Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241823EnglishN-0001November30General SciencesMOLECULAR DIVERSITY BASED CLUSTERING OF CHICKPEA (CICER ARIETINUM L.) GERMPLASM
English0105Ajaya PaliwalEnglishThe present investigation undertaken at National Research Centre Plant Biotechnology, Pusa, New Delhi in 2005 with the aim of studying inter-relationship existing between the chickpea genotypes. 46 genotypes from chickpea and its wild relatives were collected that include 45 from Cicer arietinum and one from interspecific hybrid (Cicer arietinum x Cicer reticulatum). A total of 11 STMS loci were analyzed, covering various bin locations on different linkage groups. All the 11 STMS loci, in the genetic material under study were found to be highly polymorphic except TA-72. The STMS data was utilized for preparing genetic similarity matrix and further analyzed using UPGMA clustering algorithm. The dendrogram clearly showed 4 large clusters which were divided in sub clusters. Pant G114 and BG 1107 showed remarkable genetic similarity (0.77) followed by II, III and IV. The cluster 1 to 4 comprised of 8, 25,7 and 6 genotypes respectively. The study support greater resolving power of Sequence Tagged Microsatellite Sites molecular markers for chickpea germplasm where very little morphological distinction can be found between the different cultivars.
EnglishChickpea, Molecular Markers, STMS, Diversity, ClusteringINTRODUCTION
Chickpea is a self-pollinated crop with 16 diploid chromosome number. It is third most important grain legume in the world after dried beans and dry pea. Its cultivation is mainly confined to Asia with 90 per cent of the global area and production. Chickpea is cultivated on about 10.4 m ha area adding 8.57 m tones of grains to the global food basket with the average productivity of 825 Kg per ha. As many as 42 countries grow chickpea but a dozen countries namely India, Pakistan, turkey, Canada, Mexico, Iran, Ethiopia, Myanmar, Syria, Bangladesh, and Spain contribute 97% of the global production (FAOSTAT 2004).
India is the largest producer and consumer of chickpea, presently it is grown in 6.5 million hectare area in India with 5.77 million ton production at the productivity of 887.7 Kg/ha, which represent 32 and 42% of the national pulse acreage and production respectively. Chickpea production has gone up from 3.65 to 5.77 million tons between 1950-51 and 2003-04, registering a modest growth of 0.1% annually during the period, while the area has declined (FAOSTAT, 2004).
In recent years plant breeding has benefited from DNA marker technologies that were used to establish saturated genetic maps in major crop species, accessions genotyping and diversity analysis. Molecular technology can complement the conventional breeding efforts in effective characterization and utilization of the genetic resources. The DNA based markers are being increasingly utilized in plant breeding because of their phenotypic stability, high polymorphism, ease of development and diverse applications in breeding. Chickpea mapping is severely hampered by extraordinary little genetic polymorphisms in cultivated genotypes, and earlier used molecular markers such as isozymes, Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) failed to reveal intraspecific variation (Kazan and Muehlbauer 1993; Udupa and Baum 2001; Simon and Muehlbauer 1997). Among a range of DNA-based markers, Sequence Tagged Microsatellite Site (STMS) markers are widely preferred in various crop plants, including chickpea due to their abundance, genomic coverage, genetically codominant nature and high polymorphism (Powell, 1996). STMS molecular markers first time developed by Huttel et al in 1999 in chickpea, revealed polymorphism up to desirable extent in Chickpea accessions.
Recent experiments on a STMS in the pulse family revealed that some of the STMS identified in one species are also capable of revealing polymorphism in other pulse species, it means once developed in chickpea they can also be used in other chickpea related species (Choumane et al, 2000). Such studies are required to ascertain the potential utility of these markers in analyzing molecular polymorphism in wild relatives of chickpea particularly Cicer reticulatum.
Keeping the above information in background the present study has been proposed with the following objective of fingerprinting based grouping of elite germplasm or cultivars in terms of genetic relatedness.
MATERIALS AND METHODS
The present investigation undertaken at Genetics division of IARI, Pusa, New Delhi in 2005. 46 genotypes from chickpea and its wild relatives were collected that include 45 from Cicer arietinum and one from derivatives of interspecific hybrid of Cicer arietinum x Cicer reticulatum from the Pulse Block of the division. As chickpea is highly self-pollinated crop therefore samples can be bulked. DNA from the germplasm was extracted with the help of CTAB method of Doyle and Doyle, 1987 with minor modifications. The STMS primers were synthesized by the Gene Script Corporation under the DBT funded project on molecular mapping of chickpea genome. The most polymorphic primer sequence were selected from the what available in the public domain (Winter et al, 1999 and Huttel et al 1999). STMS marker analysis was completed in the following steps, 1). Genomic DNA isolation and quantification. 2). DNA amplification in PCR with help of STMS primers. 3). Gel electrophoresis of the amplified products. 4). SSR data entry and verification. STMS markers used in the study was CaSTMS 25, TA 2, TA 8, TA 21, TA 43, TA 72, TA 80, TA 125, TR 58 and TS 45.
RESULT
In the present study, a total of 11 STMS loci were analyzed, covering various bin locations on different linkage group (Table 2). All the 11 STMS loci, in the genetic material under study were found to be highly polymorphic except TA-72. Excellent polymorphism was revealed by rest of the STMS markers employed for this analysis (Fig. 1). Data from 9 STMS loci were only utilized for further statistical analysis due to missing data (more than 30%) in 2 STMS loci. Similarly data of two lines EC 90039 and FCI-105 were not included in the analysis as these entries showed more than 30% missing data across the STMS loci analyzed.
