Diagnostic molecular microbiology and its applications: Current and future perspectives

Infectious diseases are a major cause of morbidity and mortality throughout the world. Although the field of diagnostic microbiology has substantially evolved over the past few decades, it is still heavily reliant on cultures and serology, which while being cost effective is largely time consuming and sometimes less sensitive and specific. Molecular biology on the other hand has developed with rapid and confirmatory diagnosis. However, the merger of molecular diagnostics in routine diagnostic microbiology labs has been slow paced mostly due to higher costs and lack of infrastructure. Molecular methods have progressed beyond identification to detect antibiotic marker genes, fastidious bacteria, and uncultivable microbes. It has found a scope in mycology and parasitology, in which the basic conventional techniques may sometimes be unable to make a definitive diagnosis of the concerned pathogens. In the field of virology where culturing may be impractical in some clinical settings, it has developed various multiplexing procedures which can detect and quantitate the viral copies present in the crude specimen. Besides the field offers different types of permutations and combinations like direct sequencing for finding the variants, whole genome sequencing, epidemiological testing like plasmid profiling, RFLP etc. which can point out the infection and different types of PCR customisation such as nested, multiplex and Real Time (RT) PCR. In the present review, we have described the various molecular typing techniques and their application in microbial testing with a briefing of the tests that have been already standardised with relevant sample acceptance and rejection criteria for commonly encountered pathogens and pointed towards future directions converging automated closed DNA, RNA extraction/amplification platforms, Next Generation Sequencing (NSG), Microarrays and Digital PCR (dPCR) into its arsenal. Correspondence to: Vivek Bhat, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, India, E-mail: vivekbhat2005@yahoo.com


Introduction
Infectious disease is the main cause of mortality and morbidity worldwide. The different types of infection caused by various types of pathogens including bacteria, viruses, parasites and fungi are ever increasing [1]. Increasing age, diseases such as cancer, immunosuppressions etc are contributing factors. Although the era of antibiotic, antiviral, antimycotic, and antiparasitic has led to alleviation from these infectious agents, the rampant use of these agents has led to the emergence of multi drug resistant (MDR) pathogens which if not diagnosed and contained early, could spread to large geographical areas [2]. The role played by diagnostic systems is the 'golden standard' for enacting rapid treatment regimes. Routine clinical& microbiological procedures such as cultures, serology, and microscopy still remains the procedures of choice in terms of diagnosis and are also cost effective [3]. However the routine microbiological cultures are by themselves, not confirmatory tests for the accurate diagnosis of the pathogen. Even with cultivable bacteria, cultures fail to reveal an organism in many patients with sign and symptoms consistent with infectious disease. Due to the above reasons, there is a large scope for molecular biology procedures in the diagnostic clinical microbiology laboratory.
With the advent of PCR technology about 30 years ago came the era of molecular diagnostics. It is due molecular testing that the phenotypes exhibited by a pathogen can be genetically confirmed [4]. It ensures rapid diagnosis at a cost effective price, thus increasing the diagnostic arsenal for pathogen identification. This review enlists the various types of molecular typing methods which have been inculcated in molecular microbiology diagnostic labs. It also describes the need for more advanced molecular methods for increasing the sensitivity and specificity of diagnosis and also the routine protocols for various bacterial, viral, fungal, and parasites that have been already standardised at the genus and species levels with a briefing on sample acceptance and rejection criteria for the enlisted pathogens.

Molecular typing techniques
The advent of nucleic acid amplification techniques has led to the advancement of molecular typing techniques [5]. It offers the advantage of rapid confirmatory diagnosis. Since the start of the field of medical microbiology, the main techniques have relied on the identification of phenotypic characters like biochemical characterisation, morphological view, and cultures. The inert nature of the DNA molecule makes it the most suitable marker for confirmatory diagnosis [6]. The various types of molecular techniques have been reviewed in the following sections.

