|Year : 2022 | Volume
| Issue : 6 | Page : 274-279
Revisiting conventional microbiology techniques in the era of molecular testing
Ashima Jain Vidyarthi1, Arghya Das1, Ankit Gupta2
1 Department of Microbiology, National Cancer Institute and All India Institute of Medical Sciences, New Delhi, India
2 Department of Microbiology, Pushpawati Singhania Hospital and Research Institute, New Delhi, India
|Date of Submission||01-Jul-2022|
|Date of Decision||16-Oct-2022|
|Date of Acceptance||23-Nov-2022|
|Date of Web Publication||29-Dec-2022|
Dr. Ashima Jain Vidyarthi
National Cancer Institute and All India Institute of Medical Sciences, New Delhi
Source of Support: None, Conflict of Interest: None
The diagnosis of infectious diseases has always been a matter of concern for clinicians worldwide. Although the conventional techniques like microscopy and culture have served the purpose since ages, they have been found to be inadequate now with the advent of modern technology including automated identification systems and molecular testing. The newer technology including Matrix-Assisted Laser Desorption Ionization – Time of Flight and molecular tests such as multiplex polymerase chain reactions and Next Generation Sequencing, has undoubtedly paved the way for diagnosis and discovery of various novel pathogens. This has subsequently given rise to a mindset that the conventional techniques are redundant and hence, should be abandoned. The authors, through the current review, would like to present a perspective in favour of the conventional techniques which still are one of the simplest, most inexpensive methods for diagnosing infectious diseases and provide us with the precious antimicrobial susceptibility data to guide our antimicrobial stewardship programmes. Further, considering the limited availability of the state-of-the-art molecular testing facilities across the country, it is recommended that rather than using them exclusively or separately, conventional methods and the molecular tests be used in conjunction wherever available and feasible.
Keywords: Conventional, culture, laboratory, microbiology, microscopy, molecular
|How to cite this article:|
Vidyarthi AJ, Das A, Gupta A. Revisiting conventional microbiology techniques in the era of molecular testing. Curr Med Res Pract 2022;12:274-9
| Introduction|| |
A vast plethora of micro-organisms including bacteria, virus, fungi and parasites has to be targeted for the successful diagnosis of infectious diseases. However, unfortunately, there is not a single definite method with 100% sensitivity and specificity for diagnosing them. A debate is pertinent and underway regarding the advantages and disadvantages of molecular methods over the conventional methods and vice versa. The gradual advent of artificial intelligence and new technology has led to the conventional techniques being disregarded. Not only the microbiologists and scientists are getting increasingly dependent on the latest technology, including Matrix-Assisted Laser Desorption Ionization –Time of Flight and multiplex polymerase chain reaction (PCR)-based equipment for diagnosis, they are also getting reluctant about using the conventional techniques.
Considering the fact that the Indian diagnostic services are still struggling to provide molecular testing, especially in remote areas, it is important that we try to understand and depend on the conventional microbiological techniques more for the diagnosis of infectious diseases. These techniques including microscopy and cultures have formed the cornerstone for diagnosing infections since years. The development of automated platforms for blood cultures and identification-susceptibility testing has not only made things easier and faster but has also made detection of various rare pathogens possible. Further, the molecular techniques, including multiplex PCRs and next-generation sequencing, have been instrumental in identification and discovery of novel pathogens.
This review will highlight the utility of the conventional microbiological methods in the era of molecular techniques towards the diagnosis and management of infectious diseases, especially in resource-limited settings.
| Relevance of Conventional Techniques in Clinical Microbiology: Indian Perspective|| |
India has approximately >1,000,000 laboratories (small and large) running in the country currently. Despite this, when the country needed emergency molecular testing for COVID, only 52 labs could rise up to the occasion immediately (as on 6th March 2020). Although over a period of 2 years, the number gradually and steadily increased to 3382 (as on 29th June 2022), it unmasked the scale of molecular testing in India and the fact that we, as a country, are still not ready for molecular testing to be created as a mandate for infectious disease diagnosis. Hence, we will gradually have to scale up our molecular testing while we certainly rely on the conventional tests for diagnosing our infections meanwhile.