A total of 40 alleles were found for the 9 STMS loci with an average of 9.4 per locus. The highest number of alleles were observed in TA21 (six alleles) followed by TA-2 (5 alleles), TA-8 (5 alleles), TA-64 (5 alleles), TA80 (5 alleles), TA125 (5 alleles), CaSTMS (4 alleles) and TA72 (2 alleles). Out of these 2 alleles of TA72 a single allele was present in most of the entries i.e. a2 allele in 45 entries out of the selected 46 entries without any null allele.
Similarity verses Dissimilarity Analysis
The STMS data was utilized for estimating pair wise genetic similarities among various entries using Jaccard’s coefficient (1908) method. The genetic similarity matrix was further analyzed using UPGMA clustering algorithm by software programme NTSYS pc version 2.11. The dendrogram derived from this analysis was depicted as Fig. 2. The dendrogram clearly showed 4 large clusters which were divided in sub clusters. Pant G114 and BG 1107 showed remarkable genetic similarity (0.77) followed by II, III and IV and 6ILC 3279 and Mexico local. The cluster I comprised of 8, 25,7 and 6 genotypes respectively as depicted in Fig.3. (Table 1)
DISCUSSION
Molecular markers have been utilized for variety of purposes including construction of linkage maps examining genetic relationships between individuals and identification of crop cultivars. These are considered as valuable tools for plant breeding programmers as well as for studies related to evolution and biodiversity conservation. Among the various DNA based markers, the microsatellite or STMS markers have become highly popular and the ‘The markers system of choice in diverse crop plants owing to their abundance in the genome, robustness, reproducibility, hypervariability and codominance (Powell et al., 1996). Application of STMS markers in genetic analysis of chickpea, started with an initial study of Huttel et al. (1999). Since then, the power and potential of Simple Sequence Repeat markers for a wide range of applications in genetic and breeding of chickpea has been well demonstrated by several researchers (e.g. Huttel 1999, Winter et al, 1999 and Flandez-galvez, 2003, W Choumane 2000 etc.), but still substantial number of chickpea microsatellites are not available in public domain. Microsatellite genotypic data from a number of loci have potential to provide unique allelic profiles or DNA fingerprints for establishing genotypes identity. While carrying out STMS profiling, due consideration was given to stratified sampling of polymorphic STMS loci covering bin location on various chromosomes. The STMS polymorphism were assayed using a DNA pooling strategy, although it is not supposed to do as all the genotypes under study are supposed to be pure lines.
When genetic relationship pattern obtained by STMS matrix data was composed with the pedigree information available for 46 genotypes/ varieties of genus Cicer, only few generalizations could be made. Of course, BG1103 a derivative from Pusa256 X Pusa362 Cicerreticulatum belonged to cluster I exhibiting closer relation with Cicer reticulatum. Apart from this no clear cut pattern, especially for different clustering (i.e. genetic dissimilarities) and source population diversity could be found one of the reason for apparent non-conformity of genetic relationship and pedigree information might be attributed to the base of source population; from which the genotypes were derived. Pedigree information might not be a clear cut indicator of diversity and large variation between the genotypes derived from the same source may be expected.
Cluster analysis revealed erratic pattern of clustering in aforesaid mentioned example of BG 1103 as three parents involved were grouped many genotypes of different in one cluster. This could be possible due to: (1) No trait observed (2) less number of STMS markers (3) possibility of negative / erroneous scoring. In addition, DNA marker may be affected by selection, drift and mutation, factors that are ignored when estimating association among genotype based on pedigree or origin. It may represent alleles identical in state which may not be identical by descent (Senior et al, 1998). Finally, it can also be outcome from the clustering process whatever clusters are non-overlapping. Because of the latter, a genotype which is related to other genotypes from separate cluster wise only be grouped with one it is most closely related.
The STMS (Sequence Tagged Microsatellite Sites) molecular marker proved to be highly polymorphic for chickpea germplasm and can be reliably used for resolving differences existing at DNA level between two genotypes including the extent of their similarity with each other.
ACKNOWLEDGEMENT
Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed. The author fondly appreciate the financial support provided by the DBT under project ‘Molecular Mapping of Chickpea Genome’. The present research work was carried out at NRC Plant Biotechnology, Pusa, New Delhi as part of Master’s programme with Junior Research Fellowship by the author.
Table : Jaccard’s Similarity Coefficients
Englishhttp://ijcrr.com/abstract.php?article_id=143http://ijcrr.com/article_html.php?did=143
FAOSTAT Database 2004. http://apps.fao.org/accessedon 01/05/2005.
Kazan, K., Muehlbauer, F.J., Weeden, N.F., Ladizinsky, G. (1993) Inheritance and linkage relationships of morphological and isozyme loci in chickpea (Cicer arietinum L.). Theor Appl Genet86:417–426.
Udupa, S.M., Baum, M. (2001) High mutational rate and mutational bias at (TAA)n microsatellite loci of Chickpea ( Cicer arietinum) Mol Gen Genet. 265:1097-1103
Simon, C.J., Muehlbauer, F.J. (1997) Construction of a chickpea linkage map and its comparison with maps of pea and lentil. J Hered 88:115–119.
Powell, W., Marchy, G.C. and Provan, J. (1996). Polymorphism revealed by simple sequence repeats. Trends in Plant Science. 1.215-222.
Huttel, B., Winter, P., Weising, K., Choumane, W., Weigand, F., Kahl, G. (1999) Sequence-tagged microsatellite site markers for chickpea (Cicer arietinumL.). Genome 42:210–217.
Choumane, W., Winter, P., Weigand, F., Kahl, G. (2000) Conservation and variability of STMS from chickpea within the genus Cicer. Theor Appl Genet. 101:269-278.
Jaccard, P. (1908) Nouvelles researches sur la distribution florale. Bull. Soc. Vaudoise Sci. Nat. 44:223-270
Winter, P., Pfaff, T., Udupa, S.M., Huttel, B., Sharma, P.C., Sahi, S., Arreguin-Espinoza, R., Weigand, F., Muehlbauer, F.J., Kahl, G. (1999) Characterization and mapping of sequence tagged microsatellite sites in the chickpea (Cicer arietinum L.) genome. Mol Gen Genet 262:90–101.
