Molecular Diagnostic Strategies Used for the Detection of Microbial Pathogens in Human Beings: A Review

Introduction

The healthcare system is highly dependent on diagnostic techniques. The use of these techniques is essential for the right treatment and decision-making in healthcare systems. At every stage, medical diagnostic techniques provide critical insights regarding treatment, detection, diagnosis, and better management of health conditions [1]. There are many conventional methods used in diagnostics for the diagnosis of diseases caused by pathogens. However, many pathogens are difficult to grow in the laboratory using conventional methods of cultivation. Studies have indicated that the cultivation ability of bacteria from natural samples is less than 1% [2]. There are many different factors affecting cultivation, such as lack of nutrients required for microbial growth, culture medium toxicity towards the desired organisms in a mixed population, production of harmful substances which inhibit the growth of desired microorganisms, and metabolic dependence of desired strain on other organisms for growth [3]. Many rapid immunological detection techniques can also be utilized for either direct identification of microorganisms or with the help of the immune responses of the host. In this scenario, enzyme assays such as enzyme-linked immune sorbent assay (ELISA) remains the most commonly used diagnostic technique. This technique is quick, cheap, and easy to standardize, although its sensitivity is very low, with almost 104 cells needed to produce results. The specificity of the process is varied and depends upon the antibody type used and the dead and live microorganisms [1]. Due to the underlying issues, rapid diagnostic techniques based on genetic material, such as RNA, DNA, and antibodies have been formulated for the identification of disease-causing microbes. Ideally, pathogen detection methods must be accurate, robust, less costly, sensitive, reproducible, and easy to operate. The hybridization methods for nucleic acids and polymerase chain reaction (PCR) assay exhibit these properties for the better identification of pathogens [4].

Molecular diagnostic techniques are advantageous over conventional diagnostic protocols as the former are highly insensitive, specific, and less costly for microbial identification, as compared to traditional methods. Better identification and discrimination of pathogens is carried out currently based on the high specificity of molecular diagnostic methods. These methods detect disease-causing microorganisms due to their high sensitivity directly from the clinical and environmental samples without the need for cultivation [5].

The term ''molecular disease'' was invented in 1949 by Pauling and his colleagues by observing diseases such as sickle cell anemia caused due to alternation in the β-globin chain. Their findings led to the foundations of molecular diagnostics, although during the course of years several big transformations have occurred in this field. New and advanced molecular diagnostic methods have revolutionized the field of practical clinical microbiology. There are many molecular diagnostic methods being used for the diagnosis of infections since the 1980s. Furthermore, Koch's postulates have been applied to identify the pathogenicity of bacteria at the gene level, instead of the whole organism level [6].

This brief review gives an understanding of advanced diagnostic techniques used for the identification of the pathogens in clinical microbiology. Molecular diagnostic methods used for the diagnosis of diseases might be categorized into numerous major groups [7]. These involve PCRbased techniques (such as traditional PCR, real-time PCR, RT-PCR, nested PCR, and broad range 16S rRNA gene PCR), RNA/DNA probe-based methods (including blotting techniques and direct hybridization techniques, such as in-situation hybridization, fluorescent in situ hybridization, PNA FISH), amplification-based techniques (included array-based techniques, loop-mediated isothermal amplification (LAP)) [1] sequence-based methods (included Sanger based bideoxy sequencing, pyrosequencing, multilocus sequencing typing (MLST), MALDI-TOF MS, nanopore technology), and other diagnostic methods for post-amplification [8]. Each molecular method has its advantages and limitations, illustrated in Table 1.

In this review article, advancements in molecular techniques used for the diagnosis of microbial pathogens in human beings are discussed, as shown in Figure 3. The focus remains on the emerging molecular techniques used for the identification of microbial pathogens to monitor human diseases.

Table 1. Advantages and Limitations of Molecular Techniques for the Diagnosis of Microbial Pathogens in Human Beings [9].

