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TB claims a victim every 10 seconds, it rose silently, slowly, sleeping into homes of millions. With the increased prevalence of HIV, TB became the leading cause of HIV-related deaths, Among 1.37 million new case of T.B among HIV patients in 2007, 456,000 death was due to T.B. the scenario is even getting worth by the increased rate of emerging multi-drug resistant strains of Mycobacteria.
This project aims to develop a new highly specific and sensitive diagnostic system utilizing gold nanoparticles technology for i) Detection of Mycobacteria strains in clinical samples. ii) detection of Isoniazide (INH) and Rifampin (RIF) resistance of Mycobacteria strains. This project will address the need of developing countries which represent about 80% of HBCs (High Burden Countries) by providing a low cost detection system. The proposed system should be able to be performed with minimal equipment, eliminating the need of sophisticated expensive laboratories, equipments and highly trained personnel , thus it can be readily applied in developing countries and positively impact the global TB control efforts.
Tuberculosis (TB) is a contagious disease caused by Mycobacterium tuberculosis. It spreads through the air like the common cold, left untreated, each person with active TB disease will infect on average between 10 and 15 people every year. Mycobacterium tuberculosis claims more human lives every year than any other single pathogen with estimated 1.3 million deaths in 2008 . Global T.B burden magnitude increased with the increased prevalence of HIV. WHO estimated that of the 1.3 million deaths in 2008, 0.5 million deaths occurred among HIV positive cases . In Africa, HIV is the single most important factor contributing to the increase the incidence of TB since 1990.
There were an estimated 11.1 million (range, 9.6-13.3 million) prevalent cases of TB in 2008 equivalent to 164 cases per 100 000 population . In 2007 out of 13.7 million estimated prevalent cases an estimated 687 000 (5%) were HIV-positive. According to the WHO 2009 T.B report four out of six worldwide regions are on track to at least halves the prevalence rate by 2015, these regions are Eastern Mediterranean Region, the Region of the Americas, the South-East Asia Region and the Western Pacific Region. In African and European regions the prevalence rates substantially increased during the 1990s, by 2007 the prevalence rates in African regions are above that of the 1990, while back to the 1990s rate in the European region. Projection indicates that neither regions will achieve halving the prevalence rate by 2015, and in African regions it is unlikely that the prevalence rates will be back to the 1990s rates .
Multi Drug resistance T.B
The emergence of drug resistance strains of T.B threats the T.B control effort. Multi Drug Resistance (MDR) strains were defined by the WHO as strains resistant to at least isoniazid (INH) and Rifampin (RIF), the two most powerful anti-TB drugs; rates of MDR-TB are high in some countries, especially in the former Soviet Union. Furthermore, the development of extensive drug resistant tuberculosis (XDR) due to improper use of antibiotics causes more obstacles in treating TB patients.
Incidence of MDR strains
In 2007, 0.5 million case of MDR-TB were reported, 85% of these case were accounted for by 27 countries (15 in the European region). These countries were termed 27 high MDR-TB burden countries, countries ranked from first to fifth in terms of total MDR-TB cases were India (131 000), China (112 000), the Russian Federation (43 000), South Africa (16 000) and Bangladesh (15 000). By November 2009, 57 countries and territories had reported at least one case of XDR-TB. (extended drug resistance)
Isoniazid (INH) resistance
Isoniazid is one of the first-line multidrugs therapy of tuberculosis (TB). INH resistance does not affect mycobacterium virulence but instead it delays the clinical response and increases the threat of treatment failure . Mutations in several genes and genomic regions are involved in the INH resistance .
Resistant to INH usually occurs due to point mutations in either KatG gene encoding catalase peroxidase which is required for the activation of INH prodrug , or in the inhA gene or its promoter reigon of the fabG1-inhA operon fabG1 (also known as mabA) .
KatG mutations occur mainly in codon 315 AGC to either ACC or ACA (Table 1) changing the coding amino acid from serine into threonine mostly and that represent about 30 - 90% of INH resistant strains according to the geographic regions [4, 8].
inhA regulatory region mutations most frequently occurs 15 nucleotides upstream of the contiguous mabA gene the mutation consists in a C -> T transition usually indicated as C (-15) T. G (-24) T, and T (-8) G/A also can occur but less frequently (Table 1) [4, 9-10].
There is also mutation occurs in the promoter of the ahpC and KasA gene which are reported to represent about 12 - 24% and 10 - 14% of the INH resistance, respectively .
Table : Multidrug resistant Mycobacterium tuberculosis resistance codons
G to C / C to A
G to T
C to T
T to G / T to A
A to T
C to T& A to G
C to T
Red in color means the same amino acid type, from purines to purines or from pyrimidines to pyrimidines
Rifampin (RIF) resistance
Rifampin (RIF) is another one of the five first-line drugs in the treatment of tuberculosis. Resistance to Rifampin has been associated to amino acid changes in the ß- subunit of DNA dependent RNA polymerase; the drug target; encoded by M. tuberculosis rpoB . More than 95% of RIF resistant isolates harbor mutation in 81 bp hot spot region within the rpoB known as rifampin-resistance determining region (RRDR) with 60 - 70% of these mutations are in codons Ser531Leu (TCG - TTG) and His526Cys (CAC - TGC) (Table 1) [12-13]. More than 90% of rifampin resistance TB isolates are also isoniazid resistant so RIF resistance has a great contribution to MDR TB development. The most common mutations so far in the RRDR region that is known from DNA sequencing of the resistant isolates include codons 531, 526, and Asp516Val (GAC - GTC) [14-15].