| Scope of Microscopes|| |
A common myth these days is that microscopy is an ancient technique and that it is a redundant technique now. However, the reality is that microscopy is the oldest yet the simplest and one of the most inexpensive methods of diagnosis even today. Since ages, and even today, microscopy may single-handedly be used to make the diagnosis and initiate treatment. In a bacteriology laboratory, Gram staining followed by microscopy is the first laboratory test conducted on the clinical samples. This often helps in the reduction of unnecessary processing of specimens (especially respiratory specimens such as sputum and endotracheal aspirate) and found to significantly increase the detection rate of pathogens in clinical specimens and also reduce the unnecessary cost of the testing laboratories. The application of different grading systems such as Murray and Washington criteria, Bartlett's grading systems have demonstrated satisfactory performance in distinguishing commensal/colonizing bacteria from true pathogens.,
Furthermore, the age-old Ziehl–Neelsen staining is still the cornerstone for early diagnosis and management of tuberculosis (TB) in resource-limited settings. In the remote areas with no facility for Mycobacterium tuberculosis culture, microscopically performed sputum smear conversion remains a key indicator for predicting response to anti-tubercular treatment among bacteriologically confirmed TB patients., The demonstration of Gram-negative bacilli or budding yeast cells seen in cerebrospinal fluid or blood with microscopy may also provide life-saving diagnosis and help in initiating timely treatment. Simple light microscopy with potassium hydroxide was found to be one of the most efficient techniques for early diagnosis of Mucormycosis patients, especially in resource-limited settings, during the outbreak in 2021 in India. This simple technique could demonstrate the presence of broad, aseptate, hyaline hyphae [Figure 1]a in 70% clinically suspected mucormycosis cases during second wave of COVID-19 pandemic in India. Similarly, the importance of microscopy cannot be concluded without mentioning its role in the diagnosis of parasitic infections. Given the fact that culture of most of the pathogenic parasites is labour-intensive, requires stringent bio-safety measures and molecular techniques for parasites are costly and technologically demanding, microscopy remains the preferred diagnostic method for the detection of Plasmodium spp., microfilaria, etc., in peripheral blood smears as well as for detection of amoebae and helminths in stool wet mount preparations.
|Figure 1: (a) Broad, aseptate, hyaline fungal hyphae seen on KOH mount (×400), (b) Gram negative filamentous bacilli seen on Gram stain (×1000). KOH: Potassium hydroxide|
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From a stewardship perspective too, microscopy has enormous application benefits but not frequently acknowledged and utilised. The recently published GRam stain-guided Antibiotics ChoicE-ventilator-associated pneumonia (VAP) trial indicated that Gram stain-based treatment regimen had non-inferior results as compared to guidelines-based treatment regimen in patients with VAP. Further, it was successful in restricting the use of broad-spectrum antibiotics in these patients. Moreover, the usefulness of microscopy has been demonstrated through various other studies as well. For instance, the observation of filamentous forms of certain bacteria [Figure 1]b correlates well with sub-optimal dosing of antibiotics and thus may serve as an indicator for chronic and recurrent infections. Therefore, there is an absolute need to reiterate and re-emphasise the practice of primary staining and follow a certain recommended workflow for processing of all samples received in a microbiology laboratory [Figure 2].
