Infectious diseases are significantly different than other diseases given the fact that it can easily be dispersed and may end up in an epidemic form. Hence, a rapid detection and diagnosis followed by immediate treatment is extremely crucial. Traditional methods of diagnosis of infectious diseases are often slow, lacks real-time information and in many cases quite difficult to carry out. In view of this, the newly emerged field of molecular biology calls for proper attention. Molecular technologies, based on gene and nucleic acid-based tools, put forward the solution to those problems in a more improved and efficient manner. The following discussion will depict the developments in the field of molecular biology in connection to the clinical diagnosis of infectious diseases.
Concept of Molecular Biology and Infectious Diseases
Molecular biology, a multidisciplinary branch of biology closely associated with theoretical and practical areas of chemistry, particularly genetics and biochemistry, deals with the molecular basis of biological activity. This relatively new discipline is involved in understanding the processes and interactions within and between various systems of a cell, especially the mechanisms at macromolecular levels such as synthesis of DNA, RNA and proteins as well as processes of genetic expression, replication or mutation (Alberts et al. 2002). Enlightened with molecular level biological processes and interactions, in the course of its development, molecular biology has emerged as the basis a very crucial approach in fight against infectious diseases - one of the greatest challenges in medical biology.
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At this point, it is important explain the concept of 'infectious disease' to justify the context of discussion. According to World Health Organization (WHO), "infectious diseases are caused by pathogenic microorganisms, such as bacteria, viruses, parasites or fungi" that can be spread, directly or indirectly, from one person to another, and even from an animal to a person. Thus, infectious diseases, often called as communicable diseases or transmissible diseases, are defined as illness that are visible clinically through medical symptoms and are resulted from the transmission and presence of pathogenic biological agents. Nevertheless, in some cases infectious diseases may be asymtomatic. Causative agents of such diseases, known as 'infectious pathogens', include various strains of viruses, bacteria, fungi, protozoa and multi-cellular parasites. Often unusual proteins (i.e. prions) may cause infections (Ryan and Ray 2004). In connection to this, it is noteworthy to mention that infectious diseases can be transmitted through physical contact, contaminated food, body fluids, objects, airborne inhalation, or through vector organisms. In fact, diseases that transmitted through contact with an ill person or their secretions, or objects touched by them are referred to as contagious diseases, whereas there are other communicable diseases which usually require a more specialized route of infection, such as vector transmission, blood or needle transmission, or sexual transmission.
Molecular biology, as was mentioned earlier, has emerged as a readily adaptable and promising tool for use in the field of treatment of infectious diseases (Pfaller 2001). Now-a-days it is one the contemporary approach for laboratory/ clinical diagnosis, therapy, and epidemiologic investigations and infection control (Cormican and Pfaller 1996, Pfaller 2000). However, this still immature application has still a long way to go that requires further development with concerns such as ease of performance, reproducibility, sensitivity, and specificity of molecular tests are important, cost and potential contribution to patient care (Kant 1995). Nevertheless, given the ever growing threat from infectious diseases, for which traditional routine growth based culture and microscopy methods may not be adequate, molecular methods have the potentiality to be the arsenal over conventional microbiologic testing in diagnosis of infectious diseases (Tang and Persing 1999, Woods 2001).
A Brief History of Molecular Biology
On the way to discuss the potential uses and advancements of molecular biology in diagnosis of infectious diseases, an important point of departure should be the historical genesis of the field and chronology of developed methods. Despite its prominence in the contemporary life sciences, molecular biology is a relatively young discipline, originating in the 1930s and 1940s, and becoming institutionalized in the 1950s and 1960s. In its early days, mechanisms involved in gene expression, reproduction and mutation were undisclosed where as such issues were more discussed through Mendel's law of segregation  and the law of independent assortment  (Darden 1991, Darden and Maull 1977). Later on, a major step forward in the field of molecular biology was achieved through experiments conducted by Hershey and Martha Chase (1952) on tracking the chemical components of bacteriophage entering bacteria which revealed that it is not the proteins but the deoxyribonucleic acid (DNA) is the building block of gene. On this occasion, the concept of macromolecules in information transmission at the bio-celluler levels cut the age of molecular biology in detection and diagnosis of diseases.
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Development of Molecular Tools against Infectious Diseases
Since the revolution in concepts of molecular biology, practitioners and researchers have distinguished, isolated, and manoeuvred the macromolecular components of cells and in particular cases, organisms. These macromolecular components included DNA, which is the storehouse of genetic information, ribonucleic acids (RNAs), another information carrying tool that helps DNA synthesis and replication, and proteins, a key macromolecule of a biological entity providing the basis of important operators such as enzymes.
