Fabrication of dual drug-loaded polycaprolactone-gelatin composite nanofibers for full thickness diabetic wound healing.

Aim: Design of moxifloxacin and ornidazole co-loaded polycaprolactone and gelatin nanofiber dressing for diabetic wounds. Materials & methods: The composite nanofibers were prepared using electrospinning technique and characterized for in vitro drug release, antibacterial activity, laser doppler and in vivo wound healing. Results: The optimized nanofiber demonstrated an interconnected bead free nanofiber with average diameter <200 nm. The in vitro drug release & antimicrobial studies revealed that optimized nanofiber provided drug release for >120 h, thereby inhibiting growth of Escherichia coli and Stapyhlococcus aureus. An in vivo wound closure study on diabetic rats found that optimized nanofiber group had a significantly higher wound closure rate than marketed formulation. Conclusion: The nanofiber provided prolonged drug release and accelerated wound healing, making it a promising candidate for diabetic wound care.

This article is about making a wound dressing material of tiny fibres that have antibiotic properties to kill microbes at the wound site and make wounds heal faster. This is particularly important for people with diabetes, whose wounds often take longer to heal. The designed nanofibrous dressing releases antibiotic drugs at the wound site for more than 120 h, killing harmful microbes and thus avoiding their invasion at wound site. Also, animal experiments showed that the nanofibers shorten the time wounds take to heal by providing a suitable surface and a favourable environment for wound healing. The study concludes that the fabricated nanofiber dressing helps complex wounds heal faster, and could be a strong new dressing material for diabetic wound care.

Scrub typhus: A rare cause of secondary nephrotic syndrome.

Scrub typhus is an important etiological agent in acute febrile illness in the post-monsoon season in tropical countries. It leads to dreaded complications if left untreated. Acute kidney injury is one such complication. Malaria, syphilis, and HIV have been associated with secondary nephrotic syndrome in pediatric age group. Scrub typhus has been reported only once with nephrotic syndrome. We report a case of scrub typhus-associated nephrotic syndrome with acute kidney injury in a five-year-old female with uneventful outcome.

Vitality of Proteinase K in rRTPCR Detection of SARS-CoV2 Bypassing RNA Extraction.

This study aimed to detect the SARS-COV2 viral component directly from inoculated VTM without RNA extraction. Inoculated VTMs of already tested 50 positive and 50 negative samples were divided into three groups. Group I was treated with Proteinase K (PK) followed by 3-step-heat treatment at different temperatures (25°C, 60°C, and 98°C) and stored at 4°C. Group II was directly subjected to 3-step-heat treatment without PK exposure and stored at 4°C. And group III was set-up as standard group; it was processed using Qiagen's column based QIAamp Nucleic Acid kit and the obtained nucleic acids were stored at 4°C. These stored samples were used as a template to execute real-time polymerase chain reaction, and results were noted. Group I demonstrated 96% and 88% sensitivity for N and ORF1ab genes respectively, whereas group II demonstrated 78% and 60% when compared to the results of standard group III. Overall group I showed better results than group II when compared to group III. Thus, in situations where gold-standard reagents are not available, PK exposure and heat treatment can be employed to carry out molecular detection of SARS-CoV2 viral component.

Assessment of Successful qRT-PCR of SARS-CoV-2 Assay in Pool Screening Using Isopropyl Alcohol Purification Step in RNA Extraction.

The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50 µL of one COVID-19 positive nasopharyngeal/oropharyngeal (NP/OP) specimen and 50 µL each of 4 known negative specimens. Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73 ± 1.49 ng/uL, which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR.

Galactose-Clicked Curcumin-Mediated Reversal of Meropenem Resistance among Klebsiella pneumoniae by Targeting Its Carbapenemases and the AcrAB-TolC Efflux System.

