Effectiveness Of Genotype Mtbdrplus Assay Biology Essay

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Tuberculosis is an infectious disease caused by mycobacterium; TB is a serious major cause of morbidity and mortality worldwide mostly in developing countries according to the world health organization (WHO). The effort for TB control globally has been a challenge by the steady increase in drug resistant TB. (Bwanga. F, et al 2009). The development of the new Genotype MTBDRplus assay is life saving from the multidrug resistant-TB. This molecular assay achieves satisfying rapid reduction in diagnostic delay. Furthermore the cost is also lower than the conventional testing and resources can be supplied to developing countries such as Africa. Equally, it has been noted that by and large the usefulness of the assays in detecting both Rifampicin and Isoniazid resistance strains is dependent on the amount of probes and the number of InhA gene that are present in the assay. This observation was derived from the increased sensitivity, and additional detection of 11.6% of INH resistant strain achieved by the newly GenoType MTBDRplus assay with three additional probes and the second InhA gene. Having said that, the conventional methods should not be completely replaced by either of the GenoType MTBDR assays, largely because in comparative study none of these two assays could identify KatG gene with several mutations in its region, which suggests that it may not be favorable for genes with several mutations (Motto et al 2008). Therefore, it is recommended that these assays are run side by side with the conventional methods occasionally in clinical laboratories to validate susceptibility results. On the other hand, the new MTBDRplus assay is a major advancement among assays for the daily detection of RMP- and INH-resistant MTBC strains, since the therapeutic management of patients can be optimized with this fast and consistent tool. This is of the utmost importance for MDR strains and highly infectious patients, since one of the challenges of the present and future is the prevention of transmission of resistant strains

The major threat to tuberculosis (TB) control worldwide is drug resistant mycobacterium strain, as there are emerged strains which are resistant to major anti - TB drugs. The most important first line anti-tuberculosis drugs are Isoniazid (INH) and Rifampicin (RMP), and resistance to these drugs usually result in treatment failure and fatal clinical outcome (Akos et al., 2006, and Brossier et al., 2006). A rapid easy to perform, cost effective diagnostic test is considered urgently essential due to the inadequacy of the detection of resistance by predictable methods such as the staining, biochemical analysis, proportional method and culture, this is as a result of the slow growth rate of M. tuberculosis which can take as much as two to three months to produce results and the subsequent prolonged suitable treatment which usually result in fatal clinical outcome (death) (Akos et al., 2006 and Neonakis et al., 2008); lately a Polymerase chain reaction (PCR) based test have been developed and have just been adapted to increase its potential for detecting INH resistant TB strains (Miotto et al., 2008). The new DNA strip assay have been demonstrated by various studies to be an improved version when compared to the old version (Hillemann et al., 2007, Miotto et al., 2008 and Neonakis et al., 2008)

The Genotype MTBDR (DNA strip assays)

The rising efforts by the Hain Life science company to develop a rapid easy to perform, and cost effective assays based on molecular techniques that are suitable for use in clinical mycobacteriology laboratories worldwide resulted in the development of the new PCR based Genotype MTBDR (DNA strip assays) (Brossier et al., 2006 and Neonakis et al., 2008) .

The methods of these DNA strip assays are based on the facts that resistance to Rifampicin and isoniazid in M. tuberculosis is usually ascribed to mutations in the rpoB, KatG, and inHA genes of a specific strain. RMP inhibits the RNA polymerase at the level of the beta subunit encoded by the rpoB gene. Many researchers point out that Rifampicin resistance is due to mutation in comparatively small fragments (81bp) of the rpo gene encoding for the β - subunit of the RNA polymerase, while INH resistance is a result of mutations in one of the numerous regions of the KatG gene, the inhA regulatory and coding region, and the ahpC - oxyR, ndh, and Kas A genes of the strain (Brossier et al., 2006 and Akos et al., 2006).