DOYLE, J.J.; DOYLE, J.L. (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, v.19, p.11-15.
Flandez-Galvez H, Ford R, Pang ECK, Taylor PWJ (2003) An intraspecific linkage map of the chickpea (Cicer arietinum L.) genome based on sequence tagged microsatellite site and resistance gene analog markers. Theor Appl Genet 106:1447–1456.
Senior, M.L., Murphy, J.P., Goodman, M.M., and Stuber, C.W. (1998) Utility of SSRs for determining genetic similarity and relationship in maize using an agarose gel system. Crop Sci. 38:1088-1098
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241823EnglishN-0001November30HealthcareEXTRACORPOREAL MAGNETIC INNERVATION (EXMI) IN THE TREATMENT OF URINARY INCONTINENCE IN WOMEN - A REVIEW OF RESEARCHES
English0610Weber-Rajek MagdalenaEnglish Agnieszka Radzimi?skaEnglish Marta PodhoreckaEnglishBackground: According to the World Health Organization and the International Continence Society, urinary incontinence is defined, in an objective manner, as a lack of control over urination. In women urinary incontinence is a common problem in peri- and postmenopausal period and a general symptom associated with pregnancy as well. Physical methods are used in the conservative treatment of urinary incontinence. A relatively new physical method used in the treatment of urinary incontinence is Extracorporeal Magnetic Innervation.
Aim: The aim of this study is a systematic review of researches assessing the efficacy of magnetic therapy in the treatment of urinary incontinence in women.
Methods: Clinical studies searching was based on a detailed report in accordance to the guidelines of the Cochrane Collaboration. The following databases were searched through: Pub Med., Medline, Embase, The Cochrane Library (Central). The key words were: urinary incontinence, Extracorporeal Magnetic Innervation, ExMI, magnetic stimulation and related expressions. The databases from 1998 till the year 2016 were searched.
Results: The above keywords were found in the databases of 27 studies assessing the efficacy of ExMI in the treatment of UI in women. These studies were evaluated by two independent reviewers who selected two studies that met the established criterion of the inclusion in the review.
Conclusion: Magnetic Stimulation used in the treatment of urinary incontinence is a non-invasive, painless and comfortable for the patient method, and therefore there is a need for studies with a well-designed research protocol
EnglishExtracorporeal Magnetic Innervation, Urinary incontinenceIntroduction
According to the WHO (World Health Organization) and the ICS (International Continence Society), urinary incontinence (UI) is defined, in an objective manner, as a lack of control over urination [1]. In women urinary incontinence is a common problem in peri- and postmenopausal period and a general symptom associated with pregnancy as well. Epidemiological data on the prevalence of the disease is vague, ranging from 7% to 53% [2,3]. According to the Standarisation Steering Comitee ICS there are three main types of NTM: stress (SUI -stress urinary incontinence), urgency (UUI-urge urinary incontinence and mixed (MUI-mixed urinary incontinence) [4]. Physical methods are used in the conservative treatment of urinary incontinence. The most commonly used means is electrical stimulation, which was first applied in 1963 by Cadwell [5]. Electrical stimulation strengthens the muscles of the pelvic floor - contraction of the external urethral sphincter, the increase of intraurethral pressure and the contraction of the levator ani that contributes to the elevation of the bladder neck and to the extension of the initial urethra [6]. Additionally, triggered impulses running through the nerves in labia segments S2-S4 of spinal cord contribute to better functioning of that reflex route [6]. Although the efficacy of electrical stimulation is confirmed by numerous scientific studies, not all patients opt for this form of therapy (discomfort, excessive psychological stress [6,7,8]. A relatively new physical method used in the treatment of urinary incontinence is Extracorporeal Magnetic Innervation (ExMI). Electromagnetic pulsing technology for pelvic floor muscle stimulation was approved as treatment for UI by the U.S. Food and Drug Administration in June 1998 [9] and by the European Commission in January 2011 [10].
It is a completely non-invasive and painless method. The patient sits on a specially designed treatment chair, where a therapeutic head is attached. It produces magnetic field penetrating the organs of the pelvis minor [9,11]. The magnetic field acts directly on the motor fibers of pudendal and visceral nerves. The activation of sodium-potassium pump and the regulation of motor neurons depolarization cause pulses priming in neuromuscular plates that force muscle contraction in the innervated area [9,11].
The aim of this study is a systematic review of researches assessing the efficacy of magnetic therapy in the treatment of urinary incontinence in women.
Method:
Clinical studies searching was based on a detailed report in accordance
to the guidelines of the Cochrane Collaboration [12]. The following were taken into account: the criteria of inclusion of the research for the review, search strategy and the method of tests selection.
The analysis was based on the clinical studies that met the following criteria:
population: women with various forms of urinary incontinence;
intervention: a therapy using Extracorporeal Magnetic Innervation with the parameters of magnetic field stated;
methodology: randomized controlled clinical studies; clinical studies with blind, clinical studies with the control group;
endpoints: efficacy - evaluation of bladder function, quality of life, safety (side effects).
The following databases were searched through: Pub Med., Medline, Embase, The Cochrane Library (Central). The key words were: urinary incontinence, Extracorporal Magnetic Innervation, ExMI, magnetic stimulation and related expressions. The databases from 1998 (the year when electromagnetic technology for pelvic floor muscle stimulation was approved as treatment for UI by the U.S. Food and Drug Administration) till the year 2016 were searched through.
Study selection and data extraction:
The above keywords were found in the databases of 27 studies assessing the efficacy of ExMI in the treatment of UI in women. These studies were evaluated by two independent reviewers who selected two studies that met the established criterion of the inclusion in the review.