In over eighty years, despite successive antibiotics discoveries, the rapid advent of multidrug resistance among bacterial pathogens has jolted our misapprehension of success over them. Resistance is spreading faster than the discovery of new antibiotics/antimicrobials. Therefore, the search for better antimicrobials/additives becomes prudent. A water-soluble curcumin derivative (Curaq) was synthesised, employing a Cu (I) catalysed 1, 3-cyclo addition reaction; it has been evaluated as a potential treatment for multidrug-resistant isolates and as an antibiotic adjuvant for meropenem against hypervirulent multidrug-resistant Klebsiella pneumoniae isolates. We also investigated its solubility and effect over carbapenemase activity. Additionally, we investigated its impact on the AcrAB-TolC system. We found that Curaq inhibited bacterial growth at a minimal concentration of 16 µg/mL; at a 32 µg/mL concentration, it killed bacterial growth completely. Only nine (9.4%) Klebsiella isolates were sensitive to meropenem; however, after synergising with Curaq (8 µg/mL), 85 (88.54%) hvKP isolates became sensitive to the drug. The Curaq also inhibited the AcrAB-TolC efflux system at 1 µg/mL concentration by disrupting the membrane potential and causing depolarisation. The kinetic parameters obtained also indicated its promise as a carbapenemase inhibitor. These results suggest that Curaq can be an excellent drug candidate as a broad-spectrum antibacterial and anti-efflux agent.

Making of water soluble curcumin to potentiate conventional antimicrobials by inducing apoptosis-like phenomena among drug-resistant bacteria.

The upsurge of multidrug resistant bacterial infections with declining pipeline of newer antibiotics has made it imperative to develop newer molecules or tailor the existing molecules for more effective antimicrobial therapies. Since antiquity, the use of curcumin, in the form of Curcuma longa paste, to treat infectious lesions is unperturbed despite its grave limitations like instability and aqueous insolubility. Here, we utilized "click" chemistry to address both the issues along with improvisation of its antibacterial and antibiofilm profile. We show that soluble curcumin disrupts several bacterial cellular processes leading to the Fenton's chemistry mediated increased production of reactive oxygen species and increased membrane permeability of both Gram-positive and Gram-negative bacteria. We here report that its ability to induce oxidative stress can be harnessed to potentiate activities of ciprofloxacin, meropenem, and vancomycin. In addition, we demonstrated that the soluble curcumin reported herein even sensitizes resistant Gram-negative clinical isolates to the Gram-positive specific antibiotic vancomycin, thereby expanding the antibacterial spectrum of this drug. This work shows that the soluble curcumin can be used to enhance the action of existing antimicrobials against both Gram-positive and Gram-negative bacteria thus strengthening the antibiotic arsenal for fighting resistant bacterial infections for many years to come.

MoS2-Modified Curcumin Nanostructures: The Novel Theranostic Hybrid Having Potent Antibacterial and Antibiofilm Activities against Multidrug-Resistant Hypervirulent Klebsiella pneumoniae.

The recent emergence of hypervirulent clinical variants of Klebsiella pneumoniae (hvKP) causing community-acquired, invasive, metastatic, life-threatening infections of lungs, pleura, prostate, bones, joints, kidneys, spleen, muscles, soft-tissues, skin, eyes, central nervous system (CNS) including extrahepatic abscesses, and primary bacteremia even in healthy individuals has posed stern challenges before the existing treatment modalities. There is therefore an urgent need to look for specific and effective therapeutic alternatives against the said bacterial infection or recurrence. A new type of MoS2-modified curcumin nanostructure has been developed and evaluated as a potential alternative for the treatment of multidrug-resistant isolates. The curcumin quantum particles have been fabricated with MoS2 via a seed-mediated hydrothermal method, and the resulting MoS2-modified curcumin nanostructures (MQCs) have been subsequently tested for their antibacterial and antibiofilm properties against hypervirulent multidrug-resistant Klebsiella pneumoniae isolates. In the present study, we found MQCs inhibiting the bacterial growth at a minimal concentration of 0.0156 µg/mL, while complete inhibition of bacterial growth was evinced at concentration 0.125 µg/mL. Besides, we also investigated their biocompatibility both in vitro and in vivo. MQCs were found to be nontoxic to the SiHa cells at a dose as high as 1024 µg/mL on the basis of the tested adhesion, spreading of the cells, and also on the various serological, biochemical, and histological investigations of the vital organs and blood of the Charles Foster Rat. These results suggest that MQCs have potent antimicrobial activities against hvKP and other drug resistant isolates and therefore may be used as broad spectrum antibacterial and antibiofilm agents.

