Introduction
The article, “Genotyping and identification of mycobacteria by fingerprinting Techniques” by Khosravi and Seghatoleslami (2009) shows how fingerprinting can be used to identify mycobacteria in combating Tuberculosis (TB) which for a long time has been a serious health concern by the World Health Organization (WHO). Some of the techniques used in molecular epidemiology like MIRU-VNTR and spoligotyping are described in the article.
Summary
The article has deeply illustrated the various ways of carrying out Genotyping of mycobacteria for carrying out epidemiological studies. It has also shown that through molecular typing of MTB the disease can be controlled. The study of the epidemiology of TB has always been described as a complex exercise which faces many limitations (Khosravi & Seghatoleslami, 2009). Therefore, genotyping plays a critical role in clinical identification purposes whereby cultivable members in the mycobacterium genus are classified into two distinct groups: MTC and non-MTC atypical or NMT.
This article is of great importance as it addresses the challenges posed by TB and its treatment and provides a solution to the problem using fingerprinting techniques. From the analysis it has emerged that science can impact human beings in different ways like the use of strains in treatment of TB. Some of the science procedures illustrated in the article include the REA which is used in fingerprinting. The article also shows the use of the PCR based restriction enzyme analysis (PRA) and the PCR-RFLP and the restriction-fragment-length polymorphism (RFLP) analysis genotyping MTB isolates. This method uses IS6110 copy numbers in strains which range from 0 to 25 (Thorne et al., 2007).
Conclusion
The article provides an insight into the problem of TB and gives essential information to epidemiologists on different strains of mycobacteria. The application of fingerprinting techniques shows the current development achieved in combating the health problems that are of serious concern worldwide.
References
Khosravi, A., & Seghatoleslami, S. (2009). Genotyping and identification of
mycobacteria by fingerprinting techniques. Jundishapur Journal of Microbiology, 2(3), 81-91. Retrieved from EBSCOhost.
Thorne, N. N., Evans, J. T., Smith, E. G., Hawkey, P. M., Gharbia, S. S., & Arnold, C. C.
(2007). An IS 6110-targeting fluorescent amplified fragment length polymorphism alternative to IS 6110 restriction fragment length polymorphism analysis for Mycobacterium tuberculosis DNA fingerprinting. Clinical Microbiology & Infection, 13(10), 964-970. doi:10.1111/j.1469-0691.2007.01783.x
Appendix
ARTICLE CUT
Genotyping for Epidemiological Purposes by Khosravi, A., & Seghatoleslami, S. (2009).
The molecular typing of MTB has greatly improved the knowledge and control of TB by allowing the detection of unsuspected transmission, the identification of false positive cultures, and the distinction between re-infection and relapse [3]. Molecular markers provide an important method for detecting TB transmission that has been followed by rapid progression to active disease [4]. The understanding of TB transmission dynamics has been greatly enhanced by the development of various DNA typing methods [5,6]. Genotyping can also be used to evaluate an outbreak of TB. If epidemiologic data suggest the occurrence of an outbreak, genotyping of the isolates, in combination with an epidemiologic investigation, can help determine whether an outbreak has occurred or whether there is a coincidental occurrence of a large number of cases. This strategy can delineate the extent of the outbreak and guide public health measures to reduce disease transmission [3].
There is growing evidence that the genetic diversity of MTB may have important clinical consequences. Nearly 50 years ago, Mitchison and others compared the consequences of infecting guinea pigs with isolates of MTB from either British or Indian patients with pulmonary TB.