Results:
221 patients took part the studies analyzed for the purpose of this review. 136 women underwent the therapy of ExMI and 85 were in a sham group. Gilling et al. [13] examined 70 women with SUI that had been randomly divided into two groups. The first group (n = 35) was treated with ExMI (3 sessions per week, for 6 weeks), the second group (n = 35) was sham. The following magneto fields parameters were used: 10 Hz for 10 minutes, three minute brake, and then at 50 Hz for 10 minutes. The intensity of electromagnetic stimulation was adjusted to the maximum value tolerated by the patient. The sham therapy group had the same treatment plan, however a thin deflective aluminium plate was put in the chair. That prevented the magnetic field penetration into the patient. Moreover, the noise and sensation similar to proper active session were produced. The women were not told which treatment they had undergone till the end of the study. In order to assess the therapy efficacy the following means were applied: The Urinary Incontinence Quality of Life Scale (I-QOL), King’s Health Questionnaire (KHQ), 3-day bladder diary, 24-h pad-test, the number of pads used and PFM strength and perineometry. The women were re-examined 8 weeks after they had started the treatment, then at 6 months they were evaluated with the use of a diary, pad-test and questionnaires. No disturbances were noticed and sent to the data monitoring centre.
The results showed that, in the total group of 70 patients there were significant improvements measured, both in primary and secondary examinations. Furthermore, in the group that had undergone the active therapy, significant improvements were noticed in primary and secondary measures in comparison to baseline measure. At 8 weeks, the mean (SD) values for the 20-min pad-test, the 24-h pad-test, the number of pads/day; the I-QOL score and KHQ score were improved. However, no statistically significant difference was noticed when both groups’ improvements had been compared. In women with poor pelvic floor contraction at the first examination there was a significant reduction in the 20-min pad-test leakage in comparison to the sham group. As a result of examinations the authors concluded that the whole applied therapy was not more effective than the sham treatment in the group described above. However, in women unabled to perform an adequate pelvic floor muscle contraction, significant improvements occurred in provocative pad testing, in comparison to the sham treatment.
Yamanishi et al. [14] examined the group of 151 women with SUI and MUI. The women were randomly assigned to two groups: treated group (n = 101) and sham group (n = 50). The treatment was carried out twice a week for 6 weeks. The treatment group used the following parameters of magneto fields: 10 Hz, 560 mT, pulse duration of 300 microseconds, the time of the session - 25 minutes. The maximum intensity of sham stimulation was set at a lower level in comparison to the active stimulation. In that situation no stimulation effect would appear (114 mT, 300 μs, 1 Hz, 5-s „on” 5-s „off” pulsing manner). In order to assess the efficacy of the therapy the following factors were considered: changes in the number of urinary incontinence episodes per week stated in the patient’s bladder diary, changes in the mean number of voids per 24 h, urgency episodes per 24 h, mean voided volume per micturition (ml) and maximum voided volume per micturition (ml) in the bladder diary, Overactive Bladder Symptoms Score (OABSS), International Prostate Symptom Score, Quality of life index (IPSQOL index). Differences from baseline in the active and sham group, were in leaks/week (p = 0.038), in number of urgency episodes/24 h (p = 0.011), in mean voided volume (p = 0.0056). Two types of side effect were observed – diarrhoea and constipation.
Discussion:
The results of this review show that there is a need for studies with a well-designed research protocol, that evaluates ExMI efficacy in the treatment of UI.
Similar conclusions were drawn by Lim et al. [10] who noticed the lack of randomized, sham-controlled trials and the lack recommendations on the use of magnetic stimulation for stress urinary incontinence. Lim et al. [10] suggested the following research protocol (Table I).
Tab. I. The research protocol - Magnetic stimulation for stress urinary incontinence by
Lim et al. [10]
Type of study
randomized, double-blind, sham-controlled parallel-group
Examined group
120 subjects with SUI assigned in a 1:1 to either active or sham group
Inclusion criteria
female aged 21 years and older;
demonstration of urine leak on coughing at a bladder volume of approximately 200 to 250 ml;
International Consultation on Incontinence Questionnaire for Urinary Incontinence Short Form (ICIQ-UI-SF) score of at least 6 points (range: 0–21);
patients must be able and agree to carry out 1-hour pad test and;
voluntary participation and signing of the written consent after being informed.
Exclusion criteria
patients with UUI, MUI, overflow UI;
acute severe infections;
severe cardiac arrhythmia;
cardiac pacemaker or other implanted metallic devices;
neurologic conditions (e.g., stroke, epilepsy, Parkinson’s disease, multiple sclerosis);
random blood sugar above 10 mmol/L;
pregnancy or active trying to conceive;
previous surgery for SUI;
pelvic or gynecological surgery for less than 3 weeks or within next 8 weeks;
previous treatment with MS;
history of pelvic irradiation;
concurrent medications with α-adrenergic antagonists (e.g., terazosin, tamsulosin, doxazosin), diuretics, serotonin-norepinephrine reuptake inhibitors or any other medications known to worsen incontinence;
stage III or IV of pelvic organ prolapse according to Pelvic Organ Prolapse Quantification System ;
severe urethral sphincter weakness and/or defect;
suspected urethral and/or vesical fistula;
urinary tract infection or hematuria; and/or
postvoid residual volume greater than 200 ml.
Therapy parameters
Active magnetic stimulation
2 sessions of magnetic stimulation per week for 8 weeks;
magnetic field parametrs: 50 Hz, „on” – 8 s,
„of” – 4 s, the intensity increased from 20% to100%;
Sham magnetic stimulation
using the same equipment and the same parameters as in the active group, but a magnetic coil was leaned at the angle of 22° and it induced magnetic field of lower intensity: starting at 20% of intensity at the first session, and subsequently increased by 20% after every five sessions, rising to a maximum of 60% of intensity.