|Figure 2: Placement of conventional methods in the diagnostic workflow in a clinical microbiology laboratory|
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| Culture of Cultures|| |
For years, our understanding of microbial pathogenicity circled around the principle of successfully isolating a microbe from the lesions of the disease. Eventually, the Koch's postulates provided an undisputed basis for the diagnosis of infectious diseases. However, a paradigm shift gradually happened over the last two decades and raised some fundamental questions on the relevance of the traditional Koch's postulates in modern-day infectious diseases diagnosis. A present-day microbiologist may find culture as outdated. Besides being slow (more turnaround time), different optimum growth requirements must be met for different microbes to allow their successful isolation in suitable culture media. Unsurprisingly, there is a genuine zeal to replace traditional culture-based methods with fully automated, rapid and molecular-based platforms. However, in reality, traditional culture-based techniques are more economical and less technically demanding than most of the molecular tests. By assessing the cultivable microbes, culture-based methods provide useful insight into recognising viable cells in a clinical specimen. Semi-quantitative urine culture using the calibrated loop technique is still widely practiced in routine microbiology laboratories and employed as the reference to other emerging techniques. Culture also remains the gold standard for the diagnosis and follow-up of many bacterial infections, for example, gonococcal infections. The culture process also allows selection or suppression of specific pathogen as per the diagnostic relevance of that particular pathogen in a clinical specimen. The choice of the selective agents depends on the target pathogen as well as the microbial diversity in the clinical specimen from which the target organism has to be isolated. There is a wide list of such selective agents including antibiotics (e.g. cycloserine cefoxitin fructose agar for Clostridioides difficile), antiseptics (e.g. cetrimide agar base for Pseudomonas aeruginosa), salts (e.g. Mannitol salt agar for Staphylococcus aureus), chemicals (e.g. potassium tellurite blood agar for Corynebacterium diphtheriae) and dyes (e.g. Lowenstein–Jensen medium for M. tuberculosis containing malachite green).
An important aspect of culturing a pathogen in a routine microbiology laboratory is to allow performing the antimicrobial susceptibility testing on the isolated organisms. Although the modern genotypic methods appear promising for rapid and accurate detection of the resistance determinants, there are several factors for which phenotypic methods will continue to be preferred for antimicrobial susceptibility testing. First, microbial resistance to a particular antimicrobial agent may be the result of multiple mechanisms for which a plethora of genetic determinants may be responsible. The diverse nature of the underlying genetic mechanism may actually exceed the diagnostic capacities of the molecular techniques currently under use. For the same reason, a molecular technique can predict resistance but can never guarantee the susceptibility of a pathogen to antimicrobial agents. Second, molecular techniques are expensive and sensitive to contamination by carryover of genetic materials in a targeted molecular assay. Third, sometimes the normal flora in a clinical sample may also harbour some resistance determinants. Molecular methods have poor discriminatory power in such situations, and their results must be cautiously interpreted. Other practical situations where phenotypic methods based on culture may appear indispensible are the determination of minimum inhibitory concentrations of an antimicrobial agent against a particular isolate and the preparation of hospital antibiogram. Both of these are considered crucial in modern-day clinical practice of infectious diseases.,
Furthermore, microbial culture serves as the basis for obtaining antigens for serological assays conducted in vitro and in vivo disease models. Thus, culture has irreplaceable benefits for studying emerging pathogen, and also allows further genetic studies to be carried out to elucidate microbial pathogenesis.
| Logic of Serology|| |
Serological diagnosis has proved to be useful in situations where direct microbiological evidence for the presence of the pathogen cannot be obtained easily and quickly by culturing the pathogen or morphological methods (microscopy-based diagnosis). Some investigators may argue that serological tests seek to demonstrate the patient's immune response to an invading pathogen and thereby can only provide indirect evidence of infection, which is prone to considerable variance at individual's genetic and immunological makeup. These tests also pose difficulty in interpreting the results owing to the cross-reactions, antigenic similarities between the same and different bacterial genera. On the other hand, molecular tests like PCR may be highly sensitive and specific for the diagnosis of majority of infections, including those caused by the relative unusual pathogens. However, serological tests continue to be relevant in situations where the pathogen has been quickly eliminated by the host. In addition, by demonstrating seroconversion (rising or falling antibody titre), these tests may provide useful information on the natural history of diseases. The new generation of serological tests like Enzyme-Linked Immunosorbent Assay has the ability to differentiate between Immunoglobulin (Ig) M and IgG types of antibodies, thus may also serve as an indicator for recent or remote infection.
| The Genomic Edge in Modern Microbiology Laboratory|| |
The molecular tests have proven their efficiency time and again. Their importance towards the diagnosis of infectious diseases is irrefutable. They have been the main pillars of diagnosis for some of the most recent outbreaks such as H1N1, Zika virus and now SARS-CoV-2. Further, molecular diagnosis has been the mainstay for diagnosing different viral, parasitic and culture-negative bacterial infections.,, Molecular techniques are also useful tool for typing of specific strains. The analysis of nucleotide sequences further help in accurately delineating the position of a microbial species in the phylogenetic tree. The quantitative PCR platforms also provide quantitative estimation of pathogenic load in the clinical specimens. In addition, the commercially available cartridge/chip-based nucleic acid amplification tests have made groundbreaking advancements in the field of infectious diseases diagnostics. These cutting-edge technologies not only provide rapid, highly accurate and precise diagnosis but also involve less manual handling of specimens limiting the risk of contamination.