Classification of Clinical Isolates of Klebsiella pneumoniae Based on Their in vitro Biofilm Forming Capabilities and Elucidation of the Biofilm Matrix Chemistry With Special Reference to the Protein Content.

Klebsiella pneumoniae is a human pathogen, capable of forming biofilms on abiotic and biotic surfaces. The limitations of the therapeutic options against Klebsiella pneumoniae is actually due to its innate capabilities to form biofilm and harboring determinants of multidrug resistance. We utilized a newer approach for classification of biofilm producing Klebsiella pneumoniae isolates and subsequently we evaluated the chemistry of its slime, more accurately its biofilm. We extracted and determined the amount of polysaccharides and proteins from representative bacterial biofilms. The spatial distribution of sugars and proteins were then investigated in the biofilm matrix using confocal laser scanning microscopy (CLSM). Thereafter, the extracted matrix components were subjected to sophisticated analysis incorporating Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, one-dimensional gel-based electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), and MALDI MS/MS analysis. Besides, the quantification of its total proteins, total sugars, uronates, total acetyl content was also done. Results suggest sugars are not the only/major constituent of its biofilms. The proteins were harvested and subjected to SDS-PAGE which revealed various common and unique protein bands. The common band was excised and analyzed by HPLC. MALDI MS/MS results of this common protein band indicated the presence of different proteins within the biofilm. The 55 different proteins were identified including both cytosolic and membrane proteins. About 22 proteins were related to protein synthesis and processing while 15 proteins were identified related to virulence. Similarly, proteins related to energy and metabolism were 8 and those related to capsule and cell wall synthesis were 4. These results will improve our understanding of Klebsiella biofilm composition and will further help us design better strategies for controlling its biofilm such as techniques focused on weakening/targeting certain portions of the slime which is the most common building block of the biofilm matrix.

Multiplex nested polymerase chain reaction targeting multiple genes for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens.

OBJECTIVES: The objective of this study was to design and evaluate a novel multiplex nested polymerase chain reaction (PCR) protocol for simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens obtained from symptomatic patients clinically suspected of sexually transmitted infections (STIs), targeting two different genes each for these pathogens. MATERIALS AND METHODS: A total of 116 genitourinary specimens were collected from men (n = 12) and women (n = 104). Direct microscopy, culture isolation, and antimicrobial susceptibility testing for N. gonorrhoeae were performed. Multiplex nested PCR was performed on clinical samples using novel designed primers targeting porA pseudogene and opa gene of N. gonorrhoeae and momp gene and cryptic plasmid of C. trachomatis simultaneously. DNA sequence analysis of nested PCR amplicons for each of four gene targets was carried out for the validation of in-house designed primers and PCR protocol. RESULTS: A total of 51.72% (60/116) patients were detected to have either of the two STIs. About 35.35% (41/116) of patients were positive for C. trachomatis and 33.62% (39/116) for N. gonorrhoeae by employing multiplex nested PCR. Coinfection with N. gonorrhoeae and C. trachomatis was detected in 17.24% (20/116) patients. 31.5% endocervical swabs (n = 54), 64.4% speculum-assisted high vaginal swabs (n = 45), and 80% self-collected vaginal swabs (n = 5) were detected positive for either of two STIs. CONCLUSIONS: The multiplex nested PCR protocol designed and employed in the present study may be used in the diagnosis and management of both symptomatic as well as asymptomatic cases of N. gonorrhoeae and C. trachomatis, particularly among high-risk groups.

Development, optimization and evaluation of curcumin loaded biodegradable crosslinked gelatin film for the effective treatment of periodontitis.

