), enteroviruses, parvoviruses, respiratory viruses, SARSCoV, and poxviruses, as well as general or specific bacterial screening, such as Neisseria meningitidis, Streptococcus pneumoniae, Bordetella pertussis, and Borrelia spp. Ligase chain reaction (LCR): is a probe amplification technique first described in 1989 by Wu and Wallace , LCR is a modification of PCR, where two adjacent probes hybridize to one strand of the target DNA. The small gap between the two adjacent primers is ligated by a highly specific
3 thermostable DNA ligase to form a single probe. The ligated products then serve as templates for the amplification process. LCR allows the detection of only single base pair mutations, and it is very specific. The most common application of LCR is in the diagnostics of Chlamydia trachomatis in cervical and urine samples[1-3]. Isothermal amplification techniques are based on a distinct enzyme that does not require thermocycling (heating and cooling cycles), which can take several hours. Isothermal amplification is rapid; it usually requires less than an hour to perform. However, the technique is currently fairly expensive. NASBA analysis is performed in isothermal conditions, the target RNA is converted to doublestranded DNA by using T7 RNA polymerase, RNaseH, and a primer with a T7 promoter. The DNA acts as a template to produce multiple copies of RNA with a polarity opposite that of the target, which can be used for the production of additional DNA templates. Clinically, NASBA technology is used in clinical diagnostics of many infectious disease such as Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens. Transcription-mediated amplification (TMA): The principle of TMA is similar to NASBA, except that it uses the RNaseH activity of reverse transcriptase, whereas NASBA uses a separate enzyme for that. In 1999 TMA was used in the detection and diagnosis of West Nile virus (WNV) in the US, TMA technology is heavily employed in the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine and urogenital swab specimens. SDA is another non-PCR nucleic acid amplification technique, developed in 1991. In this system, DNA polymerase initiates DNA syntheses at a single-stranded nick and displaces the nicked strand during DNA synthesis. The displaced single-stranded molecule then serves as a substrate for additional simultaneous nicking and displacement reactions. This isothermal DNA amplification procedure uses specific primers, a DNA polymerase, and a restriction endonuclease to achieve exponential amplification of target. SDA has two important advantages. Except for the initial denaturation step, SDA is isothermal and requires no specialized thermocycler. In addition, SDA can be applied to either single- or double-stranded DNA[2, 3]. SDA found application in the diagnosis of N. gonorrhea infection from female endocervical swabs and male first void urine samples. Nucleic Acid Hybridisation Use of solution-phase hybridization has allowed tests to be performed singly or in batches in a familiar microwell format. Although direct detection of organisms in clinical specimens by nucleic acid probes is rapid and simple, but it suffers from lack of sensitivity. Most direct probe detection assays require at least 104copies of nucleic acid per microliter for reliable detection, a requirement rarely met in clinical samples without some form of amplification. Probe hybridization is useful for identifying slow- growing organisms after isolation in culture. Identification of mycobacteria and other slowgrowing organisms such as the dimorphic fungi (Histoplasma capsulatum, Coccidioides immitis, and Blastomyces dermatitidis) has certainly been facilitated by commercially available probes. The commercial probe systems that use solutionphase hybridization and chemiluminescence for direct detection of infectious agents in clinical material include the PACE2 products of Gen-Probe and the hybrid capture assay systems of Digene and Murex (Table 1 above). These systems are user friendly, have a long shelf life, and are adaptable to small or large numbers of specimens. The PACE2 products are designed for direct detection of both Neisseria gonorrhoeae and Chlamydia trachomatis in a single specimen (one specimen, two separate probes). The hybrid capture systems detect human papilloma virus (HPV) in cervical scrapings, herpes simplex virus (HSV) in vesicle material, and cytomegalovirus (CMV) in blood and other fluids. All these tests have demonstrated sensitivity exceeding that of culture or immunologic methods for detecting the respective pathogens but are less sensitive than PCR or other target
4 amplification-based methods Beside being used in the traditional Southern, Western and Northern blot hyridisation which are applied to detect or identify the DNA, Protein and RNA sample respectively, there are highly modified hybridization techniques that are currently used in molecular diagnostics, Among them are: Flouricence in situ Hybridisation (FISH): This technique allows the detection of microbial nucleic acid directly from the sample (or cultured sample) without prior nucleic acid amplification. FISH is based on the use of flouresence-labelled oligonucleotide probes specifically attach to their complementary DNA sequence target on the genome and label that region with fluorescence color (e.g., Texas red, FITCI green, acridine orange). The labeled region can then be easily visualized under a fluorescence microscope. the technique consists of specimen fixation on a microscope slide, hybridizing the prepared sample with a specific fluorescent-labelled probe, and visual detection of the hybridization with a fluorescent microscope. In molecular diagnosis, the technique is used, e.g. in detection of Helicobacter pylori from gastric biopsy specimens. Identifying bacteria from CSF, or Staphylococcus aureus from blood cultures. Line probe assay The line probe assay (LiPA) is another nucleic acid hybridization test, in which specific oligonucleotide probes are attached at known locations on a nitrocellulose strip as parallel lines and hybridized with biotin-labelled PCR products. One of the widely used LiPA applications is rapid detection of rifampicin resistance in Mycobacterium tuberculosis. The LiPA technique is also applied in the detection of antiviral drug-resistant mutations of HBV. Molecular Genotyping: Laboratory characterization of microbial pathogens as biologically or genetically related is frequently useful in investigations. Molecular typing is performed to determine whether different isolates give the same or different results for one or more tests. Epidemiologically related isolates share the same DNA profile or fingerprint, whereas sporadic or epidemiologically unrelated isolates have distinctly different patterns. If isolates from different patients share the same fingerprint, they probably originated from the same clone and were transmitted from patient to patient by a common source or mechanism. This of course, aid in quick diagnosis of the infectious disease most especially in an endemic or epidemic situation. The most widely used molecular typing methods include plasmid profiling, restriction endonuclease analysis of plasmid and genomic DNA, Southern hybridization analysis using specific DNA probes, and chromosomal DNA profiling using either pulsed- field gel electrophoresis (PFGE) or PCR- based methods.