The ensuing knowledge has allowed a PCR application to be considered very practical as it amplifies gene of interest (mutational sequence) from the DNA genome of the MTBC strains as long as correct primers, Taq polymerase and ensuring that each stages (denaturation, annealing and elongation) is performed at suitable conditions e.g. time and temperatures,(Miotto et al., 2008 and Hillemann et al 2007).The amplification process is followed by the hybridization and detection process which takes place in an automated washing and shaking device where reverse hybridization of the amplified nucleic acids to specific DNA probes bound on strips and finally the DNA - strips complex can then be evaluated by either the omission of a wild type probe and/ or the staining of a mutant probe which are indications for resistant strains (Hillemann et al., 2007). See fig 1.

Figure 1: GenoType® MTBDR test procedure (DNA strip assays)

Evaluation;

DNA - strip ensuing colour formation - determinant whether a strain is resistant to specific anti tuberculosis

Hybridisation;

Reverse hybridisation of amplified nucleic acids to specific DNA probes bound on strips

Amplification by PCR

DNA extraction from NaLC (NaOH) processed sputum

(Hellmann. et al, 2007)

This molecular procedure can be directly applied to smear positive and negative specimens and have revealed to have a turnaround time of about 6 hours from specimen receipts to reporting of results of vulnerability testing, these procedures saves several weeks, which is necessary for primary isolation and conventional DST (Drug susceptibility test) (Hellmann et al., 2007., and Miotto et al 2008). nevertheless, the main fundamental limitations of the MTBDR system was its low sensitivity for the isoniazid (INH) resistance detection (70%) as a result of the fact that the test only target the KatG S315T mutation, even more so the test performance on clinical specimens was suboptimal because of the lack of a clear hybridization signal (Hellmann et al 2007 and Miotto et al 2008).

The newly equipped Genotype® MTBDRplus assay

On the 11th of January 2007, The foundation for innovative New diagnostics (FIND) and Hain Lifescience (Hain) developed the new Genotype® MTBDRplus assay and this has been approved all over Europe. This was developed as a means to improve the assays sensitivity for Isoniazid resistance and direct detection of drugs resistance in clinical specimens which will increase the dependability of the GT - MBTDR vulnerability test results. The new MTBDRplus strip is equipped with additional probes to all probes in order to enlarge the detectable rpoB DNA fragments to amino acids 503 to 533. In addition, the promoter region of the inhA gene is included on the new strip (figure 2 (B). (Brossier et al., 2007, Hilleman et al., 2007 and Miotto et al., 2008).

Figure 1: The older version - Genotype MTBDR assay

A) 2 1

(http://jcm.asm.org/content/vol45/issue8/images/large/zjm0080774630001.jpeg

The older version of the GenoType MTBDR assay, comprising of the following targeted genes and specificity shown above from top to bottom in fig 1(A);(A), amplification control (UC); conjugate control (CC); M. tuberculosis complex-specific control (Tub); rpoB amplification control; rpoB wild-type probes WT1 to WT5 located in the hot spot region WT 1 to WT 5, respectively; rpoB mutant probes (probes MUT1, MUT2A, MUT2B, and MUT3) with mutations in codons rpoB D516V, H526Y, H526D, and S531L, in that order; katG amplification control; katG codon 315 wild-type probe (WT); and katG codon 315 mutation probes (T1 and T2, respectively) with AGC-to-ACC (S315T1) and AGC-to-ACA (S315T2) exchanges, respectively.(Hillemann. D,et al,2007)

Figure 2: showing a representative example of a pansusceptible strain in (row1) and an MDR strain (row 2) obtained by the new GenoTypeplus MTDBR