They reported that the British isolates were more virulent: they caused more-severe and more-widespread disease and killed a higher proportion of animals. However, the investigators were not able to characterize the genetic diversity of the infecting isolates, and they could not find any association between virulence in guinea pigs and the severity and outcome of disease in humans. A further understanding of the relationship between mycobacterial genotype and clinical phenotype came with the advent of mycobacterial genotyping [7]. Genotyping of isolates from patients is useful in several situations. The results can be used to confirm the occurrence of crosscontamination in the laboratory. Approximately three percent of patients from whom MTB is apparently isolated in clinical laboratories do not have TB; the positive cultures are due to cross contamination. The occurrence of cross contamination is most likely when acid-fast smears are negative and only one specimen is culture-positive [8]. When MTB is isolated from a specimen but the clinical findings do not suggest the presence of TB, genotyping of the isolate and other MTB strains handled concurrently in the laboratory can strongly suggest the occurrence of cross contamination and lead to the discontinuation of anti-tuberculosis medications [3]. There is broad variability in the genotypes of MTB isolates from patients with epidemiologically unrelated TB, whereas the genotypes of isolates from patients who were infected by a common source are identical. Therefore, clustered cases of TB, defined as those in which the isolates have identical or closely related genotypes, have usually been transmitted recently. In contrast, cases in which the isolates have distinctive genotypes generally represent a reactivation of infection acquired in the distant past. Molecular epidemiologic studies have shown that the dynamics of the transmission genotyping for investigation drug resistance.
Until about a decade ago, the only markers available to study the epidemiology of TB were drug susceptibility profiles and phage types. The use of either method had serious limitations. The drug susceptibility profile of MTB strains is a highly unstable feature, because strains frequently gain resistance to anti-tuberculous drugs during treatment.
The predictive value of phage typing to link tuberculosis cases is also limited, because only a few phage types can be distinguished amongst MTB isolates. In most areas, one phage type predominates amongst MTB isolates; related and unrelated cases cannot be distinguished on this basis [18]. Genotyping permits the evaluation of isolates with different patterns of drug susceptibility. Such an evaluation may be helpful in cases in which the original organism developed drug resistance during or after anti-tuberculosis therapy, the patient was re-infected with a different MTB strain, or cross-contamination is suspected. Genotyping can be used to distinguish the patient isolates from others. If the original organism developed resistance, the cause could be non-adherence to therapy or reduced concentrations of anti-tuberculosis drugs as a result of malabsorption or drug interactions. If the cause was re-infection, however, public health authorities should attempt to identify the source [44].
Genotyping for clinical identification purposes
The cultivable members of the genus mycobacterium can be distinguished into two groupings: the MTC and the non-MTC (atypical) or NTM [18]. While TB due to MTC strain is the most common mycobacterial infection in developing countries, many NTM are also of medical relevance, particularly for immunocompromised patients. The NTM infections occur more frequently in developed countries where the incidence of TB is low [45]. There is a need for rapid diagnosis since increased morbidity and mortality is associated with NTM infections. As few clinical and radiological findings differentiate NTM infections from TB, microbiological identification to the species level is necessary. The distinction between species has not only epidemiological implications but is also RFLP.
The standard approach to genotyping MTB isolates is restriction-fragment-length polymorphism (RFLP) analysis of the distribution of the insertion sequence IS6110 in different strains, and large databases of IS6110-based genotypes are available. This method is based on differences in the IS6110 copy numbers per strain, ranging from 0 to about 25, and variability in the chromosomal positions of these IS6110 insertion sequences [18]. Isolates from patients infected with epidemiologically unrelated strains of tuberculosis have different RFLP patterns, whereas those from patients with epidemiologically linked strains generally have identical RFLP patterns. Strains with fewer than six IS6110 insertion sites have a limited degree of polymorphism, and supplementary methods of genotyping are used in these cases [12].
Another main target for DNA fingerprinting purposes is hsp65 sequences, which are highly conserved within a species and thus can be used for taxonomic studies. hsp65 is a promising target because the method based on this target has been widely used since 1992 and the technique employs only two restricted enzymes for broad identification of mycobacterial species [21]. PCR based restriction enzyme analysis (PRA) of the hsp65 or PCR-RFLP, is one of the simple and rational methods developed by Plikaytis et al. and later modified by Telenti et al. [22] for rapid identification of the most mycobacterial isolates. In this method, DNA is cleaved with particular restricted enzymes, the resulting DNA restriction fragments are separated on agarose gels and analyzed by eye [21-24]. Later on, repetitive DNA elements were cloned that could be used as probes to visualize only those restriction fragments that contain the DNA sequence complementary to the probe: RFLP typing [21]. Telenti et al. [22] demonstrated that a 439-bp portion of the hsp65 gene could be used for PRA and showed the patterns for REA.