Diagnosis of T.B
"Early case detection through quality-assured bacteriology" is recommended by WHO as a part of Stop TB strategy. Sputum smear microscopy is the primary tool for the detection of TB while culture identification is recommended for smear negative samples and Drug Sensitivity Testing (DST). The average number of laboratories capable of performing smear microscopy exceeds the target of at least 1 lab per 100,000 populations in four worldwide regions, while the number of laboratories in western pacific region is 0.5 per 100,000 populations.
Laboratories capable of providing culture and DST service are essential either for the testing of negative smear sample especially in the settings where HIV prevalence is high or for diagnosis of drug resistance. The capacity to perform culture services is seriously limited in most of the 22 HBCs, only seven HBCs out of 22 have at least one culture laboratory per 5 million population as recommended by WHO . While only five HBCs reported having 1 laboratory with DST capability per 10 million .
The emergence of extensively Drug Resistant TB (XDR-TB) raises the needs to access laboratories capable of performing DST of second line drugs. According to the WHO report these services were available to 63 out of 142 reporting countries in 2007 either within or outside the country.
In summary there is a general shortage in laboratories capable of providing culture and sensitivity testing services especially in the high burden countries. This shortage raises the need for rapid introduction of new diagnostics tools capable of fulfilling this gap.
Current challenges in TB diagnosis
Smear microscopy remains the primary identification tool especially in the developing countries. Although smear microscopy is very selective, its accuracy depends on the bacterial load and the quality of the sputum specimen and the training of the laboratory technicians . In its best situation smear microscoby can be considered insensitive as it can roughly detect 50% of all positive cases, in 2007 the incidence of TB were 9.27 million case with only 4.06 million smear positive cases.
Isolation and culturing of Mycobacterium on liquid or solid media is more sensitive method and allows for testing antibacterial sensitivity. However culturing Mycobacterium requires Expensive biosafety facility that is expensive to build and maintain and require highly trained laboratory technicians. Some developing countries doesn't have TB culturing facility at all while in other TB culture is performed in National references laboratories or in hospitals in large cities. As previously mentioned only few developing countries have the access to high quality sensitivity testing of First-line Drugs and even fewer for testing second line drugs.
Even when capacity exist TB diagnosis by culture still can take weeks because of the slow growth rate of mycobacteria. In most countries TB culturing take place in central laboratories so specimen often had to be sent to distant laboratories which increase the specimen processing time and affects the results .
Gold Nanoparticles for ultrasensitive colorimetric detection
Gold nanoparticles (AuNPs) possess a unique phenomenon known as surface Plasmon resonance (SPR) which is responsible for their intense red color. This color changes to blue when the nanoparticles in the colloidal solution are close to each other enough to aggregate . These unique optical properties have allowed the use of AuNPs in simple and rapid colorimetric assays for nucleic acids detection offering higher sensitivity and specificity than current detection techniques [17-18]. Nucleic acid detection using gold nanoparticles is based on two main mechanisms: (i) the unmodified AuNPs method, and (ii) modified AuNPs method. The latter requires covalent modification of the AuNPs with an oligonucleotide specific to the target needed to be detected.
The unmodified AuNPs method is based on the fact that single-stranded DNA (ssDNA) adsorbs on citrate-coated AuNPs preventing their salt induced aggregation, and retains their red color even in the presence of sodium chloride, while double-stranded DNA (dsDNA) does not adsorb on AuNPs due to the repulsion between its negatively-charged phosphate backbone and the negatively-charged coating of citrate ions on the surfaces of the AuNPs. This method has been used to detect single nucleotide polymorphisms in PCR-amplified genomic DNA extracted from clinical samples .
The modified AuNPs method is based on mercaptoalkyl oligonucleotide modified gold nanoparticle probes which align in a head to tail fashion onto target polynucleotide. Hybridization of gold nanoparticles modified with mercapto alkyloligonucleotide and target sequence (HCV RNA) result in the binding of an oligonucleotide probes to their complementary sequence, and change in color from red to blue occurs indicating the presence of the target. On the other hand, in absence of the target, the solution remains red in color.
Soo et al have recently reported the use of gold nanoparticles derivatized with thiol modified oligonucleotides for identification of Mycobacterium tuberculosis (MTB) and differentiation of MTB from other members of M. tuberculosis complex (MTBC) from clinical sputum samples. The reported assay showed a 96.6% sensitivity and 98.9% specificity towards detection of MTBC, and a 94.7% sensitivity and 99.6% specificity for detection of MTB .
National & Global impact
Although TB claims huge number of lives each year, it is curable and preventable and we can still face it and achieve the No TB goal by 2050. According to the WHO report there is an urgent need for new diagnostic tools for either positive / negative detection of TB and for sensitivity testing. The challenges facing the conventional TB diagnostic methods directly impacts the efforts of controlling TB worldwide especially with the MDR-TB strains are emerging.
Molecular detection lines based on PCR, Real time PCR and microarray were used for the identification of Mycobacterium [21-22] and detection of resistance . Molecular methods might prove advantages regarding sensitivity and processing time, however performing these method needs highly equipped laboratories with highly trained staff. This will limit the benefits of the low income countries which represent the majority of HBCs which in turn will limit the impact of these new methods on the global TB control efforts. Table 2 list some of the commercially available molecular diagnostic kits for Mycobacteria.
The current facts regarding TB incidence and prevalence, the emerging MDR-TB and XDR-TB together with socioeconomic status of the HBCs, raise the need of developing new diagnostic tools. Optimum new diagnostic tool should be highly specific, highly sensitive, require low cost laboratory and minimal skilled labor, thus it can be widely used in low income countries and positively impact the global TB control efforts.