One of the basic mechanisms involved in molecular biology is the expression cloning which is basically a process of protein synthesis of certain type using DNA. A more delicate mechanism involved here is called the polymerase chain reaction (PCR) which is a technique for replicating DNA or a part of DNA (any particular sequence) as much as one wants, or alter in a desired fashion. For an instance, PCR method can be employed to mutate particular nucleotide or bases of a DNA, or to introduce restriction enzyme sites, so on and so forth. Use of such technologies in diagnosis of infectious diseases will be discussed in later sections. Another important tool is called the gel electrophoresis that is used to isolate DNA, RNA or proteins utilizing an electric field. Hybridization, bottling and probing of DNA are methods that involved manipulation and transportation of a DNA sequence and probing it to a target DNA. Allele specific oligonucleotide (ASO) is another technique that recognises even a single base mutation without involving other isolation techniques such as PCR or gel electrophoresis (Abir-Am 1985).
Techniques of molecular biology, more specifically, diagnosis techniques utilizing DNA/ RNA -based tests in detection of infectious diseases use all the above mentioned methods of isolating nucleic acids from cellular components from clinical samples. Restriction endonuclease enzymes, gel electrophoresis, and nucleic acid hybridization techniques are involved in these cases (Tang and Persing 1999). Due to the fact that quite often an extremely small amount of target DNA or RNA exists in the clinical samples, different signal amplification methods are associated along with the standard nucleic acid isolation techniques in detection of infectious agents. This dual approach is not only making it possible to detect infectious agents which are previously difficult due to uncultivatable nature, but also has made it readily curable through the characterization of antimicrobial resistance gene mutations (Bergeron and Ouellette 1998). Oligonucleotide probe arrays known as DNA chips are also of great potentiality in characterizing infectious pathogens. A number of techniques, with appropriate examples, currently being involved in molecular diagnosis of infectious diseases are described in Table 1.
Table 1: Examples of commercially available methods for detection of infectious diseases
Polymerase chain reaction (PCR)
Detection of Chlamydia trachomatis, Mycobacterium tuberculosis; HIV quantitation
Ligase chain reaction (LCR)
Detection of C. trachomatis, N. gonorrhoeae
Transcription-mediated amplification (TMA)
Detection of C. trachomatis, Mycobacterium tuberculosis
Strand displacement reaction (SDR)
Screening/detection of C. trachomatis/ N. Gonorrhoeae
Nucleic acid strand-based amplification (NASBA)
Detection of cytomegalovirus (CMV)
Detection of human papillomavirus (HPV); detection of C. trachomatis, N. gonorrhoeae
Group A strep detection; detection of Gardnerella, Trichomonas vaginalis, and Candida; Culture confirmation of Bacteria and fungi
Source: Compiled from Pfaller (2001).
Despite the fact that these commercially available methods are quite popular, however, some clinically important infectious agents  need investigator-designed methods. In a similar way, molecular strain typing or genotyping has been promising against several viral pathogens and against diagnosis and control of some epidemics (Pfaller 1999). In view of this, the following discussion will focus on some of the recent developments of molecular approach in diagnosis of infectious diseases.
The diagnosis and detection of infectious pathogens using nucleic acid probes is simple and rapid. However, it seriously lacks sensitivity requiring at least 104 copies of nucleic acid per microliter for reliable detection which is not always found in clinical samples. To overcome such problems amplification of the detection signal is required after probe hybridization to improve the sensitivity. This method is widely used in quantitative assays of virus such as HIV, hepatitis B virus and hepatitis C virus. On the other hand, PCR based methods, a target amplification-based approach, is much more analytically sensitive. The former method (signal amplification-based probe method) for diagnosis of such viruses employs branched chain DNA probes and QB replicase methods. Despite being less sensitive, the quantification by these methods is quite useful in diagnosis and monitoring response to therapy (Nolte 1999). The PCR method, a signal amplifying method to make detection highly sensitive, is one of the first of its kind and currently widely used in clinical laboratories because of its flexibility and ease of operation. Currently, PCR amplification based diagnostic methods for infectious diseases are commonly used for detecting N. gonorrhoeae, C. trachomatis, M. tuberculosis, and certain viral infections such as hepatitis B and C, HIV, cytomegalovirus (CMV), and enterovirus. The adaptability of the PCR method has also promoted researchers to develop investigation-developed assays that have successfully detected numerous other pathogens.
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In the field of diagnosis of infectious diseases, antimicrobial-drug resistance is another important issue. Traditionally this is tested through broth and agar based antimicrobial susceptibility analysis methods which reveal a phenotypic profile of the response of a certain pathogen against a certain antimicrobial agent; however, this process is extremely time consuming and not flawless. Molecular methods, in the other way round, are being able to detect antimicrobial-drug resistance in clinical settings very rapidly and have substantially contributed to our understanding of the spread and genetics of resistance (Cockerill 1999). This method can be directly applied to clinical samples and can simultaneously detect and identify infections by detecting specific antimicrobial-drug resistance genes (Bergeron and Ouellette 1998), and even by the detection of specific point mutations associated with resistance to antibiotic agents (Stuyver et al. 2000, Courvalin 1991).