Restriction analysis
The microbial DNA consists of various sites containing sequences which are repetitive in nature. These repeats are tandemly repeated after constant intervals [6]. Also restriction enzymes or endonuclease (R.E) a class of DNA-cleaving enzymes isolates from bacteria are used to cleave these DNA at a particular and specific sequence which results in the fragmentation of DNA molecule. This technique thus can detect the fragmentation pattern of the target and compare it with the in house pathogenic strains and handout results whether the isolated pathogens are similar or dissimilar in origins. Thus, in this technique

Plasmid profiling
In these technique plasmids which are extrachromosomal DNA elements are cut with restriction enzymes (RE). Transfer of plasmids is very common between members of the family Enterobacteriaceae. It is mainly useful in epidemiological outbreaks in which the plasmids are cut with the same RE which may result in same restriction patterns from plasmids isolated from various pathogens in the outbreak area thus showing the profiles in outbreak associated strains [8]. It has been carried out in opportunistic multidrug resistant Pseudomonas aeruginosa isolated from wound infections, which carried a pattern of resistance to various antibiotics [9]. A similar study was used for screening of Neisseria gonorohoeae [10] and Shigella spp. [11] among others [12,13]. It has been also widely carried out to identify multidrug resistant isolates present in sewage associated with health care centres [14]. A study carried out used this technique for the study of the emergence of drug resistance in diarrheagenic E. coli in paediatric population in a developing country [15,16].

Restriction fragment length polymorphism (RFLP)
RFLP has been mainly used in multi drug resistant Mycobacterium tuberculosis outbreaks for simultaneous identification and differentiation in which polymorphisms present in DR locus which is characterised by repetitive sequence interspersed between non repetitive sequences has been hybridised by a probe known as spacer oligotyping or spoligotyping.

Pulse field gel electrophoresis (PFGE)
With the advent of RFLP, there arose a need to perform high resolution separation on agarose gel to look out for similarity in banding patterns obtained after RE digestion. In 1982, Schwartz introduced the concept that DNA molecules larger than 50kb can be separated by using two alternating electric fields. This led to the technique of PFGE. It is based on the digestion of bacterial DNA with RE that recognises few sites along the chromosome, generating large DNA fragments (30-800Kb). The basis for PFGE separation is the size-dependent time-associated reorientation of DNA migration. When visualised electronically bacterial isolates with identical or very similar band patterns are more likely to be related genetically than with more divergent band patterns. PFGE has been used for the detection of Listeria monocytogenes and Campylobacter spp. which are food borne pathogen difficult to culture [22,23]. It is also widely applied to the diagnosis of eukaryotic DNA like parasites which are dense and complex in nature [24].

PCR amplification of gene target
This method is the basis of all types of PCR technology. In this method designed primers which amplify a unique region of microorganisms are used. Thus, this method is target specific.

Multiplex PCR
Multiplexing is one of the most widely applied techniques in the field of diagnostic microbiology. It is a variant of PCR, which enables simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. Its main use is in the field of virology in which primers are designed to detect different types of viruses from a sample [

Nested PCR
Involves two consecutive PCR reactions of 25 cycles. The first PCR uses primers external to the sequence of interest. The second PCR uses the product of the first PCR in conjunction with one or more nested primers to amplify the sequence within the region flanked by the initial set of primers. It has been designed for various bacterial [30-33], fungal [34,35], and parasitic infections.

Real time PCR (RT-PCR)
It allows viewing the increase in the amount of DNA as it is amplified. All real time PCR systems rely upon the detection and quantitation of the fluorescent reporter, the signal of which increases in direct proportion of the amount of PCR product in a reaction. The most economical reporter is the double strand DNA specific dye SYBR green, which upon excitation emits light. However SYBR green has its limitations as it will bind to any double stranded DNA in a reaction [36]. Two most popular alternatives to SYBR green are TaqMan and molecular beacons. Both technologies depend on hybridisation probes relying on fluorescence resonance energy transfer (FRET) and quantization [

Direct sequencing
Direct sequencing is the most reliable method for the detection of sequences at the molecular level. The first whole genome sequenced was that of bacteriophage φX174. Given below is a briefing of some of the methods applied in microbial sequencing. It is noteworthy that sequencing has helped to differentiate fungi and parasites at the species level which is difficult to achieve by current morphological and phenotypic techniques [41].

Whole genome sequencing (WGS)
WGS has changed the landscape of molecular biology with its advent in 2005 by Roche. It is the most cost effective approach for solving many epidemiologic outbreaks and confirmation of pathogens after cultures [42,43]. The near market platforms available are Roche 454, Pacific Biosciences, Ion Torrent, and IlluminaMiSeq which can perform operations in 48hrs [44]. In terms of sample preparation for pathogens Oxford Nanopore is also very reliable [45].

