Assessment of the Viability of Mycobacterium Leprae by 16S rRNA Using Different Methods for Rapid Extraction of rRNA

INTRODUCTION

It is a chronic systemic infection disease, caused by the rod-shaped bacterium Mycobacterium leprae, first described by G. Armauer Hansen (so that this disease is also known as Hansen‘s disease) a Norwegian physician, in 1874. The word leprosy derives from the ancient Greek words ―lepros‖, scale and ―lepein‖, which mean ―Defilement‖. Historically the term Tazaarth‖ from the Hebrew Bible is commonly translated as leprosy (Hansen 1874). Leprosy is a fully treated disease with worldwide use of WHO recommended multi drug treatment (MDT) there is major decline in prevalence of disease. Hence the mode of transmission is not fully understood. It is thought to be transmitted by nasal discharges and skin sores, possibly also by contaminated objects, by direct skin it skin contact, mother to child through placenta, biting insects and arthropods (Euzeby JP;2008). Leprosy or infectious disease, is a chronic, mildly infectious malady capable of producing, when untreated, various deformities and disfigurements. Leprosy is

the upper respiratory tract muscle, bone and testis. (Bratschi, M. W:2015, Katoch V.M.; 2007). The first known leprosy hospitals were established by Christians in Rome and Caesarea in 4th century. Leprosy affected humanity at least 600 B.C and was well recognized in the civilization of ancient China, Egypt, and India also located in tropical and subtropical zone (Katoch V.M.; 2007). M.leprae has never been grown on any acceptable media but can be maintained in axenic cultures. As a result propagation of M.leprae has been restricted in animal models, including the armadillo and normal, athymic and gene knockout mouse. Growth of M.leprae on mouse footpads also provides a tool for accessing viability of preparation of bacteria and testing the drug susceptibility of clinical isolate. M.leprae stored at 33oC in 7H12 media has been shown to remain viable for weeks. Other than human the bacteria affects armadillos, mangable monkeys, Rabbies, mice, Chimanzees and cynomologous Macaques (Araujo, S;2016). The disease is portrayed by unusual changes of the skin. These changes, called injuries, are at first level and red. After developing, they have sporadic shapes and a trademark appearance. The sores are regularly darker in shading around the edges with stained pale focuses. Since the life form develops best at lower temperatures the Mycobacterium leprae highlights an inclination for the skin, the mucous films and the nerves. Contamination in and devastation of the nerves results in tactile misfortune. The loss of sensation inside the fingers and toes will expand the opportunity of damage. Insufficient consideration causes contamination of open wounds. Gangrene can likewise pursue, perpetrating body tissue to pass on and become deformed.

In 1886 Lehman and Neuman used the term Mycobacterium Hansen discovered lepra bacillus and Robert Koch (1882) isolated mammalian tubercle bacillus. Johne (1895) described M. paratuberculosis (Johne's bacillus). Mycobacterium is a bacterial genus containing 130 different species (Euzeby 2008) and includes M.tuberculosis and M.leprae the causative agents of tuberculosis and leprosy respectively. The genus includes obligate parasites genes as well as saprophytes, which are atypical nontuberculous mycobacteria (NTM). Atypical mycobacteria cause pulmonary and generalized infections in immuno compromised individuals and other conditions like lymphadenitis in children (Good 1985). In immunocompromised individuals the infections due to non tuberculous Important species of NTM associated with disease in human are M.scrofulaceum, M.malmoense, M. kansasii, Mxenopi, M.ulcerans, M.avium, M. intra cellulare complex (MAIG) and M.simiae. One important category person with NTM diseases is patient with AIDS and disseminated disease M.avium is now recognized as one of the most common cause of opportunistic infection in patient with AIDS in the United States of opportunistic infection inpatient with AIDS in the United States (Wallace et al 1990).

M.leprae is a straight rod about 1-8 um long and 0.3 um in diameter. In infected tissue the rods are often stacked or clumped together in globi. Electron microscopy shows an ultra-structure common to all mycobacteria:

Cell Wall- This is composed of two layers: i) Outer Layer: It is electron transparent and contains lipopolysaccharide composed of branching chains of arabinogalactan esterified with long chain mycolic acids.

be specific to M.leprae although the peptide is too scanty to be used as a diagnostic antigen. iii) Plasma Membrane: It is composed of lipid and proteins.The proteins are mostly enzymes and in theory constitute good targets for chemotherapy.They may also constitute the ‗surface protein antigens‘ that have been extracted from the cell walls of disrupted M.leprae and extensively analysed.

Unusual genetic structure and repetitive elements.