OBJECTIVE: Aim of the present study was to prepare curcumin (CUR) loaded biodegradable crosslinked gelatin (GE) film to alleviate the existing shortcomings in the treatment of periodontitis. SIGNIFICANCE: Gelatin film was optimized to provide anticipated mucoadhesive strength, mechanical properties, folding endurance, and prolonged drug release over treatment duration, for successful application in the periodontitis. METHODS: The film was developed by using solvent casting technique and "Design of Experiments" approach was employed for evaluating the influence of independent variables on dependent response variables. Solid-state characterization of the film was performed by FTIR, XRD, and SEM. Further, prepared formulations were evaluated for drug content uniformity, surface pH, folding endurance, swelling index, mechanical strength, mucoadhesive strength, in vitro biodegradation, and in vitro drug release behavior. RESULTS: Solid state characterization of the formulation showed that CUR is physico-chemically compatible with other excipients and CUR was entrapped in an amorphous form inside the smooth and uniform film. The optimized film showed degree of crosslinking 51.04 ± 2.4, swelling index 138.10 ± 1.25, and folding endurance 270 ± 3 with surface pH around 7.0. Crosslinker concentrations positively affected swelling index and biodegradation of film due to altered matrix density of the polymer. Results of in vitro drug release demonstrated the capability of the developed film for efficiently delivering CUR in a sustained manner up to 7 days. CONCLUSIONS: The developed optimized film could be considered as a promising delivery strategy to administer medicament locally into the periodontal pockets for the safe and efficient management of periodontitis.

Correction: Quantum curcumin mediated inhibition of gingipains and mixed-biofilm of Porphyromonas gingivalis causing chronic periodontitis.

[This corrects the article DOI: 10.1039/C8RA08435A.].

Hidden burden of chikungunya in North India; A prospective study in a tertiary care centre.

BACKGROUND: Arboviral diseases, such as chikungunya, dengue and now zika represent a public health problem, especially in tropical countries. Epidemiology of chikungunya and dengue is well known, including its social and climatic factors associated, but only few data and reports of chikungunya are available from North India. The clinical differentiation of chikungunya from dengue is no doubt challenging since both diseases can share clinical signs and symptoms leading to potential misdiagnosis of chikungunya in areas where dengue is endemic. The aim of this study was to know the seroprevalence, seasonal trends, clinical presentations of chikungunya and its co-infection with dengue virus. METHODS: This was a prospective study conducted in Varanasi, from January to December 2016. All serum samples were tested for both chikungunya and dengue IgM antibodies by MAC ELISA test. RESULTS: Total of 186 samples, out of which 108 (58%) samples were total seropositive, 23 (12.37%) samples positive for chikungunya IgM antibodies, 57 (30.65%) samples positive for dengue and 28 (15.05%) samples positive for both chikungunya and dengue. The most affected age group was 20-30 years and males were more affected than females. A seasonal peak for chikungunya and its co-infection with dengue were seen in November. CONCLUSION: In India, the seroprevalence of chikungunya is increasing. India is a rapidly developing country where adequate sanitation is required. More aggressive intervention and vigilance by health authorities is needed to decrease vector born diseases.

Quantum curcumin mediated inhibition of gingipains and mixed-biofilm of Porphyromonas gingivalis causing chronic periodontitis.

Periodontitis is a biofilm-associated irreversible inflammation of the periodontal tissues. Reports suggest the role of Porphyromonas gingivalis specific Arg- and Lys-specific proteinases in the orchestration of the initiation and progression of periodontal diseases. These proteinases are precisely termed as gingipains R and K. Curcumin is an active polyphenol that is extracted from the rhizomes of Curcuma longa. However, the molecule curcumin owing to its high hydropathy index and poor stability has not been able to justify its role as frontline drug modality in the treatment of infectious and non-infectious diseases as claimed by several investigators. In the present study, at first, we synthesized and characterized quantum curcumin, and investigated its biocompatibility. This was subsequently followed by the evaluation of the role of quantum curcumin as an antimicrobial, anti-gingipains and antibiofilm agent against Porphyromonas gingivalis and select reference strains. We have successfully synthesized the quantum curcumin utilizing a top-down approach with the average size of 3.5 nm. Apart from its potent antimicrobial as well as antibiofilm properties, it also significantly inhibited the gingipains in a dose-dependent manner. At the minimal concentration of 17.826 µM, inhibition up to 98.7% and 89.4% was noted for gingipain R and K respectively. The data was also supported by the in silico docking experiments which revealed high exothermic enthalpies (-7.01 and -7.02 cal mol-1). Besides, the inhibition constant was found to be 7.24 µM and 7.1 µM against gingipains R and K respectively. The results suggest that quantum curcumin is a potential drug candidate which needs further clinical validation.