2. CATEGORIZATION OF MOLECULAR TECHNIQUES

2.1. PCR-based Diagnostic Methods

The invention of polymerase chain reaction (PCR) has revolutionized the field of molecular diagnostics in terms of the identification, differentiation, and characterization of microorganisms, especially pathogens. PCR-based techniques have been highly specific and sensitive in their function and produce multiple copies from a single specific fragment of DNA. During the last 2 decades, many varieties of the standard PCR technique have been introduced. However, those mostly used for the identification of microbial pathogen detection are conventional PCR, nested PCR, real-time PCR, and broad range 16S rRNA gene PCR.

2.1.1. Conventional PCR. This amplification process runs with the help of specific primers via alternate cycles of denaturation, annealing, and elongation, as shown in Figure 1. This technique depends upon the efficiency of DNA extraction and concentration of deoxynucleoside triphosphate. At the beginning, PCR was only used for the detection of bacterial and viral diseases, although it is now used also for the detection of plant pathogens [10]. Sometimes, the function of PCR is compromised due to inhibitors present in the tested sample. However, PCR-specific primers designing is needed for the replication of DNA, which hinders its practical application in field sampling. Sometimes, a single primer pair is insufficient and produces faulty results. DNA primers and probes are widely used to limit this problem [11]. This is a much more specific technique but remains costly and needs a lot of labour in traditional PCR.

Figure 1. Overview of Conventional PCR [12]

2.1.2. Nested PCR. Nested PCR comprises two rounds of amplification. The first round is used to make copies of a large region of DNA via a single set of primers. Then, the product of the first round itself is used as a template for the second round by using two internal primers. Due to the two rounds of amplification, the cycle's sensitivity is enhanced, diluting any inhibitor present in the sample and increasing specificity. In the first round of amplification, if non-specific products are generated, then such products do not act as templates for the second round [13]. Due to an alternative round of amplification in separate tubes, there are chances of significant contamination. So, false positive results generated due to contamination and intense struggle are the main disadvantages of nested PCR [14]. Laboratories must take precautions and follow some advanced protocols to limit these drawbacks [15].

2.1.3. RT-PCR. Reverse transcription PCR was invented after the development of a large number of changes in traditional PCR. Today, RT-PCR is used in many medical and biological fields due to its diagnostic capabilities [16]. RT-PCR technique can identify the presence of any pathogens or nonpathogenic genetic material including viruses. In this molecular diagnostic technique, RNA is converted into complementary DNA (cDNA) reversely and then copies are made through the PCR technique with the help of random primers and reverse transcriptase enzyme. RT-PCR is widely used for the detection of infection-causing RNA viruses, such as Retroviruses. The diagnosis of RNA viruses is very useful for the development and checking of antimicrobial vaccine therapy [17]. Other conventional PCR could not distinguish between live and dead pathogens. However, with the discovery of RT-PCR, this drawback was eliminated. In fact, mRNA disintegrates in dead cells. So, mRNA is detected by RT-PCR for checking the viability of cells [18]. Fluorescent dyes, such as SYBR Green 1 or Taq Man probe, are used for the monitoring of reaction during the amplification process. The signal is produced when dye intercalates into the DNA. After each cycle of amplification, the amount of DNA increases with the increase of signal from the fluorescent dye [19]. RT-PCR is a less costly but a nonspecific technique. The disadvantage of this method is the formation of primer dimer when dye intercalates into the DNA. As a result, false positive results can be produced.

2.1.4. Real-time PCR. Real-time PCR techniques have been widely used in clinical microbiology. This method functions with the help of specific probes, which are labelled by fluorescent in addition to amplification techniques as shown in Figure 2. There are numerous different probes formats and instruments for the detection of pathogens are present, such as TaqMan hydrolysis probes, Scorpion probes, molecular beacons, and minor groove binding probes [20]. In this technique, the risk of contamination is minimized to a greater extent due to the closed tube system in real-time PCR. One of the major benefits of real-time PCR using fluorescence technology is that quantify genetic variations e.g., single nucleotide polymorphisms with the help of a set of precise probes for each possible SNP within the same species [21].