Target gene sequencing
It is the most common method of sequencing used which relies on Sanger dideoxy chain terminator method. In this the primers complementary to the gene of interest are used to amplify the band containing the gene of interest and sequenced using an automated sequencer like the Applied Biosystems 8180xl genetic analyser. It is used for diagnosing multi drug resistance in microorganisms like presence of acquired carbapenamase resistance geneNDM-1 gene [28,46].

Universal gene target
These are specific sequences used in microbial diagnostics which are highly conserved and also impart certain phylogenetic traits at the genus and species levels.

Ribosomal RNA (rRNA)
The bacterial pathogens which are unidentifiable with routine testing, slow growers, uncultivable can be identified by amplification of DNA encoding ribosomal RNA genes followed by DNA sequencing. In bacteria there are three genes that make up rRNA functionality, i.e., 5S, 16S, and 23S rRNA. The 16S has been employed for identification purposes due to it being highly conserved and having a moderate copy number depending on the genus. Besides 16S rRNA genes are found in all bacteria and accumulate mutations at a slow, constant rate over time, hence serving as "molecular clocks" [47]. The use of this technique has led to the discovery of novel clinical isolates and culture negative infections [48]. It has been widely used to identify tuberculosis and non-tuberculosis infections [49] and biothreat agents like Brucella spps [50]. Recently, there have been several reports regarding the use of the large subunit (23S rRNA) which is specific for bacterial species recognition. It has been used frequently for the detection of Stenotrophomonas maltophila from patients with cystic fibrosis [51].

Heat shock proteins
The use of 16S rRNA although applied routinely bears some limitations. Strains with less than 97.5%16S rRNA gene sequence identity are unlikely to be related at the species level. However, there are a number of strains that share less than 50% DNA similarity by reassociation and therefore are classified as distinct species, but share 99% to 100% 16S rRNA gene sequence identity. For example, Mycobacterium chelonae and Mycobacterium abscessus have more than 99% 16S rRNA gene sequence identity, but their DNA similarity by reassociation is only 35% [52]. In such circumstance sequencing of essential genes such as the heat shock proteins (HSP; HSP60, HSP65, groEL, groER, etc.), have been shown to be useful [53,54]. The heat shock response is an important homeostatic response that enables the microbial cells survive the conditions of stress. This phenomenon is observed in all microorganisms from bacteria, fungi, to parasites [55]. A polymorphism in HSP 70 of Leishmania spps has been used for the differentiation of neotropical Leishmania species, as well as Trypanosoma cruzi [56].

Antibiotic resistance gene
Antibiotic resistance among microorganisms has become a worldwide issue. A review published by Fluit et al. has highlighted on the increasing numbers of multidrug resistant bacteria and its molecular detection [4]. Molecular detection by Xpert MTB/RIF automated system (GeneXpert, Cepheid) has found a wide scale acceptance for the detection of rifampicin resistant tuberculosis, which is on the rise in developing countries [57]. Also the detection of NDM-1 and its variant has been carried out by conventional PCR against the NDM-1 gene with members of the enterobactericeae family [58]. For detection of H pyroli a slow growing ulcerative pathogen resistance markers amplification strategies are used [59]. In parasitology it has found applications for detection of chloroquine resistant Plasmodium falciparum by using nested PCR followed by mutation-specific, restriction-endonuclease digestion for detection of mutations in pfcrt and pfmdr gene for treatment regimens [60].

Molecular standardised diagnostic protocols
In this section we review some of the standardised protocols employed in a diagnostic molecular microbiology laboratory at the genus and species levels for various clinically important pathogens including bacteria, fungi, parasites and viruses. We also include the sample acceptance and rejection criteria as cited in the literature.