However, the inherent limitation of PCR to detect non-viable organisms too along with viable ones cannot be ruled out. The possibility of false positives due to contamination and false negatives due to inhibitors in the sample also makes the test interpretation challenging especially when done as stand-alone tests for diagnosis. The distinction between colonisers and pathogens amongst the organisms identified in multiplex systems is another dilemma to be resolved., The organisms reported in these tests might even not correlate with the clinical condition of the patient and the culture results. Further, the resistance determinants detected might be from a non-viable/non-significant organism. In that scenario, it would be unnecessary to expose the patient to higher antibiotics which may subsequently contribute towards antimicrobial resistance.
| Interdependence of Molecular and Conventional Methods|| |
In a diagnostic microbiology laboratory, both molecular methods and conventional techniques are not mutually exclusive but should be utilised in conjunction to serve the ultimate purpose of pathogen detection. Besides choosing an appropriate medium and ensuring ideal incubation conditions, the main critical issue with culture is the selection of unique cultivable clones. To achieve this, not only specimens with a high load of the suspected pathogen needs to be inoculated, but also an optimum volume of the specimens needs to be sampled from anatomic sites relatively free from commensals or, colonisers. However, in a real clinical scenario, the former may not be always possible. Conversely, the most recent molecular methods have the potential to detect all culturable, not-yet-culturable microbes and previously uncultured microbes from mixed microbial population in specimens based on a metagenomic approach. With metagenomics, the recovery and analysis of nucleotide sequences from samples have become entirely independent of microbial growth in culture. However, whether the DNA sequences recovered from the clinical specimen is actually representative of the pathogen depends on all the physical, chemical and biological steps involved in the molecular analysis. Ironically, the retrieval of DNA also depends on the appropriateness of sample collection, stringency in sample transportation and efficiency in nucleic acid isolation. The latter is also influenced by many biological and chemical factors (presence of PCR inhibitors and nucleases in clinical specimens, cell wall density of the microbes).,
Thus, although the molecular technology tends to be superior to traditional techniques like culture, the use of traditional methods is seemingly advantageous for some obvious reasons. Molecular diagnostic testing requires expensive reagents, sophisticated semi-automated or automated equipment, and expertise, whereas traditional methods tend to be cost-effective. The use of selective media for culture also tends to yield faster results for screening of key pathogens from mixed microbial population than metagenomic analysis. Furthermore, metagenomics may lack the desired sensitivity in some scenarios (low concentration of enteric pathogens in stool specimens). Molecular methods also fail to distinguish between viable and non-viable organisms. They have the potential to completely replace conventional techniques, but whether all positive results represent true infection is a matter to debate. The studies till date have shown molecular tests to complement culture-based methods and frequently detected additional microorganisms which are missed by culture., It has also been suggested that while the currently available rapid diagnostic tests, including the automated molecular assays are outstanding in making early diagnosis and facilitating timely initiation of treatment, they should not replace conventional methods considering they are not as comprehensive and are plagued with varied sensitivity and specificity. Further, the results generated do not directly provide antimicrobial susceptibility patterns and hence, may not contribute towards antimicrobial stewardship programs. The clinical and economic impact of these tests also needs to be investigated further.
Therefore, more exhaustive research works are solicited to decide the investigation of choice to the satisfaction of the clinical microbiologists between molecular and conventional methods for most if not all of the pathogenic microbes. Till then, it will be prudent to use both the methods in conjunction with each other based on the available evidence.
We are grateful to Prof. Rama Chaudhry, Head of Department, Department of Microbiology, AIIMS, New Delhi, for giving us the opportunity and inspiring us continuously to work towards our goals.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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