Genomics and Infectious Diseases: Microbes, Diagnosis, and Sequencing

This report provides a comprehensive overview of microbial infectious diseases, covering the main classes of pathogens: bacteria, fungi, protozoans, and viruses. It details how these microbes are currently diagnosed and typed using various laboratory methods, including microscopy, culture, and s...Read More

Genomics and Infectious Diseases
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The main classes and types of microbes causing infectious diseases
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

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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.
Describe how these microbes are currently diagnosed and typed
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

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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.

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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).
Potential for whole genome sequencing to replace or improve upon current methods
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

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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.
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

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understand genome sequencing should be able to decipher information useful for clinical
practice.

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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 http://www.pathogenica.com:
http://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.