Bacteriology
Most medically important classes of pathogens belong to the class of bacteria and their molecular detection is the need of the hour for rapid confirmatory diagnosis [61]. Molecular standardised protocols have been cited in Table 1. Whipple's disease is a rare but fatal infection caused by Tropheryma whipplei the diagnosis of which until recently only relied on histopathology and electron microscopy, often from post morterm material. PCR now allows the diagnosis of neuro-Whipple's disease and endocarditis by the detection of T. whipplei from noninvasive specimens [62]. Molecular diagnosis can also help to diagnose uncultivable pathogens like that of cat scratch disease Bartonella henselae, Q fever due to Coxiella burnetti, and male urethritis caused by Mycoplasma genitalium [63].

Virology
The field of virology has already adopted diagnosis by molecular methods and Table 2 sites the medically important viruses encountered in clinical labs. Genotypic mutation analysis of the virus which correlates with phenotypic resistance has been designed for retroviruses like HIV in treatment naïve and for viral rebound in patients already on treatment, to establish if resistance has developed which requires a change in treatment [64,65].

Mycology and parasitology
The molecular diagnosis of parasites and fungi has been a revolution as they are difficult to diagnose at the species level [24]. Although still in a developing phase in parasitology, the lack of culturing techniques has necessitated diagnosis by molecular techniques. The medically encountered parasites and fungi are elaborated in Table 3. In mycology the most common testing employed is for molecular detection of Pneumocystis jiroveci (Pneumocystis carinii) an opportunistic fungus causing severe pneumonia in HIV-infected patients, which has made the microscopic and silver staining of tissue specimens a thing of the past [66].

Future applications
The introduction of PCR technology by Nobel Prize winning scientist Dr Kary Mullis in 1983 has revolutionised the field of medical diagnostics including microbiology. A number of automated extraction/detection systems are available in the market which will become essential in molecular labs in the future. This system, such as QiaSymphony (Qiagen) offers automated DNA extraction, PCR setup, including reagent preparation, dilution series and sample pipetting.
In future a diverse array of next generation sequencing (NSG) platforms like Roche 454 will be routinely used as it can sequence ~500 bp approaching that of Sanger sequencing at lower cost [67]. For large bacterial sequences IlluminaHiSeq platform sequence analysers are the most useful, as it delivers ~300 Gb of raw data per eight-lane flow cell in the form of a 100 bp reads and provide rapid sequencing [68]. The future is promising for bench top NGS analysers, like Ion PGM and the IlluminaMiSeq than the larger counterparts [43]. A new platform of NSG released in 2012 by UK startup Oxford Nanopore Technologies has led to landmark improvement by introducing two sequencing platforms (the GridION and MinION) capable of delivering highthroughput, ultra-long sequence reads at low cost. It offers chips that are configured to read 2,000 or 8,000 pores simultaneously and that reads can be up to tens of kilobases in length. Because it reads native DNA, the Oxford Nanopore technology is anticipated to work with fairly crude samples and low DNA concentrations this makes colony processing a thing of the past [45]. Another future application lies in Micorarrays which are designed to simultaneously monitor wholegenome, host and pathogen gene expression, providing a complete view of disease progression [69]. The most recent generation of microarrays performs sequence analysis often performing WHG in a single experiment. One such array the Gene Chip Customseq Arrays (Affymetrix, CA, US) can sequence up to 300 kb of a genome in 48hrs with minimal amplification of the genomic target thus enabling pathogen detection and identification [70]. The limit of detection in Gene Chip is as low as 10 femtograms for pathogenic DNA much below the detection limits of existing technologies. Thus the flexibility and high-throughput nature of microarrays offers an unprecedented opportunity for infectious disease diagnosis. Also the platform which holds a huge scope is digital PCR (dPCR) which is still in its early stage of development. The sample is diluted and partitioned into hundreds or even millions of separate reaction chambers so that each contains one or no copies of the sequence of interest. By counting the number of 'positive' partitions (in which the sequence is detected) versus 'negative' partitions (in which it is not), scientists can determine exactly how many copies of a DNA molecule were in the original sample [71]. In one type of platform manufactured by Fluidigm and Life Technologies, reactions are created in within specially designed chips or plates. While in other platforms developed by BioRad and RainDance, reagents are sequestered into individual droplets i.e., Droplet Digital PCR (ddPCR).
Thus the future of diagnostic microbiology holds a tremendous scope offered by molecular diagnostics which has the potential to transform the precision, sensitivity and specificity of pathogen detection in a rapid and cost effective manner. 16