[9, 12] Genotyping of PCR amplicons can be performed by direct sequencing, by reverse hybridization to genotype-specific probes, by restriction fragment length polymorphism, or by detection of single nucleotide polymorphisms with mass spectrometry. Genotyping plays an important role in the management of HCV infection, as response to treatment considerably varies between different genotypes; patients with HCV genotype 2 or 3 infection respond better to treatment than those with genotype 1 infection. Microarrays: Microarrays consist of a two-dimensional matrix of biomolecules which are printed or synthesized on a glass, silicon, plastic or nylon membrane. Microarrays can detect both nucleic acids and antibodies, which attach to the immobilized biomolecule, which can be, e.g., an oligonucleotide or a protein. Positive reaction can be detected with highly advanced scanners by use of target labeling with fluorescent probes or antibodies. In molecular diagnostics serological microarrays play an important role, several applications have already been proposed and introduced, including detection of rifampicin- and isoniazid-resistant M. tuberculosis, detection of chloroquine-resistant Plasmodium falciparum, and herpesvirus detection from Crebro spinal fluid (CSF) .
5 Mass spectrometry: Mass spectrometry allows the measurement of the molecular mass of a sample, which can be utilized in various clinical diagnostic settings. In this technique, the sample is first ionized, the ions are separated according to their mass-to-charge ratios, and finally the separated ions are detected according to the ion current. Ionization of the sample can be done in various ways, of which matrix-assisted laser desorption/ionization (MALDI) is widely used in the genotyping of single nucleotide polymorphisms. MALDI mass spectrometry is an important tool in typing of bacteria and viruses, which is useful, e.g. in detecting hypervirulent or antimicrobial drug resistant strains. The technique has been introduced, e.g. for genotyping of HCV and monitoring of quasispecies (a cloud of variant RNA viruses that arise from mutations over time within a viral isolate) in chronic HCV infection. It is also used in the detection of hypervirulent N. meningitidis strains[1, 5, 9, 10, 13]. In Conclusion, based on what I have cited above, I learnt that among the most highly touted benefits of molecular diagnostics techniques for infectious diseases is the promise of earlier and quick detection of certain pathogens. For instance, the rapid detection of M. tuberculosis directly in clinical specimens by PCR or other amplification-based methods is quite likely to be cost-effective in the management of tuberculosis. Other examples of infectious disease that are amenable to molecular diagnosis and for which diagnosis was improved by this technology, the inability to culture and analyze the principal etiologic agent of non-A, non-B hepatitis limited medical advances in this area. Using various molecular methods, however, investigators have been able to isolate hepatitis C virus (HCV) nucleic acid. Analysis and cloning of the HCV genome has provided the viral antigens necessary for the development of specific serologic tests. Another unculturable microbe that has been specifically detected by PCR and probe analysis is Tropheryma whippelii, the causative agent of Whipple disease. Because of the inability of this organism to grow on conventional media and the lack of a serologic test, diagnosis of Whipple disease is usually based on clinical and specific biopsy findings. But now, due to the advances in the molecular detection of T. whippelii this dilemma has become a case of history. Therefore, in my opinion, I believed that molecular diagnostic techniques contribute more to the rapid diagnosis of infectious diseases, for example, Accurate and early diagnosis of a disease state such as a bacterial or viral infection, or in a more complicated situation hemorrhagic diarrhea, means live saving because proper medical interventions can be applied in a timely manner before it is too late to treat the disease. DNA-based diagnosis is now done mostly with amplification technology breakthroughs such as polymerase chain reaction (PCR). Moreover, protein based diagnosis such as blood antibody test for a disease specific antigen (e.g., HIV and HPV virus infections; prostate-specific antigen for prostate cancer) are more accurate, convenient, and noninvasive. Issue like viral load testing and genotyping of HCV that are useful in determining the use of expensive therapy, can be used to justify decisions on extent and duration of therapy. With AIDS, viral load determinations plus resistance genotyping have been used to select among the various protease inhibitor drugs available for treatment, improving patient response and decreasing incidence of opportunistic infections. Nonetheless, Molecular techniques have been successfully used in the investigation and control of classical and emerging nosocomial pathogens, such as the enterobacteriaceae, Pseudomonas aeruginosa, Staphylococcus aureus, coagulase-negative staphylococci, enterococci, Candida albicans, M. tuberculosis, and Chlamydia pneumoniae. Application of DNA probe-based assays allows the diagnosis of other nosocomial infections caused by respiratory syncytial virus, varicellazoster virus, herpes simplex virus, and legionella to be made in only a few hours.
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5. 6. 7. 8.
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