Normal flora and pathogenic microbes

Our bodies are akin to an ecosystem because several species of normal flora are natural inhabitants of the body. Approximately 10¹⁴ microbial cells of various kinds of bacteria, fungi and protozoa are part of this ecosystem and live on the skin, mouth, vagina, or the colon. Even viruses inhabit our cells, though they may not always be able to cause disease symptoms. But pathogenic microbes are different from these natural inhabitant. While the normal flora can cause disease only in immuno-compromised patients or when they gain entry into normally sterile tissues or part of the body, pathogens cause infections the moment they inhabit our bodies by invading cells and biochemical barriers. The main classes of pathogens that cause infections are:

• Bacteria

Bacterial infections are common could be caused by endogenous or exogenous bacteria. Endogenous bacteria are the normal flora of the human body and may gain access to the sterile parts of the body through the respiratory tract or when the skin or mucosa suffers from trauma or is exposed during a surgical procedure.

• Fungi

Fungi are rarely pathogenic and are not known to cause many diseases though we constantly come in contact with the propagules. Most fungal infections are caused in patients with low immunity, examples include Candida albicans, Histoplasma casulatum, Blastomyces dermatitidis, Penicillium marneffei, Coccidioides immitis and Paracoccidioides brasiliensis. Most pathogenic fungi are dimorphic in nature and change morphology from yeast to filamentous or vice versa. Most fungal pathogens that infect the hair, nail or skin tissue are able to produce the enzyme keratinase. Fungal infections can be cutaneous, sub-cutaneous or systemic.

• Protozoans

Like fungi, the protozoans are also eukaryotes and are therefore more difficult to treat. Plasmodium vivax, the malaria parasite is an example and has a complex life cycle with several stages, part of the life cycle is completed in a vector, which adds to the complexity of finding a vaccine (Alberts, 2003).

• Viruses

Several viruses such as the inflenza virus that causes flu infect human beings. Other viral diseases include, the chicken pox virus, Hepatitis A, Hepatitis B and Hepatitis C viruses. Viruses that have virulent characteristics are able to cause infection in cells of the body. Once infection occurs, the virus can spread in the body and replicate within cells to an extent that the target organ’s function gets impaired. When a virus is virulence, it is able to replicate even during the occurrence of fever and inflammation.

Bacterial infections and fungal infections

Diagnosis of microbial infectious disease requires the history of patient’s illness, physical exam, radiographic tests, if relevant and laboratory tests. In the present context it will be pertinent to focus on the methods of laboratory tests. The specimen is collected on the basis of the symptoms and signs and then it is processed. The collection must be collected before the antibiotic therapy is started. Depending on the nature of the infection, specimen collection could be invasive or non-invasive. Non-invasive collection, usually involves collection of  urine sample or a sputum sample. Invasive collection, may involve collection of blood using a syringe, collection of swab from a site of infection, collection of cerebro-spinal fluid or even a surgery to collect samples of pus and local tissue in case of a deep seated abscess.

Sensitivity and specificity of tests is an important aspect of laboratory testing of pathogens. The sensitivity of a test depends the number of microbial cells present in a specimen. Whereas, specificity depends on how close the isolated microorganism is morphologically or structurally to the pathogen, or how specific the reaction between its antigen and antibody are during an immunoassay or how well the DNA or RNA probe binds to that of the test microbe’s. Specificity also depends on the stage of the disease at which the specimen was collected or the method used to collect the specimen. Usually a low power 10X or 40X  and an oil immersion 100X objective lens and a 10X ocular lens, is enough in a binocular light microscope is to observe a specimen. An ultraviolet light source is required when doing fluorescence microscopy.

Several diagnostic kits are used for carrying out immunoassays, such as, ELISA and RIA. When presence of pathogens needs to be determined or confirmed through studying the growth on culture media. The specimen has to be inoculated on culture media. Enumeration of bacterial colonies may be required in some cases. Viruses have to be cultured in cell culture systems. At times viruses may have to be cultured in living organisms.

The above mentioned techniques and many others may be required before diagnosis can be confirmed. But these procedures for diagnosis are time consuming and require different kinds of equipment and chemicals or kits for each type of pathogen

Employment of techniques for direct examination of the specimen may be done. Primary technique employed is microscopy, immunofluorescence assay, immuno-peroxidase staining, and others. In case of DNA or RNA sequences, genetic probes are used for identification of the genus and species. Culture media can be used to isolate organisms from the specimen. Selective and specialized media are required for the isolation and identification of the infectious agent, while non-specific media support the growth of many type of micro-organisms. Selective media contain inhibitory substances that allow the growth of specific microorganisms only.