Figure 2. Schematic Representation of Real-time PCR [1]

2.1.5. Broad Range16s rRNA Gene PCR. Broad range 16S ribosomal ribonucleic acid gene polymerase chain reaction is used for pathogen detection and identification of clinical specimens [22]. This technique is usually applied for cases where bacterial culture gives negative results, despite suspected infection [23]. This technique is mainly used for fastidious bacteria that do not grow easily in the laboratory, such as Mycobacterium genovese, Coxiella burnetii, Ehrlichia chaffeensis, and Tropheryma whipplei [24]. Conserved regions of the 16S rRNA are ideal for designing primers, sequencing based on PCR, and alignment of sequences. Species identification is done by this gene because of having species-specific variable regions [25].

Universal primers have been utilized for the amplification of PCR products and gene sequencing of the 16S rRNA gene when a tested specimen is suspected to have nonculturable pathogens. Unknown bacteria were detected by comparing the gene sequence of 16S rRNA with the sequences already present in the database. Samples detected via PCR showed negative results specific for C. burnetii. However, when these samples were detected via 16S rRNA gene sequences, about 98% showed similarity to that of C. burnetii [26].

2.1.6. DiversiLab Repetitive Sequence-based PCR (DL rep-PCR). DiversiLab is a program based on a repetitive sequence PCR system for bacterial typing. It has a high degree of standardization, specifically at the electrophoresis step achieved through Bioanalyzer [27]. In contrast to conventional gel electrophoresis, this method via microfluidic capillary electrophoresis overwhelms the low duplicability of prior rep-PCR methodologies [28]. Also, it provides internet-based, user friendly, and computer supported data evaluation [29]. The consistency of data achieved through this system has been species-dependent [30].

2.1.7. Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). It is a process (based upon PCR) that has been used to subtype the strains of microorganisms through the variable copy number of tandem repeats. MLVA applies to DNA sequences that differ in the number of tandem repeats obtained on multiple loci in bacterial genomes identified through PCR by utilizing flanking primers [31]. It is a more promising method as compared to other typing techniques due to greater diversity and comprehensive discrimination ability [32]. It is a rapid method with great resolution. MLVA microbial cells from the agar plate are taken and DNA is released by boiling the cells. For the detection of the DNA region, a variable number tandem repeat array is performed. PCR is used to combine DNA with chemicals for the amplification of VNTR. After PCR, the size of the PCR product must be determined. Then, the PCR product is loaded into the sample analysis plate and blended with chemicals which help to determine the product size. Capillary electrophoresis is used to determine the size of DNA fragments. Data outputs of MLA are expressed in the form of an electropherogram [33].

Figure 3. Overview of Molecular Techniques [9]

Table 2. Advantages and Limitations of PCR-based Techniques

2.2. DNA/RNA Probe-based Method (Sequencing Independent Method)

2.2.1. Southern Blotting. This technique is a detection method for the target DNA sequence. Firstly, restriction enzymes, such as restriction endonucleases, are used to cut the target DNA into short pieces. Then, gel electrophoresis technique is used to segregate these pieces. On gel, these fragments move according to their molecular weight. After gel electrophoresis, DNA fragments are shifted onto a membrane called the nitrocellulose membrane or blotting paper. These fragments are then incubated on blotting paper with selective probes. The probes are highly selective in their function and are radioactively labelled. Moreover, bind with the resolution 1 in a million fragments. In molecular biology, this technique is usually used for the detection of viral and bacterial infections. Autoradiography has been used for detection purposes after the completion of the incubation period, as shown in Figure 4 [39].

Figure 4. Procedure for Southern Blotting [40]

2.2.2. Northern Blotting. Northern blot is also a hybridization-based technique used for the detection of specific RNAs for several purposes. This technique is similar to southern blotting, except that the target sequence is an RNA molecule. Control over structure and function is achieved via northern blotting by identifying the level of expression of genes during differentiation and morphogenesis, as well as an abnormal condition. One of the major drawbacks is the degradation of mRNA during gel electrophoresis. Secondly, multiple probe detection makes the process less efficient [41].