Viability Methods: Sensitive techniques for determination of viable units of an organism in the lesions are needed to monitor the effect of any antimicrobial treatment. A variety of in-vivo and in-vitro techniques have been developed. of an organism in the lesions are needed to monitor the effect of any anti-microbial treatment. Chemotherapy‘s effect is first on viability and much later on the above parameters pertaining to the total bacillary load. These methods are also relevant to detect the viable M.leprae in the environment. Even though there is no acceptable in vitro method for the cultivation of M.leprae, a variety of in vivo and in vitro techniques have been developed and used for its measuring viability. Mouse food pad In vivo methods Animal models for assessing viability The demonstration of limited multiplication in the normal mouse footpad was a major break-through in leprosy research. Normal mice after inoculation with 103 to 104 organisms in their foot pad do not develop the disease like humans but show limited multiplication (Shepard 1960). This model has been extensively used for drug susceptibility testing (Colston et al 1978, Shepard 1967). It has also been used to monitor viability of M. leprae; multiplication is seen with about 5 or more viable organisms (Levy 1987). This technique has some drawbacks. In leprosy samples from patients who have had a few months of multi-drug therapy (MDT), viable organisms are difficult to detect in the normal mouse foot pad as the few viable organisms are diluted by the very large number of dead organisms. It also takes a very long time (6 to 12 months) for the results to be available.

These models are thus especially useful for studies on persisters. Another model widely used for leprosy research is the nude mouse. Nude mice are the main draw back with different types congenitally athymic hairless mutants with total absence of an immune system. This model is more sensitive than NTR and T900R mice for

(c) Biege mice Biege mice, a strain of mice congenitally deficient in macrophages and natural killer cells, are useful for studying the multiplication of M.leprae for viability testing (Gupta & Katoch 1995, Gangadharam & Dhople 1992). Multiplication of M.leprae has been observed in several other animals including armadillos, monkeys, chimpanzees, Indian pangolin, and slender loris (Gupta & Katoch 1995, Kirchheimer & Storrs 1971, Gormus et al 1993). While armadillos have been used to generate large quantities of M.leprae for research and vaccine purposes, other models are being investigated for understanding the pathobiology of disease. While the above models serve limited purposes of studying the viability from leprosy cases, their suitability to grow leprosy bacillus from environment is questionable as these will require 104 bacilli. In Vitro viability methods

It is based on the concept that solid staining bacilli are viable organisms. This has been shown to correlate with infectivity in mice and with bacillary ATP content (Katoch et al 1989). However, zero MI values have been recorded from untreated cases with positive mouse uptake (Desikan 1976) and ATP levels probably because of clumping and sampling errors. Another limitation that is common with other techniques is that besides the skin, bacilli are also lodged in other tissues like nerves, skeletal and smooth muscles, internal organs, etc. that cannot be bacteriologically examined routinely. However, the fall of MI is an important parameter for monitoring the progress of chemotherapy.

Infection of macrophages with live M.leprae produces some alteration on the surface and on the metabolism of macrophages. This fact has been used to develop various viability assays (Nath et al 1982, Nair & Mahadevan 1984). Infection with M.leprae induces changes in Fc receptors (assessed by EA rosetting and radioactive labeling) on the surface of human and mouse derived macrophages (Nair & Mahadevan 1984). The changes in the ratio of cholesterol to cholesterol esters within macrophages have been used for drug testing (Nair & Mahadevan 1984). These systems need approximately a million bacilli/test tube. Their lower limits of sensitivity are not clear. (d) Uptake of various substrates for metabolic pathways Using nucleic acid synthesis as an indicator of viability of M.leprae within differentiated macrophages, several viability testing systems have been developed (Nath et al 1982, Harshan et al 1990). Cultures of live M.leprae incorporate significant levels of 3H thymidine for DNA synthesis as compared to parallel cultures of heat killed bacilli inside the macrophages. This information has been used for the evaluation of anti-leprosy drugs, the diagnosis of dapsone resistance, and for measuring the viability of M.leprae in cell-free systems (Ambrose et al 1978,), 3H adenosine (Harshan et al 1990) and 3H uracil have been used in place of 3H thymidine but the latter is slightly less specific in macrophages because of its incorporation in the RNA of macrophages. While investigating the purine metabolism in M.leprae, it was found that incorporation of radioactive hypoxanthine was more rapid than that of

as targets for developing viability methods.

Techniques based on the measurement of oxidation of 14C-palmitic acid 14CO2 by M.leprae using Buddemeyer type counting system or BACTEC 400 system (Franzblau 1988,1989) have been described for screening of potential anti-leprosy compounds.

ATP is present in fairly constant amounts in each cell type and is rapidly lost after cell death. Hence measurement of the ATP content can be used for determining the viability of M.leprae ( Katoch 1989, Sharma et al 1992, Katoch et al 1991, 2004,). Around 100 colony forming units of cultivable mycobacteria can be detected with this technique, which is sensitive enough to detect the small number of viable bacilli even after 2 years of MDT. ATP assays are useful for monitoring the effect of chemotherapy/immunotherapy and also for confirming relapses (Katoch 1989, Katoch et al 1991, 2004,). Bacillary ATP decay profiles of M.leprae when incubated with different drugs are useful for drug screening (Katoch et al 1989). For the purpose of drug screening there is limited application potential due to requirement for at least a million bacteria. Overall measurement of bacillary bioluminescence is a cheap and rapid technology for monitoring the response to chemotherapy and for drug screening.