Protozoan infections and viral infections

Identification of microbes can be done on the basis of colony morphology, cellular morphology and growth characteristics in the presence of different substrates. Carbohydrate utilization, immunoassays, enzyme activity or genetic probes may be used for the purpose.

Serological tests that indicate a rising titre of IgM or IgG antibodies are used for confirmation of diagnosis. A study of antibiotic susceptibility helps to determine the antibiotics that the pathogens are sensitive or resistant to (Baron, 1996).

Although complete replacement of the current methods used for the diagnosis of microbial infectious agents may not be possible. The possibility of rapid, high throughput whole genome sequencing on bench top platforms is becoming a reality and the reduction in cost means that the technology can find use in public health microbiological surveillance of outbreaks is there (Torok & Peacock, 2012).  But the use of whole genome sequencing for surveillance of an outbreak particularly for microbes that are not amenable to laboratory cultures and cause outbreaks is the area where the technology can be applied beforelong (Niedringhaus, Milanova, Kerby, Snyder, & Barron, 2011). Another area where the use of whole genome sequencing can be made with immediate effect is antimicrobial susceptibility testing (Köser, et al., 2012).

Culture testing that is now automated still takes a considerably long time to identify species and in epidemiological typing, if the turnaround time is shorter, the benefits of the tests can become available to a larger number of patients when an outbreak occurs. This is more true in case of bacterial and fungal pathogens. The identification of viral pathogens is already being done by PCR methods. Current genotyping methods for bacteria use only a part of the genome, (Janda & Abbott, 2007) but whole genome sequencing gives better resolution for epidemiological studies (Köser, et al., 2012). Phenotypic testing would still be necessary to understand newer mechanisms of antimicrobial resistance but antibiotic resistance can be studied much faster through whole genome sequencing. While usual culture methods take two days for growth to occur on plates, the slow growing bacteria, for example, Mycobacterium tuberculosis takes much longer incubation period. In such cases, whole genome sequencing will shorten the time required for diagnosis.

In case of HIV viral genotyping, tests that can find the viral tropism are required to find out which cell entry inhibitor can be chosen for a patient. But the problem occurs when the infection is caused by mixed viral genotypes and this is where the current genotyping methods fall short.

Laboratory methods for diagnosis of microbial infections

However, it is true that the WGS technology may not be directly applied to the clinical specimen in case the number of pathogenic organism is very low and requires an enrichment culture before it can be detected, as has been pointed out by the website Pathogenica (Pathogenica). Inspite of the advantages, the complex sample preparation required for WGS is  hindrance that has to be overcome, several samples are required to labelled, tagged and prepared in equimolar concentrations. The potential to detect even a single base variant by WGS may one day enable the technology to be used in place of phenotypic testing. Physicians may also be able to detect the drug resistance to anti viral agents earlier than they can at present. With the ever evolving WGS technology, as it cheaper and faster forms are developed, the validation for the use of the technique by clinicians will be required (Rehm, et al., 2013).

Diagnostic microbiology will benefit from the WGS technology even though only some of the currently used techniques might be replaced by it. But a shorter turnaround time at no additional expenses will be a great benefit to clinical microbiologists. The interpretation software will have to be developed such that it is more user friendly and people whole do not understand genome sequencing should be able to decipher information useful for clinical practice.

References

Alberts, B. (2003). Molecular Biologyof the Cell. Garden Science.

Baron, S. (1996). Medical Microbiology, 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston.

Janda, J. M., & Abbott, S. L. (2007). 16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls . . Journal of Clinical Microbiology, 45(9), 2761–2764.

Köser, C. U., Ellington, M. J., Cartwright, E. J., Gillespie, S. H., Brown, N. M., Farrington, M., & Peacock, S. J. (2012). Routine Use of Microbial Whole Genome Sequencing in Diagnostic and Public Health Microbiology. . PLoS Pathogens, 8(8), e1002824. h.

Niedringhaus, T., Milanova, D., Kerby, M., Snyder, M., & Barron, A. (2011). Landscape of next-generation sequencing technologies. Anal Chem., 83(12):4327-41.

Pathogenica. (n.d.). technology.php. Retrieved from https://www.pathogenica.com: https://www.pathogenica.com/technology.php

Rehm, H., Bale, S., Bayrak-Toydemir, P., Berg, J., Brown, K., Deignan, J., . . . Commitee., W. G. (2013). ACMG clinical laboratory standards for next-generation sequencing. Genet Med. , 15(9):733-47.

Torok, M., & Peacock, S. (2012). Rapid whole-genome sequencing of bacterial pathogens in the clinical microbiology laboratory–pipe dream or reality? J Antimicrob Chemother, 67(10):2307-8.