2.2.3. In-situ Hybridization (ISH). In-situ hybridization (ISH) is an outstanding method for the identification and characterization of viral nucleic acids and other pathogens which are causative agents of different infectious diseases and cancer [42]. In-situ hybridization consists of four basic steps, namely (1) denaturation, (2) hybridization, (3) probe detection, and (4) analysis, as illustrated in Figure 5. ISH technique is used to regulate and detect the exact fixed location of different viruses, such as herpes simplex viruses, hepatitis viruses, human papillomaviruses, adenovirus, JC viruses, cytomegalovirus, and Epstein–Barr viruses [43]. The occurrence of cervical cancer is due to human papillomavirus (HPV). About 100 genomic types of HPV have been detected. Most types are not associated with the development of cervical cancer and hence are not oncogenic types. Therefore, those oncogenic types are divided into groups according to their potential to develop cancer, such as high-risk, moderate-risk, and low-risk HPVs. Currently, ISH is extensively used in cervical specimens for the differentiation and detection of HPV. There are different probes such as Dako Corporation supply probes for HPV ISH. These are named as biotinylated probes and high-risk groups [44].

Figure 5. Basic Steps of In-Situ Hybridization [45]

2.2.4. Peptide Nucleic Acid-Fluorescence In-Situ Hybridization (PNA FISH). Fluorescence in situ hybridization (FISH) using peptide-nucleic acid (PNA) probes (PNA FISH) is a unique molecular technique. PNA probes have been used for the detection of infectious diseases quickly and with more accuracy. PNA probes are synthetic compounds with nucleotide bases the same as DNA that adhere to the backbone of peptides [45]. PNA with neutral backbone make PNA probes with great properties of hybridization, with a high level of specificity, rapid kinetics, strong affinity, and better hybridization ability for rRNA which is a highly structured target. PNA FISH probes have been made and checked for the identification of various pathogens, such as S. aureus, E. coli, E. faecalis, C. albicans, coagulase-negative staphylococci, Pseudomonas aeruginosa, C. dubliniensis and K. pneumoniae. Currently, FDA has approved some PNA probes for the pathogens listed above, such as S. aureus PNA FISH, C. albicans PNA FISH, and E. faecalis PNA FISH for diagnostic purposes. A recent evaluation suggests that PNA FISH is an accurate method with high sensitivity and specificity [46]. Mycobacterium bacteria can be identified from liquid culture with the help of using PNA FISH [47]. PNA FISH has been used also for the quick detection of species from positive cultures of blood and to evaluate bacterial diversity in environmental samples [48].

2.3. Sequence-based Technologies

Due to technological advancement in equipment and the lessening of price per reaction, DNA sequencing has been introduced in the laboratory. Up till now, sequencing has been utilized for the detection of microorganisms that have been hard to recognize by traditional methods. The target considered the utmost for that application is ribosomal DNA gene [49, 50].

2.3.1. Sanger Sequencing. Sanger sequencing was the key sequencing technique utilized during the period 1975-2005 and the golden standard for all sequencing technologies. The rRNA, particularly 16S and 23S rRNA, have been the most convenient phylogenetic markers for the diagnosis of diseases in Sanger sequencing [51]. The marker 16S rRNA is a widely used unit for microbiological species identification, and it is incredibly abundant in bacteria. The length of sequence 1 is about 1.5 kb [52] and it remains remarkably intact in terms of assembly and operation in a variety of flexible areas [53]. So, this sequence can be used for the identification of various genera and species of diverse pathogenic bacteria. At present, 16S rRNA bacterial gene analysis is mainly attained through sequencing the flexible regions of this gene [54]. The specific clones from microbes attained by the culture can be recognized through the Sanger method of sequencing, subsequently after amplification by 16S general primers. Bacterial, full-length, 16S rRNA gene could be sequenced via Sanger sequencing. Nonetheless, the disadvantage of this technique is that bacterial culture must be untainted; else, the bacterium could not be recognized. The positive rate of the clinical culture is not clearly known. Furthermore, microbes cannot be distinguished and the mix of the16S genes cannot be utilized to differentiate species. Moreover, it is a time-consuming technique. First-generation DNA sequencing is highly affected by all the above highlighted flaws [55]. An overview of Sanger sequencing is represented in Figure 6.