Standardization and Classification of In vitro Biofilm Formation by Clinical Isolates of Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is Gram-positive bacterium commonly associated with nosocomial infections. The development of biofilm exhibiting drug resistance especially in foreign body associated infections has enabled the bacterium to draw considerable attention. However, till date, consensus guidelines for in vitro biofilm quantitation and categorization criterion for the bacterial isolates based on biofilm-forming capacity are lacking. Therefore, it was intended to standardize in vitro biofilm formation by clinical isolates of S. aureus and then to classify them on the basis of their biofilm-forming capacity. MATERIALS AND METHODS: A study was conducted for biofilm quantitation by tissue culture plate (TCP) assay employing 61 strains of S. aureus isolated from clinical samples during May 2015- December 2015 wherein several factors influencing the biofilm formation were optimized. Therefore, it was intended to propose a biofilm classification criteria based on the standard deviation multiples of the control differentiating them into non, low, medium, and high biofilm formers. RESULTS: Brain-heart infusion broth was found to be more effective in biofilm formation compared to trypticase soy broth. Heat fixation was more effective than chemical fixation. Although, individually, glucose, sucrose, and sodium chloride (NaCl) had no significant effect on biofilm formation, a statistically significant increase in absorbance was observed after using the supplement mix consisting of 222.2 mM glucose, 116.9 mM sucrose, and 1000 mM NaCl (P= 0.037). CONCLUSIONS: The present study puts forth a standardized in vitro TCP assay for biofilm biomass quantitation and categorization criteria for clinical isolates of S. aureus based on their biofilm-forming capacity. The proposed in vitro technique may be further evaluated for its usefulness in the management of persistent infections caused by the bacterium.

Curcumin Quantum Dots Mediated Degradation of Bacterial Biofilms.

Bacterial biofilm has been reported to be associated with more than 80% of bacterial infections. Curcumin, a hydrophobic polyphenol compound, has anti-quorum sensing activity apart from having antimicrobial action. However, its use is limited by its poor aqueous solubility and rapid degradation. In this study, we attempted to prepare quantum dots of the drug curcumin in order to achieve enhanced solubility and stability and investigated for its antimicrobial and antibiofilm activity. We utilized a newer two-step bottom up wet milling approach to prepare Curcumin Quantum Dots (CurQDs) using acetone as a primary solvent. Minimum inhibitory concentration against select Gram-positive and Gram-negative bacteria was performed. The antibiofilm assay was performed at first using 96-well tissue culture plate and subsequently validated by Confocal Laser Scanning Microscopy. Further, biofilm matrix protein was isolated using formaldehyde sludge and TCA/Acetone precipitation method. Protein extracted was incubated with varying concentration of CurQDs for 4 h and was subjected to SDS-PAGE. Molecular docking study was performed to observe interaction between curcumin and phenol soluble modulins as well as curli proteins. The biophysical evidences obtained from TEM, SEM, UV-VIS, fluorescence, Raman spectroscopy, and zeta potential analysis confirmed the formation of curcumin quantum dots with increased stability and solubility. The MICs of curcumin quantum dots, as observed against both select gram positive and negative bacterial isolates, was observed to be significantly lower than native curcumin particles. On TCP assay, Curcumin observed to be having antibiofilm as well as biofilm degrading activity. Results of SDS-PAGE and molecular docking have shown interaction between biofilm matrix proteins and curcumin. The results indicate that aqueous solubility and stability of Curcumin can be achieved by preparing its quantum dots. The study also demonstrates that by sizing down the particle size has not only enhanced its antimicrobial properties but it has also shown its antibiofilm activities. Further, study is needed to elucidate the exact nature of interaction between curcumin and biofilm matrix proteins.