Study results
Primary outcome
International Consultation on Incontinence Questionnaire for Urinary Incontinence Short Form (ICIQ-UI-SF). The primary criterion for response is defined as having a 5-point or greater reduction in the ICIQ-UI-SF score (score range: 0–21) from baseline to 8 weeks;
Secondary outcomes
cure,
stress urinary incontinence-related symptoms (incontinence episode frequency, urine loss in 1-hour pad test,
pelvic floor muscle strength);
health-related quality of life:
Patient Global Impression of Improvement; International Consultation on Incontinence Questionnaire (ICIQ) -Lower Urinary Tract Symptoms Quality of Life;
EuroQol five dimensions questionnaire (EQ-5D).
The safety of magnetic stimulation
cost-efficacy analysis using patient-reported outcomes
Conclusion:
Magnetic Stimulation used in the treatment of urinary incontinence is a non-invasive, painless and comfortable for the patient method, and therefore there is a need for studies with a well-designed research protocol.
ACKNOWLEDGEMENT
Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.
Ethical Clearance: NA
Informed Consent: NA
Source of Funding: None
Conflict of interest: NIL
Englishhttp://ijcrr.com/abstract.php?article_id=144http://ijcrr.com/article_html.php?did=144
Abrams P, Cardozo L, Fall M, Griffiths D, Rosier P, Ulmsten U et al. The standardisation of terminology of lower urinary tract function: report from the Standardisation Sub-committee of the International Continence Society. Neurourol Urodyn 2002, 21(2): 167-178.
Buckley BS, Lapitan MC. Epidemiology Committee of the Fourth International Consultation on Incontinence, Paris, 2008. Prevalence of urinary incontinence in men, women, and children—current evidence: findings of the Fourth International Consultation on Incontinence. Urology 2010;76(2):265–70.
Markland AD, Richter HE, Fwu CW, Eggers P, Kusek JW. Prevalence and trends of urinary incontinence in adults in the United States, 2001 to 2008. J Urol 2011;186(2):589–93.
National Collaborating Centre for Women's and Children's Health (UK), Urinary Incontinence The Management of Urinary Incontinence in Women, NICE Clinical Guidelines 2006, Nr 40.
Cadwell KPS. The electrical control of sphincter incompetence. The Lancet 1963, 282 (7300): 174-175.
Bø K, Talseth T, Holme I. Single blind, randomised controlled trial of pelvic floor exercises, electrical stimulation, vaginal cones, and no treatment in management of genuine stress incontinence in women. BMJ 1999; 318 (7182): 487-493.
Bø K, Maanum M. Does vaginal electrical stimulation cause pelvic floor muscle contraction? A pilot study. Scand J Urol Nephrol. Suppl., 1996; 179:39-45.
Spruijt J, Vierhout M, Verstraeten R, Janssens J, Burger C. Vaginal electrical stimulation of the pelvic floor: a randomized feasibility study in urinary incontinent elderly women. Acta Obstet Gynecol Scand 2003; 82(11): 1043-1048.
Galloway NT, El-Galley RE, Sand PK, Appell RA, Russell HW, Carlan SJ. Extracorporeal magnetic innervation therapy for stress urinary incontinence. Urology 1999;53(6):1108–11.
Lim R, Liong ML, Leong WS, Khan NAK, Yuen KH. Magnetic stimulation for stress urinary incontinence: study protocol for a randomized controlled trial. Trials 2015;16:279.
Galloway NT, El-Galley RE, Sand PK, Appell RA, Russell HW, Carlin SJ. Update on extracorporeal magnetic innervation (EXMI) therapy for stress urinary incontinence. Urology 2000;56(6 Suppl 1):82–6
Higgins JPT, Green S: Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0 [updated March 2011]. The Cochrane Collaboration, 2011
Gilling PJ, Wilson LC, Westenberg AM, McAllister WJ, Kennett KM,
Frampton CM et al. A double-blind randomized controlled trial of electromagnetic stimulation of the pelvic floor vs sham therapy in the treatment of women with stress urinary incontinence. BJU Int 2009;103:1386–1390.
Yamanishi T, Homma Y, Nishizawa O, Yasuda K, Yokoyama O. Multicenter, randomized, sham-controlled study on the efficacy of magnetic stimulation for women with urgency urinary incontinence. Int J Urol. 2014;21(4):395-400.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241823EnglishN-0001November30HealthcareA STUDY OF THYROID PROFILE AND SERUM ALBUMIN IN PREECLAMPSIA WOMEN
English1115Rahul R. ChaudharyEnglish M.G. MuddeshwarEnglishAim: The study was carried out to estimate thyroid profile and serum albumin level in preeclamptic women as compare to normotensive women. In addition to this correlation between thyroid profile and albumin in preeclamptic women was also calculated.
Material and Methods: The study comprised of 60 cases of preeclamptic women who attended in Obstetrics and Gynaecology Department in Govt. Medical College, Nagpur from February 2013 to August 2014 and 60 normotensive healthy womens as controls. Serum total Tri-iodothyronine (T3), Tetraiodothyronine (T4), Thyroid Stimulating Hormone (TSH) and serum albumin were estimated.
Results: We observed that out of 60 preeclampsia patients 13 (21.66%) women having hypothyroidism, 18 (30%) women having sub-hypothyroidism and 29 (48.33%) women were of euthyroid. The mean serum TSH in preeclamptic cases (preeclampsia women) were increased significantly (p >0.001). On the other hand serumT3 levels and Serum T4 levels were decreased significantly in preeclamptic women when compared to controls (p >0.001). Serum albumin was decreased significantly in preeclamptic women when as compared to controls (p >0.001). We found that there was statistically significant positive correlation between serum T3 and T4 and serum albumin (r = 0.8735, p = 0.0001) and statistically significant negative correlation between serum TSH and serum albumin (r = - 0.6109 p = 0.0001).
