Rapid detection of ethambutol-resistant Mycobacterium tuberculosis in clinical specimens by real-time polymerase chain reaction hybridisation probe method

Background: Early diagnosis of drug resistance (DR) to ethambutol (EMB) in tuberculosis (TB) remains a challenge. Simple and reliable method (s) are needed for rapid detection of DR Mycobacterium tuberculosis (MTB) in clinical specimens. Objectives: The aim of this study was to design fluorescence resonance energy transfer hybridisation probe-based real-time polymerase chain reaction (PCR) method for the early detection of EMB-resistant MTB direct from clinical sputa. Materials and Methods: Primers and probes were designed against 306 codon of embB gene which is commonly associated with EMB resistance. A comparative study was done between Lowenstein–Jenson (L–J) proportion and hybridisation probe-based real-time PCR method for susceptibility testing. DNA sequencing was used in nine representative isolates to validate the efficiency of real-time PCR method to detect emb306 mutation of MTB. Results: A total of 52 clinical sputum samples and corresponding culture isolates (from category II pulmonary TB cases) were included in this study. Out of 52 MTB isolates, 32 and 20 were resistant and susceptible to EMB, respectively, as determined by L–J proportion method. Real-time PCR showed 95% specificity, 75% sensitivity and 82.69% accuracy when compared with L–J proportion method. A 100% of concordance was observed by validating the real-time PCR results with DNA sequencing. Conclusions: Our real-time PCR hybridisation probe method promises for rapid detection of EMB-resistant MTB directly from clinical specimens. However, future studies and modifications of method by incorporating other potential loci along with targeted mutation (emb306) are still required to increase the sensitivity of method.

Synthesis, characterization and analytical application of new polysaccharide cation exchanger resin containing methacrylic acid for industrial waste water treatment.

TMAA (Tamarind methacrylic acid) cation exchanger resin was synthesized, based on locally available polysaccharide Tamarind Kernel powder. The resin was characterized by FTIR and elemental analysis. The resin was found to be stable in acidic as well as in basic medium. Physicochemical properties of the resin were examined. The total cation exchange capacity was measured and effect of pH and metal ion concentration on ion exchange capacity were studied. The distribution coefficients at different pH were also studied using batch equilibration method. The developed column technique has been used for the binary separation of Cu+2/Zn+2, Cu+2/Pb+2, Cu+2/Cd+2 and waste water treatment.

Synthesis, characterization and rheological properties of guaran grafted polyacrylamide (G-G-Pam) copolymer.

Guaran was transformed into grafted polymer using vinyl monomer. The vinyl monomer used for the graft was acrylamide. The grafting was initiated through the formation of free radical centers on the polymer backbone by oxidation of guaran with cerium (IV) in nitric acid medium. The degree of grafting was varied by using varying amount of acrylamide vinyl monomer. The rheological properties of the guaran grafted polyacrylamide copolymer have been studied by varying the degree of grafting, time, concentration, temperature, spindle number and shear rate. Thermal Characteristics of the guaran-grafted polyacrylamide was studied using thermo gravimetric analysis under nitrogen atmosphere. The prepared copolymer was characterized by FTIR.

Is Mycobacterium avium subsp. paratuberculosis, the cause of Johne’s disease in animals, a good candidate for Crohn’s disease in man.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease or paratuberculosis, a gastro intestinal inflammatory condition in ruminants and other animals, which is similar to Crohn’s disease (CD) that occurs in man. The role of MAP in the causation of CD has been under intense investigation in the last few decades. This review summarizes the status of MAP in animals and the food chain and its association with CD in man.

Strain diversity within Mycobacterium avium subspecies paratuberculosis — A review.

Mycobacterium avium subspecies paratuberculosis (MAP), is the etiological agent of Johne’s disease (or paratuberculosis) in animals and has also been linked with Crohn’s disease of human beings. Extreme fastidious nature of the organism (MAP) has hampered studies on diversity within the organism. Studies based on phenotypic properties like growth rate, pigmentation, lipid profile etc., are unable to provide complete information on diversity of MAP organism in nature. However, with the advent of molecular assays (IS900 RFLP, PFGE, IS1311 PCR-REA, SSR typing, VNTR typing etc.) in last 2 decades, progress has been made to differentiate MAP strains. MAP isolates have been classified into various types and subtypes using these molecular tools. Optimization of these typing assays has led to generation of new information about MAP strains, subtypes, their comparative genomics, relative evolution, comparative virulence etc. Knowledge of strain diversity is important for better understanding of molecular and sero-epidemiology, infection and patho-biology, vaccine development and planning control strategies. The present review provides available information on MAP strains, host adaptations, their virulence, comparative genomics, relative genetic evolution and differentiation.