B) 2 1

(http://jcm.asm.org/content/vol45/issue8/images/large/zjm0080774630001.jpeg

The MTBDR plus assay (A) comprises of three extra rpoB wild-type probes (which results in probes WT1 to WT8) and also InhA C15T mutation in the regulatory region of the InhA and targets the regulatory region of the inhA gene with the inhA amplification control (WT 1 to WT 8, correspondingly); inhA gene wild-type probes WT1, which covers the region from positions -9 to -22, and WT, which covers from positions -1 to -12; and inhA mutant probes MUT1, MUT2, MUT3A, and MUT3B with mutations -15C/T, -16A/G, -8T/C, and -8T/A, respectively (MUT 1, MUT 2A, MUT 2B, and MUT 3, respectively). Pansusceptible isolate 1 was positive with the wild-type rpoB and katG probes of the MTBDR assay and, additionally, with the inhA wild-type probes of the MTBDRplus assay. MDR isolate 2 shows RMP resistance in the two assays (for the MTBDR assay, the exclusion of rpoB WT 5 and positivity for rpoB Mut 3; for the MTBDRplus assay, the exclusion of rpoB WT 8 and positivity for rpoB Mut 3). Regarding INH resistance, isolate 2 had to be deduced to be INH susceptible (katG wild-type probe) by the MTBDR assay, while the MTBDRplus assay signified INH resistance (exclusion of the inhA WT1 probe and positivity for the inhA MUT1 probe). (Hillemman .D,et al. 2007).

The Effect of the additional target gene and probes to the new Genotype® MTBDR assay

In contrast with the older version, introducing the new targeted genes for isoniazid resistance and the three added probes enhances the sensitivity of GT - MTBDR plus with a smaller amount probes and devoid of inhA gene. This can also be seen in the outcome of GenoType MTBDR and MTBDR assays with smear - positive sputum specimens.

A new study which shows how the new GT - MTBDRplus assay was tested with isoniazid resistant isolates and 78 clinical specimens, that had been initially analysed by the GT - MTBDR and the outcome was then evaluated with those of sequence analysis. The analysis show that the results derived by the new GT - MTBDRplus is significant in comparison to the older version, as it could permits the correct identification of an additional 11.6% (20/173) of isoniazid resistant strains when compared to the results obtained for GT - MTDBR, with a specificity of 100% . (Miotto et al., 2008). Nevertheless, in one sample none of the tests allowed the detection of the katG gene due to the existence of numerous mutations in the same gene, as confirmed by the sequence analysis. Both the assays were interpreted as indeterminate for katG analysis. (Miotto et al., 2008). (See table 1. below)

Table1. showing the detection of isoniazid resistance in clinical isolates by the G- MTBDR and the G - MTBDRplus assays compared with the sequencing data

No (%) of Isolates carrying

Assay

Gene

Wild-type gene

Isoniazid resistant gene

Indeterminate gene

GT-MTBDR

katG

55(31.8)

117(67.6)

1(0.6)

GT-MTBDR plus

katG

InhA

55(31.8)

153(88.4)

117(67.6)

20(11.6)

1(0.6)

0(0.0)

Sequence analysis

KatG

InhA

55(31.8)

153(88.4)

117(67.6)

20(11.6)

1(0.6)

0(0.0)

The isolates tested (n = 173) were previously determined to be isoniazid resistant by drug susceptibility testing. The GT - MTBDR assay did not target the inhA gene. In one isolate, the katG gene was not detected by either the GT - MTBDR or the GT - MTBDR plus assay, which sequence analysis confirmed as being due to several mutations in the same gene. (Miotto et al., 2008)

In conclusion the Genotype MTBDR multiplex test is easy to perform on culture based isolates and directly on smear positive sputum samples from patients with TB. It also offers easy procedure that is compatible with regular workflow and can be completed within 24hrs (see fig 1.). This technique is rapid as it involves Polymerase chain reaction (PCR) required to amplify the mutant sequence from the DNA genome of the TB which can be performed under two hours and consequently saves several weeks, which is required for primary isolation and conventional DST (Drug susceptibility test) that shows a slow growth rate of M. tuberculosis and can take as long as two to three months to produce result. The rapid turnaround time of the test should enable optimization of the therapy of these patients (Akos et al., 2006 and Neonakis et al., 2008);

Recent molecular based awareness that INH resistance is due to mutations in one of the numerous regions of the KatG gene, the inhA regulatory and coding region, and the ahpC - oxyR, ndh, and Kas A genes of the strain (Brossier et al., 2006 and Akos et al., 2006), has led to the development of the newer version of this assay known as the GenoType MTBDRplus assay.

In general performance, scientists concluded that the new Genotype MTBDRplus assay was better useful tool than the usual testing in the control of tuberculosis because it is able to identify RIF and INH resistant mycobacterium tuberculosis in both strains and clinical samples. (Lacoma. A,et al 2008)

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