Conclusion: From the results all these parameters play an important role in preeclampsia hence early detection of these parameters in preeclampsia may help to improve maternal and fetal outcome.
EnglishT3 -Total triiodothyronine, T4- Tetraiodothyronine, TSH- Thyroid stimulating hormoneIntroduction
Pregnancy is a physiological state associated with many alterations in metabolic, biochemical, physiological, haematological and immunological processes. If these changes are exaggerated, they can lead to complications during pregnancy1.
Pregnancy induced hypertension presents with new onset hypertension and proteinuria after 20 weeks of gestation. Hypertensive disorders are the most common medical complications occurring during pregnancy. Pregnancy induced hypertension was reported with the incidence of about 10% of first pregnancy and 20% to 25% of previous pregnancy. The incidence of Pregnancy Induced Hypertension (PIH) varies among different hospitals, regions and countries.2
According to WHO's World Health Report 1998, Preeclampsia is defined as “the development of hypertension (>140/90 mm of Hg) after 20 weeks of pregnancy in a woman with proteinuria associated with or without edema having no previous history of hypertension"3
Recent finding related to pathophysiology of preeclampsia suggest that the endothelial cell injury and altered endothelial cell function play a pivotal role.4 It has been proposed5 that the poorly perfused placenta is the origin of a humoral factor that affects maternal systemic function, directly or indirectly, by activating endothelial cells, with the resultant vascular injury.5The characteristic pathologic lesion seen in the uteroplacental bed of patients with preeclampsia is a necrotizing arteriopathy consisting of fibrinoid necrosis, accumulation of foam cells or lipid?laden macrophages in the decidua, fibroblast proliferation and a perivascular infiltrate. This lesion has been termed "acute atherosis.6
Normal pregnancy is associated with physiological and hormonal changes. However abnormal hormonal changes especially thyroid hormone dysfunctions has also shown the relationship between developments of preeclampsia. High levels of TSH, low level of T3 and T4 were shown to be associated with development of preeclampsia and its complication.
In recent glomerular injury also has been considered as characteristic feature of preeclamptic women.7 More precisely it comprises endothelial cell swelling, obliteration of endothelial fenestrate leading to loss of proteins via proteinuria. Keeping all these facts in mind the present work was undertaken to evaluate the relationship of these parameters with preeclampsia.8,9
Serum T3, T4, TSH was measured by enzyme immunoassay on STAT FAX 4300 CHROMATE ELISA Reader using ERBA Thyroid kits by Aspen Laboratories Pvt. Ltd. The estimation of the serum albumin was done by BCG Dye binding method on fully automated XL300 analyse.
Material and methods
Hospital based cross sectional study was conducted in Department of Biochemistry with the help of Obstetrics and Gynaecology Department during period May 2013 to October 2014. The study was approved by institutional Ethics Committee for research work.
The study comprised of 60 cases of preeclamptic women who attended in Obstetrics and Gynaecology Department in Govt. Medical College, Nagpur from February 2013 to August 2014 and 60 normotensive healthy women as controls. These patients were all primigravidae aged between 18 to 30 years and in the third trimester of pregnancy
The diagnosis of Preeclampsia was done by the Department of Obstetrics and Gynaecology based on the definition given by American college of obstetrics and gynecology. Systolic blood pressure > 140 mm Hg or diastolic blood pressure >90 mm Hg; systolic blood pressure increase of > 30 mm Hg or diastolic blood pressure increase of 15 mm Hg over first trimester of pregnancy values (manifested on two occasion at least 6 hrs apart) and proteinuria ≥300 mg or greater in 24 hr urine collection or dipstick protein ≥ 1+ (on two occasion at least 6 hrs apart) is preeclampsia
The inclusion criteria for pre-eclamptic subjects include Age -18 to 30 years, primigravidae in the 3rd trimester of pregnancy, BP - 140/90 mm Hg in third trimester of pregnancy (ACOG criteria), Urine albumin ≥ | + dipstick or 300 mg per 24 hour urine. Normal pregnant women in third trimester of pregnancy were taken as controls.
The exclusion criteria for both the groups were Age < 18 years and > 30 years. (Multigravidae with more than one para, previous history of hypertension, diabetes mellitus, thyroid disorder, dyslipidemia, renal disease and convulsions, Family history of preeclampsia. After explaining all details, informed consent was taken from each subject for participation in this study. History of patient was recorded on preformed questionnaire which included detailed history about present pregnancy, family history of preeclampsia and exclusion criteria. 2 ml of fasting blood sample was collected in plane blub and serum is separated by centrifugation compare demographic, clinical and biochemical parameters between preeclampsia and control groups. Pearson’s correlation coefficient (r) was calculated to assess the correlation between serums TSH, T3 and T4 and serum albumin.
STATISTICAL ANALYSIS-
Unpaired t-test was performed to compare demographic, clinical and biochemical parameters between preeclampsia and control groups. Pearson’s correlation coefficient (r) was calculated to assess the correlation between serums TSH, T3 and T4 and serum albumin
Result
Characteristics in preeclampsia cases and normal pregnant controls
The mean age in the preeclampsia cases was 23.57 ± 2.99 years and that in controls was 22.90 ± 2.96 years. The mean gestational age of cases was 33.92 ± 2.42 weeks where as in controls 33.98 ± 2.48 weeks.(Table1) There was no significant difference in age and gestational age between cases and controls [p value > 0.05]. Study groups were well matched for age and gestational age.
In our study we found that out of 60 preeclampsia patients 13 (21.66%) women having hypothyroidism, 18 (30%) women having sub-hypothyroidism and 29 (48.33%) women were euthyroid. In normotensive pregnant controls all 60 women were euthyroid.