Evaluation of four methods of DNA recovery from Mycobacterium avium subspecies paratuberculosis present in intestine tissue of goats and comparative sensitivity of IS900 PCR with respect to culture for diagnosis of Johne's disease.

Low sensitivity of PCR reaction for detection of Mycoobacterium avium subspecies paratuberculosis (MAP) in tissues and fecal samples is mainly attributed to false negative results. Present study was undertaken to compare four methods of DNA isolation from tissues of infected animals and to determine most sensitive protocol for the recovery of DNA, suitable for IS900 PCR based detection of Johne's disease infection. Method I, the traditional van Soolingen2 method of DNA isolation was adopted for the isolation of DNA from tissues. Method II was modification (hexadecyl pyridinium chloride-HPC treatment) of van Soolingen2 method. Method III was traditional tissue DNA isolation method based on tissue lysis buffer. Method IV was modification of method III (HPC treatment). Using four methods of DNA isolation from 25 intestinal tissues of clinically infected goats, DNA was isolated from 15 (60.0%), 18 (72.0%), 13 (52.0%) and 13 (52.0%) tissues using method I, II, III and IV, respectively. All isolated DNA preparations were positive for MAP in IS900 PCR. HPC treatment enhanced the recovery of DNA from tissues of infected animals using method II. Therefore, method II can improve the diagnosis MAP infection using IS900 PCR.

Mycobacterium avium subspecies paratuberculosis diagnosis and strain typing--present status and future developments.

Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic gastroenteritis of ruminants and has zoonotic importance. We present here a review of MAP with respect to--(i) present diagnostic techniques and important developments; and (ii) MAP strain-typing tools. A summary of the findings to date is presented, and advantages and disadvantages of each of the methods are compared and discussed.

Non-chemical method of DNA recovery and characterization of Mycobacterium avium subspecies paratuberculosis using IS 900 PCR.

In the present study, two methods of DNA isolation-routine, traditional and standard DNA isolation protocol for Mycobacteria (Method 1) and a new non-chemicals and non-enzymes (physical) method (Method 2) of DNA recovery have been compared and evaluated in IS900 PCR for the specific detection of pathogen. Using the new Method 2, DNA has been recovered from few (1 - 3 colonies), extremely minute and stunted colonies. DNA, thus, isolated from these colonies (colonies PCR) and cultured for the first time from the cases of Crohn's disease in human beings, dairy cattle, raw milk and pasteurized commercial milk samples has been characterized in the present study. It is the first report from India.

Effect of Semecarpus anacardium nuts on lipid peroxidation.

Alcoholic extract of pericarp showed significant protection against FeSO4 induced lipid peroxidation, as compared with whole native nut and seeds. Mechanism of action may be through metal chelation or activation of endogenous antioxidant enzymes, because the extract did not show hydroxyl and super oxide anion scavenging property. Further in vitro experiments against FeSO4, it did not maintain the level of reduced glutathione.

Resistant coagulase negative staphylococci from clinical samples.

Antibiotic susceptibility testing against 17 antibiotics was done on 96 strains of various species of coagulase negative staphylococci by Stokes method. Hundred per cent sensitivity was found against vancomycin and cefotaxime and about 90 per cent against ciprofloxacin, clavulanate potentiated amoxycillin, cloxacillin and clindamycin. Strains showed highest resistance against cotrimoxazole (77.08%) and tetracycline (64.59%). Clavulanate potentiated amoxycillin was found to be highly active against penicillin, ampicillin and amoxycillin resistant organisms. The results highlight the importance of antibiotic resistance typing among coagulase negative staphylococci species which are increasingly being reported from serious clinical infections making empiric therapy and selection of antibiotics difficult in these infections.