Conventional phenotype based epidemiologic diagnosis of infectious agents  are slow and labour intensive and infested with too much variables. Newly developed molecular methods such as DNA-based typing techniques have eliminated much of such drawbacks and are now popular tools for epidemiological typing  (Pfaller 1999; Arbeit 1999). These methods detects the unique patterns for each plasmids and similar patterns, indicating same DNA profile, are detected for different isolates from different samples, that indicates epidemiologically related pathogens. Molecular typing methods have revealed the relationship between colonizing and infecting isolates in individual patients, distinguish contaminating from infecting strains, document nosocomial transmission in hospitalized patients, evaluate reinfection versus relapse in patients being treated for an infection, and follow the spread of antimicrobial-drug resistant strains within and between hospitals over time (Pfaller 1999; Pfaller and Herwaldt 1997).
Epidemiologically vital viruses cannot be grow or grown with difficulty in cell culture. Conventional methods of viral analysis are based on immunological, nucleic acid-based, and cell culture based techniques (Metcalf, Melnick and Estes 1995). These techniques are still slow and may take weeks to produce a reliable result. This lacking has prompted the molecular biologists to innovate more specific, rapid and sensitive methods such as nucleic acid-based amplification methods - PCR, reverse transcription-PCR (RT-PCR), or quantitative real-time PCR (qRT-PCR) (Jothikumar et al. 2005, Stellrecht et al. 2000, Wang et al. 2002). Real time monitoring of pathogenesis in living body has also become very important. In view of this, molecular beacons (MBs) can be mentioned as one of the most recent technologies currently under development for gene detection in living cells (Yeh et al. 2009). MBs are single-stranded oligonucleotide probes which have a stem-loop structure, and are labeled with a fluorophore and a quencher. The spontaneous hybridization between MBs and their target sequences is highly specific and can even distinguish a single nucleotide mismatch (Marras, Kramer and Tyagi 1999, Tyagi , Bratu and Kramer 1997, Tyagi and Alsmadi 2004). As few as 1 PFU of hepatitis A virus can be detected by MB-based reverse-transcription-PCR (RTPCR) proving its sensitivity and specificity of detection (Galil 2004). Uniformly distributed probes within the nucleus and cytoplasm can detect viruses with multiple replication and assembly strategies within different cellular compartments in their viral reproductive cycles. In this backdrop, insertion of oligonucleotides with the help of microinjection or streptolysin O have been employed (Mhlanga et al. 2005).
DNA-based assays also have greatly enhanced the capacity to explain how immune cells contribute to the defence against infectious pathogens. The flow cytometry (FC) technique is now one of the most recent and widely used methods for analysis of immune response. The basic principle involves staining individual cells with fluorescent molecules known as flurochromes which are passed through a laser beam (Figure 1). Following the exposure, the stained cells emit fluorescence that correlates with cellular response. Modification based on molecular biological theories, DNA intercalating fluorescent probes are used for staining. In the presence of any infectious agent, the immune system responses immediately and the cell DNA manufactures certain antibiotic protein. The FC detects the amount of DNA in the cell along with other produced molecules giving an extremely quick indication of infection. It is also possible to recognize a specific microorganism using complex protein probes (Cohena, Vernonb and Bergeronc 2008).
Figure 1: Principles of flow cytometry (FC)
Source: Cohena, Vernonb and Bergeronc 2008
Cohena, Vernonb and Bergeronc (2008) reported that "the Centre de Recherche en Infectiologie (CRI) of Université Laval in Québec City (Dr Michel G. Bergeron) and other researchers from U. Laval have proposed to develop an integrated, fully automated, and single-step portable micro-fluidic laboratory-on-a-compact disc (lab-CD) device for the rapid (<1h) detection of microbial nucleic acids in water". This method will also be able to identify the gene expression of the white blood cell in response to immunological activation. These two outcomes will jointly be able to detect infectious diseases rapidly.
With the ever advancing scientific knowledge and technological sophistication, the field of molecular biology is gaining more paces in running the turf of medical diagnosis. Some more recent developments includes nucleic acid extraction methods, database of conserved microbial genes, detailed phenotypic and genotypic analysis of 16,000 microbial strains, preparation of DNA/RNA-free reagents, micro-fluidic technologies, cationic polythiophene biosensors and optical detection, micro- and nanofabrication technologies, etc. More recently, the integration of nucleic acid extraction methods, real-time PCR, microarray hybridization, microfluidic technologies, optical detection, and micro- and nanofabrication led to the development of the first two FDA-approved rapid (<1h) real-time PCR assays (BD GeneOhmStrep B and BD GeneOhmMRSA) and of the first CD-based micro-fluidic device capable of performing DNA hybridization on a microarray, at room temperature in 15 minutes (Ho et al. 2005).
Molecular diagnosis of infectious diseases has been in the centre of attention in recent times because of its time-efficiency (fast), higher sensitivity and specificity, and ease of operation and automatization. However, the blessings of molecular detection methods are infested with higher costs. Nevertheless, that only limitation (in fact all other latest technology suffers the very same problem) can easily be outnumbered by the advantages of the molecular techniques. This comparatively young bio-medical field has still a long way to go and more research involving professionals from many related disciplines is required to improve and innovate newer methods of diagnosis and treatment of infectious diseases.