Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 4, 2011, pp. 325-326

Indian Journal of Medical Microbiology, Vol. 29, No. 4, October-December, 2011, pp. 325-326

Editorial

Molecular diagnostic tests: Keeping up with mutations

R Kanungo¹, D Metzgar²G

¹ Editor, IJMM, MD, PhD, A 3 No. 38, Labourdonnaise Street, Puducherry 605 001, India
² Principal Scientist, Ibis Biosciences, 2251 Faraday Avenue, Suit 150, Carlsbad California 92008, USA
Correspondence Address: R Kanungo, Editor, IJMM, MD, PhD, A 3 No. 38, Labourdonnaise Street, Puducherry 605 001, India, rkanungo1@gmail.com

Date of Submission: 07-Oct-2011
Date of Acceptance: 09-Oct-2011

Code Number: mb11082

PMID: 22120788
DOI: 10.4103/0255-0857.90152

Gaps in our knowledge of genetic diversity of microorganisms have been overcome to a great extent by advanced technology. Unraveling the mystery of the genes has opened a Pandora′s box of evolutionary changes microbes use to survive adverse conditions. The following is based on an article by the co-author (DM) published in the Journal of Clinical Microbiology July 2011 Volume 49 number 7 (page 2774-5) entitled ′Adaptive evolution of diagnostic resistance′. ^[1] The author discusses a novel concept based on selective pressure created by the use of PCR tests to determine antibiotic treatment. This pressure favors bacteria with mutations in the genetic targets of the PCR tests which cause the test to yield false-negative results, which in turn leads to a lack of appropriate antibiotic treatment and treatment failure. This phenomenon may occur when highly specific molecular diagnostic tests are used to define treatment. He calls it ′diagnostic resistance′. In the traditional culture of bacteria and virus on media and cell cultures, the same selective pressure is placed on the pathogens, but there is no easy way for the pathogen to mutate in a way that makes it unculturable. The only reason that diagnostic resistance does not arise easily when culture is used for testing is that changes that would result in resistance to diagnostic culture (that is, mutations that make the pathogen unculturable) must involve significant changes in phenotype, often at the cost of a loss of ability to grow in the host. Such changes are likely to be maladaptive and hence counter selected. On the other hand, a single silent base pair mutation can offer resistance to diagnostic testing, and these sorts of mutations have little or no adaptive cost and are not likely to result in phenotypic change. Hence, the use of highly selective molecular methods targeting specific sites in the genome may result in the pathogens evading detection due to selective mutation of the genetic loci targeted by the test, under selective pressure created by the treatment. Mutations at a base pair will prevent binding of the primer designed to detect the original base pair and therefore the pathogen will remain undetected by the test. The mutant that emerges results in ′diagnostic resistance′. The source of the selection is antibiotic treatment, not the molecular diagnostic test itself. The test does not affect the reproduction of the pathogen. However, if the antibiotic treatment is applied as a result of positive test results, then the pathogen has two distinct ways to survive - it can become resistant to the antibiotic or it can become resistant to the test (in the sense that it becomes invisible to the test through mutation in the genetic regions targeted by the test). Either way will achieve the same goal - in the first case it will survive the antibiotic through antibiotic resistance, while in the second case it will avoid treatment by yielding a false negative result on the test. The author cites the example of a single target PCR test used in 1995 to detect Chlamydia trachomatis genital infections in Sweden. ^[2] A decade later the dominant strain was replaced by a mutant (diagnostic resistance mutant) which had lost the region of DNA targeted by the test. In summary, the article stresses the need for careful designing of diagnostic assays. ^[2] It is conceded that ′evolutionary etiology can be exceptionally difficult to demonstrate′. However, with evidence to suggest ′diagnostic resistance′, adaptive evolution needs to be considered while designing molecular assays. It is advocated that ′careful monitoring be done to test efficacy and use of multiple parallel tests to identify loss of sensitivity for individual tests′. This has led CDC to continually keep PCR tests current for highly mutable pathogens, of which influenza virus is an example, through careful analysis of variability in test efficacy and direct analysis of the PCR target regions through sequence analysis. ^[1]

Diagnosis of several other communicable diseases has come to rely heavily on molecular diagnostic tests. An important case is the use of molecular kits for the detection of MDR TB. Keeping in mind the steady rise in cases with drug-resistant TB, and limitations of drug susceptibility testing (DST), rapid molecular tests appear promising. An increasing number of laboratories are adopting the techniques, especially in developing countries with a heavy burden of the disease. It has been reported that mutations outside the region of a specific gene may not bedetected by some assays designed to detect drug resistance. These strains could still be resistant, with the risk of the patient being subjected to ineffective treatment with the drug. ^[3] Another scenario is the highly mutable human immunodeficiency virus (HIV). Rapid mutations in a drug-resistant strain can impact the potential for detection. High specificity in designed primers limits the detection of resistance mutations to the currently prevalent ones, but fails to detect emerging resistance.

Another major drawback is that the mere presence of resistance genes may not correlate with expressed resistance and vice versa. The absence of the gene does not rule out resistance, which could be by another mechanism. ^[4] Similarly, resistance genes may be present but unexpressed due to mutational or regulatory silencing. As much as it is necessary to use rapid molecular tests, it is also importantto be vigilant towards emergence of mutants which might escape the detection limit. A word of caution is offered in use of narrow molecular tests in public health and clinical practice. Apparent eradication of the pathogen (due to failure in detection) and treatment failure would be an undesirable outcome. Awareness and vigilance are required to avoid this pitfall in addition to looking out for new and emerging mutants.

References

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