Figure 6. Overview of Sanger Sequencing [56]

2.3.2. MALDI-TOF MS. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is useful for the study of the entire bacterial protein profile. A few years ago, the sequencing of the nucleic acids of bacteria has been upgraded through MAL of the nucleic acid DI-TOF MS technique. Cleavage products have been studied by the MALDI-TOF MS the resultant mass patterns have been related to reference ranges for sequence purposes. Still, MALDITOF MS are probable to develop the process of choice for high-output testing. MALDITOF MS provides precise outcomes, is automatable, fast, and cost-effective [57, 58].

2.3.3. Multilocus Sequence Typing. Multilocus sequence typing (abbreviated as MLST) is an extension of MLEE. In MLST, alleles at all housekeeping loci are allocated directly through nucleotide sequencing. Interior -150 base pair fragments of the housekeeping genes are applied in MLST. Meanwhile, such fragments could be precisely sequenced on every strand with a single primers pair and, in many species, would offer adequate difference to identify numerous alleles in a population. Therefore, bacterial strains are defined by a string of integers consistent with alleles at all housekeeping loci. The use of nucleotide sequencing for the allocation of alleles at the housekeeping loci is of advantage [59]. Firstly, sequencing reveals all differences at a locus, resulting in more numerous alleles per loci as compared to those revealed by the MLEE. Therefore, MLST attains high levels of discrimination by using seven loci. Secondly, the uniqueness of alleles becomes definite through sequence data in contradiction of MLEE, where similar electrophoretic mobility might imitate identical/similar sequences of nucleotide, or totally dissimilar sequences that encode enzymes which travel on the starch gel at an equal rate. Thirdly, the electronic movability of the DNA sequences permits some testing centers to characterize bacterial isolates by presenting sequences of seven gene fragments through the Internet to the chief MLST website, which grips frequently increasing databases of the allelic profiles of the isolates of species. Finally, sequences of the seven loci from 100-1000 isolates of all species could be applied to address the features of their evolutionary biology and population [60].

2.3.4. Nanopore Technology. Nanopore technology is an electrical signal-based Seq-technology [61]. Protein-based   nanopores (such as microscopic pores that basically form the channels on the membrane) are packed in the synthetic membrane and absorbed in the electrophysiological solution, which permits the ionic current to pass through them. The flow of ionic current is restricted when the molecules such as DNA/RNA pass it, producing a distinctive modification in the current signals. In nanopore technology, signal is evaluated in real-time format to determine the strand of DNA/RNA base sequence, which is transitory through the pore that permits the whole DNA/RNA sequence to be determined [62, 63]. This technology efficiently reports the flaws of the NGS in the aetiological diagnostic field, directly sequencing DNA/RNA that reads above 1 Mb in length [64]. Nanopore Seq-technology eradicates the exertion and the time required for reverse transcription. Since RNA reverse transcription into cDNA is not essential, the nanopore technology may execute RNA sequencing unswervingly [65].

2.3.5. Pyrosequencing. Pyrosequencing is a DNA sequencing method founded on the identification of the released pyrophosphate synthesis of DNA. The reactions are based on enzymes; visible light is produced which is proportionate to the number of the unified nucleotides.  Enzymatic cascade is initiated with the polymerization reaction of nucleic acid (DNA/RNA) in which inorganic pyrophosphate remains unconfined due to the incorporation of nucleotide by the polymerase. Unconfined PPi is later converted to ATP through ATP sulfurylase, which delivers energy to the luciferase that oxidizes luciferin and also produces light. As more nucleotides come to light, then template sequence could be identified [66]. The complete reaction from polymerization to the detection of light occurs in 3–4 seconds at room temperature. Single PMOL of DNA in a pyrosequencing reaction produces 6 × 1011 molecules of ATP, which produce above 6 × 109 photons at 560 nm (wavelength). The light amount is simply detected through a photomultiplier tube, photodiode, and charge-coupled device camera. Two different strategies of pyrosequencing are now accessible, namely liquid-phase pyrosequencing [67] and solid-phase pyrosequencing [68]. Pyrosequencing has opened novel avenues for the accomplishment of sequence-based analysis of DNA. It is used for the analysis of SNPs (single nucleotide polymorphisms) and currently applied for the fast typing of a huge number of bacteria, viruses, and yeasts. Moreover, it remains the quickest technique for the sequencing of PCR products [69]. This technique is also used for the resequencing of suppressor gene (p53 tumor) where mutations are effectively determined and measured [70]. It has possible advantages of accuracy, parallel processing, flexibility, and automaticity.