Comparison of serum thyroid profile
In the present study, in preeclamptic women the mean values of T3, T4 were found to be 1.10 ± 0.34 ng/mL and 6.54 ± 2.19 ug/dL respectively while in control group it was 1.47 ± 0.36 ng/mL and 8.46 ± 1.66 ug/dL. (Table 2) The results showed that mean T3 and T4 levels in preeclamptic women were significantly decreased as compared to control group with p value Englishhttp://ijcrr.com/abstract.php?article_id=145http://ijcrr.com/article_html.php?did=1451. Soma-Pillay P, Nelson-Piercy C; Tolppanen H, Mebazaa A. Physiological changes in pregnancy. Cardiovasc J Afr. 2016;27(2):89-94.
2. Ghosh MK. Maternal Mortality: A Global Perspective. J Reprod Med. 2001;46(5):427-33.
3. World Health Organization. The World Health Report 1998 . Life in the 21st century. 1998. p. 97.
4. Roberts J, Redman C. Pre-eclampsia: more than pregnancy-induced hypertension. Lancet. 1993 Jun;341(8858):1447-51.
5. Rodgers GM, Taylor RN, Roberts JM. Preeclampsia is associated with a serum factor cytotoxic to human endothelial cells. Am J Obstet Gynecol. Elsevier; 1988 Oct 10;159(4):908-14.
6. Goode GK, Miller JP, Heagerty AM. Hyperlipidaemia, hypertension, and coronary heart disease. Lancet. 1995 Feb;345(8946):362-4.
7. Kuang-Yu J, Laszik ZG. Renal Effects of Preeclampsia [Internet]. Available from: www.intechopen.com
8. Stillman IE, Karumanch SA. The Glomerular Injury of Preeclampsia. J Am scocity Nephrol. 2007;18:2281-4.
9. Strevens H, Wide-Swensson D, Hansen A, Horn T, Ingemarsson I, Larsen S, et al. Glomerular endotheliosis in normal pregnancy and pre-eclampsia. An Int J Obstet Gynaecol. 2003 Sep;110(9):831-6.
10. Pasupathi P, Chandrasekar V, Kumar US. Thyroid Hormone Changes in Pregnant and Non-Pregnant Women: A Case-Control Study. Thyroid Sci. 2009;4(3):3-7.
11. Khandakar M, Ali AR, Kahtun M. Thyroid status of normal pregnant women in Dhaka City. Mymensingh Med J. 2002;11(1):1- 5.
12. Qublan HS, Al-Kaisi IJ, Hindawi IM, Hiasat MS, Awamleh I, Hamaideh a. H, et al. Severe pre-eclampsia and maternal thyroid function. J Obstet Gynaecol. 2003 Jan;23(3):244-6.
13. Kaya E, Sahin Y, Ozkceci Z, Pasaoglu H. Relation between birth weight and thyroid function in preeclampsia. Gynecol Obstet Invest. 1994;37(1):30-3.
14. Hubel CA. Oxidative stress in the pathogenesis of preeclampsia. Proc Soc Exp Biol Med. 1999;222(3):222–35.
15. Fantz CR, Dagogo-jack S, Ladenson JH, Gronowski AM. Thyroid Function during Pregnancy. Clin Chem. 1999;45(12):2250- 8.
16. Das S, Char D, Sarkar S, Saha TK, Biswas S. Evaluation of Thyroid Hormone Changes in Non-Pregnant , Normotensive Pregnant and Pregnancy with Preeclampsia. J Dent Med Sci. 2013;11(6):16-8.
17. Pasupathi P, Deepa M, Rani P, Vidhya Sankar K, SP satish kumar. Evaluation of Serum Lipids and Thyroid Hormone Changes in Non-Pregnant, Pregnant, and Preeclampsia Women. Thyroid Sci. 2009;4(10):2-7.
18. Khadem N, Ayatollahi H, Roodsari FV, Ayati S, Dalili E, Shahabian M, et al. Comparison of serum levels of Triiodothyronine ( T3 ), Thyroxine ( T4 ), and Thyroid Stimulating Hormone ( TSH ) in preeclampsia and normal pregnancy. Iran J Reprod Med. 2012;10(1):47-52.
19. Kumar A, Ghosh BK, Murthy NS. Maternal thyroid hormonal status in preeclampsia. Indian J Med Sci. 2005;59(2):57-63.
20. Basbug M, Aygen E, Tayyar M, Tutus A, Kaya E, Oktem O. Correlation between maternal thyroid function tests and endothelin in preeclampsia-eclampsia. Obstet Gynecol. 1999;94(4):551-5.
21. Khaliq F, Singhal U, Arshad Z, Hossain MM. Thyroid functions in pre-eclampsia and its correlation with maternal age, parity, severity of blood pressure and serum albumin. Indian J Physiol Pharmacol. 1999;43(2):193-8.
22. Sardana D, Nanda S, Kharb S. Thyroid hormones in pregnancy and preeclampsia. J Turkish Ger Gynecol Assoc. 2009;10(3):168-71.
23. Dhananjaya B, Kumaran DS, Venkatesh G, Murthy N, Shashiraj H. Thyroid Stimulating Hormone (TSH) Level as a Possible Indicator of Pre-eclampsia. J Clin Diagnostic Res. 2011;5(8):1542-3.
24. Larijani B, Marsoosi V, Aghakhani S, Moradi A, Hashemipour S. Thyroid hormone alteration in pre-eclamptic women. Gynecol Endocrinol. 2004;18(2):97-100.
25. Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: An endothelial cell disorder. Am J Obstet Gynecol. Elsevier; 1989 Nov 11;161(5):1200–4.
26. Muzammil S, Khayyam UK, Siddiqui N ali. Serum protein ratio in normal and pre-eclamptic women of primiparous and multiparous in relation to age. Int J Basic Appl Med Sci. 2014;4(2):331- 5.