Performance of nested multiplex PCR assay targeting MTP40 and IS6110 gene sequences for the diagnosis of tubercular lymphadenitis.

The conventional methods for diagnosis of tubercular lymphadenitis (TBLN) such as - fine needle aspiration cytology, Ziehl-Neelsen staining and culture have limitations of low sensitivity and/or specificity. So, it becomes essential to develop a rapid, sensitive, and specific method for an early diagnosis of TBLN. Therefore, the present study was conducted to evaluate nested multiplex polymerase chain reaction (nMPCR) targeting MTP40 and IS6110 gene sequences of Mycobacterium tuberculosis and Mycobacterium tuberculosis complex, respectively in 48 successive patients of TBLN and 20 random patients with non-tubercular lymph node lesions. Out of the 48 cases of TBLN, 14 (29.2%) were found to be positive by Ziehl-Neelsen staining, 15 (31.2%) were positive by culture and 43 (89.6%) cases were positive after first round of PCR while 48 (100%) cases were positive by nMPCR assay. The sensitivity and specificity of nMPCR was found to be 100% for the diagnosis of TBLN. The results thus obtained indicate that nMPCR assay is a highly sensitive and specific tool for the diagnosis of TBLN.

PCR and Genotyping for HPV in Cervical Cancer Patients.

AIMS: To devise nested multiplex polymerase chain reaction (NMPCR) protocol for detection of mucosal human papilloma viruses (HPVs) and typing of HPV-16 and -18 in formalin-fixed, paraffin-embedded (FFPE) tissues of carcinoma cervix (CaCx). SETTINGS AND DESIGN: Cross-sectional observational study. MATERIALS AND METHODS: NMPCR was done for simultaneous detection of HPV, targeting 134 bp L1 capsid gene employing GP+/mGP+ primers and typing of genotypes-16 and -18, targeting E6/E7 gene from 34 FFPE tissue blocks of CaCx and cervical intraepithelial neoplasia (CIN). Detection of 142 bp consensus sequence of L1 capsid gene was performed by nested PCR employing MY/GP+ primers. Sequencing of selected PCR amplicons of the later protocol obtained from control cell line DNA and 5 select samples were done for validation of the NMPCR protocol. STATISTICAL ANALYSIS USED: Calculation of percentage from the Microsoft Excel Software. RESULTS: Of 26 FFPE samples of CaCx, 17 (65.3%) samples were found positive for HPV by NMPCR. Amplicons of 142 bp L1 capsid gene employing MY/GP+ primers were observed in 11 (42.3%) samples of CaCx. Nearly 25% samples of CIN were positive for HPV. On sequence analysis, it was observed that the sample typed as HPV-16 by NMPCR was found to be the same on sequencing of amplicons obtained after MY/GP+ nested PCR. CONCLUSIONS: This study indicates the usefulness of our NMPCR protocol for detection of mucosal HPVs and typing of HPV-16 and -18 from FFPE tissue samples of CaCx. The NMPCR protocol may be used to detect HPV and type common genotypes-16 and -18 in fresh tissue of cervical biopsy or scrape samples for screening of CaCx.

Differentiation of Mycobacterium tuberculosis complex from non-tubercular mycobacteria by nested multiplex PCR targeting IS6110, MTP40 and 32kD alpha antigen encoding gene fragments.