2.4. Post-amplification Techniques

2.4.1. Array-based Techniques. Typical solid phase hybridization uses precise hybridization probes attached to the solid substance for the detection of target-labelled molecules in the solution. Primarily, assays for identification and characterization, certain probes were placed in microtiter plate wells and rendered immobile [71]. Secondarily, microarrays of the fixed manifold probes at definite locations on nitrocellulose/nylon membranes are advanced, with diverse probes applied as dot (in dot-blots)/lines (in line probe assay) [72, 73]. The diversity of numerous LIPA assays remains accessible for the detection of Enterococci that is vancomycin-resistant, the toxin of Clostridium difficile, MRSA, Neisseria meningitides, E. coli, and extended-spectrum and metallo-beta-lactamases. In comparison to microarrays, the DNA microarrays have reduced versions with spot sizes typically below 200-300 µm. These microarrays are typically restricted to less than a hundred probes. DNA microarrays differ from low-density arrays that carry 100-1000 probes, while high-density arrays comprise 10000-100000 probes. Various labelling and detection systems can be utilized in combination with array-based technologies, extending from the fluorescence-ended silver ppt to assessable enzyme-facilitated substrate alteration [74]. Peptide or protein arrays have been defined for the detection of the HCV/HIV and Mycobacterium tuberculosis [75, 76]. Microarrays antigen  are used for the identification of the negative endocarditis blood culture [77]. DNA microarrays are applied for the diagnosis of bacterial meningitis and its general representation, as illustrated in Figure 7 [78].

Figure 7. General Representation of the Microarray Experiment [8]

2.4.2. Loop-mediated Isothermal Amplification. It is a novel and strong amplification technique for nucleic acids and considered as a substitute for PCR. LAMP amplifies targeted nucleic acid with high specificity in isothermal conditions. In LAMP, there is no need for a thermal cycle for the changes made in temperature because it needs a specific temperature for DNA amplification [79]. Moreover, four primers comprising two internal and two external primers and Bst DNA polymerase are used to identify the six exclusive target sequences on the tested DNA. The two internal primers are the forward internal primer and the backward internal primer, while external primers are B3 and F3 [80]. Each FIP and BIP comprise two discrete sequences conferring to the sense or antisense strands of the targeted DNA. LAMP reaction begins with the help of one of the internal primers and the other is used for self-priming. This reaction is processed for 1 h in a water bath or heat block at 65°C. The amplification product is detected through SYBR Green 1 dye. The product yield has numerous inverted repeats of the targeted sequence that display a cauliflower-like assembly with many loops. It has 10 times better accuracy and sensitivity than traditional polymerase chain reactions [81]. Due to significant amplification efficacy, 1039 copies of the target part could be achieved within 1 hour of incubation. In LAMP, less inhibition reaction happens as compared to PCR in which molecular beacons act as detectors for the amplification product. A new process founded on MB-LAMP has been confirmed that offers direct detection of the LAMP product [82].

3. CONCLUSION

Studies about the history of evolving bacterial infections provide a new vision of the specific roles performed by various identification technologies. The old techniques are regarded as outdated due to the development of methods based on molecules. In this review, the use of direct hybridization, nucleic acid amplification, and variety of methods for postamplification analysis was made to detect microbial pathogens in human beings. PCR assists in identifying the taxonomical location of new organisms that still remain uncultured. LAMP is considered as a substitute for PCR and amplifies targeted nucleic acid with high specificity in isothermal conditions. Pyrosequencing technique is used for the resequencing of suppressor gene where mutations are effectively determined and measured. For the wide range and specific detection of pathogenic microbes, advancements in nucleic acid-based assay and sequencing have been introduced.

CONFLICT OF INTEREST

The author of the manuscript has no financial or non-financial conflict of interest in the subject matter or materials discussed in this manuscript.