The lipase content of purified M.leprae has been proposed as another indicator of viability. Although many unconventional assays for determining the viability of M.leprae have been developed, experience with most of them is limited. At present ATP estimation, single cell spectra of Na/K with LAMMA and rRNA targeting gene probes/PCR are among the most sensitive in vitro techniques available for monitoring the viability of M.leprae. Apart from their sensitivity and specificity, economic feasibility and reproducibility are other important

(b) DNA targeting probes

(c) RNA targeting probes RNA is a much more unstable molecule than DNA. After death RNAs degrade faster than DNA, therefore their demonstration and/or quantitation is likely to correlate better with the presence of live bacteria in the lesions. It is known that messenger RNA (mRNA) has the shortest half - life and thus would be an ideal targeting system for development of probes for viability determination. Probes targeting mRNA have not been very successful because of the very short half-life and technical difficulties of purification and detection of mRNA. Ribosomal RNA (rRNA) is another target molecule which is present as several copy

The appearance of continuous PCR and ongoing converse interpretation PCR (constant RT-PCR) has significantly changed the field of estimating quality articulation. Ongoing PCR is the strategy of gathering information all through the PCR procedure as it happens, in this manner consolidating intensification and identification into a solitary advance. This is accomplished utilizing a wide range of fluorescent sciences that correspond PCR item fixation to fluorescence power (Parashar et al 2005). Responses are described by the point in time (or PCR cycle) where the objective intensification is first distinguished. This esteem is generally alluded to as cycle edge Ct (In ABI System) or Crossing over point (In Roche Applied Biosystem) the time at which fluorescence force is more prominent than foundation fluorescence. Conse¬quently, the more prominent the amount of target DNA in the beginning material, the quicker a noteworthy increment in fluorescent sign will show up. Ongoing PCR innovation has improved atomic diagnostics of numerous pathogens. One of the benefits of this technique is the evaluation of bacterial DNA content in clinical examples. Finding of sickness can be founded on clinical examination, yet it might be hard to separate from infections that reason comparative side effects, particularly in zones where its rate is low (Naafs 1990). Research facility affirmation is attractive in such settings and can likewise be useful in separating a speculated reinfection or backslide from an extreme touchiness response (inversion responses) (Naafs 1990). Uses of PCR might be improved or upgraded by the utilization of novel ongoing recognition strategies.

PCR can be broken into four noteworthy stages: the direct ground stage, early exponential stage, log-straight (otherwise called exponential stage), and level stage (Tichopad et al 2003). Amid the straight ground stage (typically the initial 10–15 cycles), PCR is simply starting and fluorescence discharge at each cycle has not yet transcended foundation. Standard (generally multiple times the standard deviation of the pattern) than foundation levels. The cycle at which this happens is known as Ct in ABI Prism® writing (Applied Biosystems, Foster City, CA, USA) or intersection point (CP) in LightCycler® writing (Roche Applied Science, Indianapolis, IN, USA). This esteem is illustrative of the beginning duplicate number in the first format and is utilized to ascertain exploratory outcomes (Heid et al 1996). Amid the log-straight stage, PCR achieves its ideal intensification period with the PCR item multiplying after each cycle in perfect response conditions. At last, the level stage is achieved when response segments become restricted and the fluorescence power is never again valuable for information estimation (Bustin, 2000).

Kramme et al (2004) used real time PCR for the detection and quantification for Mycobacterium leprae from clinical specimens by Real Time PCR. They proposed that this technique help to monitor the effect of therapy in patients. When performed in untreated leprosy patients, the clinical diagnosis could be confirmed by PCR in 88.9% of MB and 33.3% of the AFB negative (PB) cases. However, Real-time detection did not increase the clinical sensitivity of PCR, despite its evidently high analytical sensitivity. In another study by TaqMan real-time PCR was used for rapidly detecting and quantifying M.leprae DNA in clinical specimens in which bacilli were undetectable by conventional histological staining. Depending upon the availability of infrastructure and resources real time PCR can be used for various effect of leprosy.

PCR analysis provides a sensitive and specific means to detect microorganisms in complex environmental samples. Soil contain abundant humic and fulvic acids that are inhibitory to Taq DNA polymerase and other enzymes used in PCR assay Soil is one of the most challenging environmental matrices from which to obtain microbial DNA that will be useful for PCR detection.

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Assistant Professor, Department of Medical Laboratory Technology, Amity Medical School, Amity University Haryana, Gurgaon, India vsmicroaiims@gmail.com