BACKGROUND: Control of the global burden of tuberculosis is obstructed due to lack of simple, rapid and cost effective diagnostic techniques that can be used in resource poor-settings. To facilitate the early diagnosis of TB directly from clinical specimens, we have standardized and validated the use of nested multiplex PCR, targeting gene fragments IS6110, MTP40 and 32kD α-antigen encoding genes specific for Mycobacterium tuberculosis complex and non-tubercular mycobacteria (NTM), in comparison to smear microscopy, solid culture and single step multiplex PCR. The results were evaluated in comparison to a composite reference standard (CRS) comprising of microbiological results (smear and culture), clinical, radiological and cytopathological findings, clinical treatment and response to anti-tubercular therapy. METHODS: The nested multiplex PCR (nMPCR) assay was evaluated to test its utility in 600 (535 pulmonary and 65 extra-pulmonary specimens) clinically suspected TB cases. All specimens were processed for smear, culture, single step multiplex PCR and nested multiplex PCR testing. RESULTS: Out of 535 screened pulmonary and 65 extra-pulmonary specimens, 329 (61.5%) and 19 (29.2%) cases were culture positive for M. tuberculosis. Based on CRS, 450 patients had "clinical TB" (definitive-TB, probable-TB and possible-TB). Remaining 150 were confirmed "non-TB" cases. For culture, the sensitivity was low, 79.3% for pulmonary and 54.3% for extra-pulmonary cases. The sensitivity and specificity results for nMPCR test were evaluated taken composite reference standard as a gold standard. The sensitivity of the nMPCR assay was 97.1% for pulmonary and 91.4% for extra-pulmonary TB cases with specificity of 100% and 93.3% respectively. CONCLUSION: Nested multiplex PCR using three gene primers is a rapid, reliable and highly sensitive and specific diagnostic technique for the detection and differentiation of M. tuberculosis complex from NTM genome and will be useful in diagnosing paucibacillary samples. Nested multiplex PCR assay was found to be better than single step multiplex PCR for assessing the diagnosis of TB.

Assessing genetic diversity of Mycobacterium tuberculosis by spoligotyping and IS6110-based restriction fragment length polymorphism in North India.

OBJECTIVE/BACKGROUND: Molecular epidemiology methods are very useful for differentiating between strains, assessing their diversity, and measuring the prevalence of the most circulating strain in an area. Various molecular typing methods using different molecular markers have been utilized worldwide, such as restriction fragment length polymorphism (RFLP), spoligotyping, Mycobacterial Interspersed Repetitive Unit - Variable Number of Tandem Repeat (MIRU-VNTR), and Double repetitive element-PCR (DRE-PCR) typing, for simultaneous detection and epidemiologic typing of Mycobacterium tuberculosis. The present study is conducted to assess the genetic diversity of M. tuberculosis by IS6110-RFLP and spoligotyping in patients attending a tertiary care hospital in eastern Uttar Pradesh, North India. METHODS: A total of 83 representative isolates of M. tuberculosis were included in this study. These isolates were subjected to spoligotyping and IS6110-RFLP DNA fingerprinting techniques as described previously. RESULTS: The spoligotype patterns were compared with SpolDB4.0; patterns of 64 out of 83 M. tuberculosis isolates were matched with the available data, while 19 isolates were found to be orphan, that is, absent in the SpolDB4.0 database. The majority of the M. tuberculosis strains (56.5%) belong to central Asian (32.5%), ill defined T (13.2%), and Beijing (10.8%) families. On IS6110-RFLP analysis, in 19.2% (16/83) of these isolates, IS6110 element was not found (0 copy number strains). Further, 15.6% (13/83) isolates were found to be low-copy-number strains having less than six copies of IS6110 element, and the remaining 65.0% (54/83) were multiple-copy-number strains with six or more copies of the element. On comparing the results of spoligotyping and IS-6110-RFLP, a total of 47 isolates were clustered by spoligotyping; out of these isolates, 40 were found to be unique by IS6110-RFLP. CONCLUSION: Spoligotype analysis resulted in the grouping of a much larger number of isolates within apparently identical clusters compared with IS6110-RFLP typing, while IS6110-RFLP was not found to effectively distinguish between zero- and low-copy-number isolates. Therefore, we concluded that, in India, the use of both the techniques simultaneously for DNA fingerprinting of M. tuberculosis could be a better approach.