27. Olooto W, Amballi A. Assessment of Total Protein, Albumin, Creatinine and Aspartate Transaminase level in Toxemia of Pregnancy. J Med …. 2013;12(8):791-6.
28. Demers L, Spencer C. The Thyroid: pathophysiology and thyroid functioning testing. In: Tietz textbook of clinical chemistry and molecular diagnostics. 4th ed. Elsevier; 2006. p. 2053-96.
29. Osathanondh R, Tulchinsky D, Chopra IJ. Total and free thyroxine and triiodothyronine in normal and complicated pregnancy. J Clin Endocrinol Metab. 1976;42(1):98-104.
30. Lao TT, Chin RK, Swaminathan R, Lam YM. Maternal thyroid hormones and outcome of preeclmaptic pregnancies. An Int J Obstet Gynaecol. 1990;97(1):71-4.
31. Tolino A, de Conciliis B, Montemagno U. Thyroid hormones in the human pregnancy. Acta Obstet Gynecol Scand. 1985;64(7):557-9.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241823EnglishN-0001November30HealthcareIONTOPHORESIS A BOON FOR TREATMENT OF DENTINAL HYPERSENSITIVITY:
CASE REPORT
English1620Irudaya Nirmala J.English Ramakrishnan T.English Sivaranjani P.English Shobana P.English Manisundar N.English Ebenezar M.EnglishAim: Dentinal hypersensitivity is an unpleasant symptom which occurs because of dentin exposure as a result of attrition, erosion, abfraction, and gingival recession. There are various treatment modalities to treat dentinal hypersensitivity. Iontophoresis is one of the popular technique which allows a concentrated application of drugs into the desired localized region of exposed dentine there by reducing the dentinal hypersensitivity.
Case Report: This article describes about treatment of patient with dentinal hypersensitivity using Iontophoresis technique.
Discussion: Immediate relief from dentinal hypersensitivity was achieved by clogging the dentinal tubules with usage of APF (Acidulated phosphate fluoride) gel by this iontophoresis technique.
Conclusion: Six months follow up study of this patient showed the long lasting effects of this technique, achieved by deeper penetration of fluoride ions. So it can be used as a first line of treatment in dentinal hypersensitivity.
EnglishDentinal hypersensitivity, Electrical dosage, Iontophoretic unit, Iontophoresis, APF gel (Acidulated phosphate fluoride gel)INTRODUCTION
Dentinal hypersensitivity is characterized by short, sharp pain arising from exposed dentin in response to external stimuli, which cannot be ascribed to any other form of dental defect or disease.[1] It commonly involves the cervical region of facial surfaces of premolars and canines.[2] It causes discomfort and pain to the patient which also deters to maintain adequate oral hygiene.[3] Dentinal hypersensitivity is triggered by thermal, chemical, and mechanical stimuli. Chemical stimuli such as acidic foods (mainly fruits), sweets, and rarely salty foods, Mechanical stimulus such as tooth brush bristles during brushing, nicking the sensitive area with a finger nail, and cold stimuli causes pain and hypersensitivity.[4]
Histologically, sensitive dentin has two times widened, larger dentinal tubules than normal tooth.[4]
The various causes of dentin exposure are Attrition due to parafunctional habits, Mastication, tooth brushing abrasion, erosion by acids, and abfractions. Gingival recession due to periodontal diseases, and faulty tooth brushing causes hypersensitivity.[5]
The Main reason for pain in Dentinal hypersensitivity was proposed in Hydrodynamic theory of Brannstrom and Astron in 1964. It was very well accepted by all. This theory states that stimuli causes displacement of the fluids within the Dentinal tubules which indirectly stimulates the extremities of the pulp nerves causing pain sensation.[6]
According to Lutin, An Ideal desensitizing technique or material should be painless, must not irritate the pulp, be easily applied, be permanently effective, be quick acting, be consistently effective, produce no discolouration.[7]
A potential desensitizing agent clogs the tubules or decreases the activity of the dentinal sensory nerves. Various measures are used to treat this condition in clinic or in home care.
The home measures include Desensitizing dentifrices or mouth washes with active compounds such as sodium fluoride, potassium nitrate, strontium chloride, stannous fluoride etc. The clinic measures include application of sodium fluoride, strontium chloride, cavity varnishes; restorative resins and also includes the use of Laser, resin, Cyanoacrylate, and Iontophoresis. Among which Iontophoresis is effective and long lasting treatment modality for treating hypersensitivity.[2]
IONTOPHORETIC UNIT:
The term Iontophoresis (Greek word) is simply defined as ion transfer.
Ionto = Ion; Phoresis = Transfer
The process of influencing ionic motion by electrical current has been termed Iontophoresis, Electrophoresis, or cataphyoresis. The method of Iontophoresis was described by PIVATI in 1747.[5]
MECHANISM OF ACTION:
Iontophoresis utilizes a low ampere direct electrical current to introduce ions or ionized drugs into the tissue. It allows concentrated application of the drug to the desired localized area without the systemic effects of the conventional oral or parenteral drug therapy.
Normally, ionized drugs will not penetrate the tissue rapidly enough to be of any therapeutic value. By applying appropriately charged direct electrical current, ionized drugs can be driven in to the tissue, based on the principle – like charges repel and opposite charges attract.
Ex: Fluoride - Negative ion.
Applying fluoride under a negatively charged electrode, fluoride can be driven with a direct electrical current into the tooth structure.[2]
ARMAMENTARIUM:
JONOFLUOR SCIENTIFIC is an apparatus for the topical application of fluorine gel for desensitization of the teeth, prophylaxis of caries, and in case of periodontitis. It is designed to use 12- Volt (8×1.5 V) batteries that supplies a direct current of 12 volts. An ammeter with graduations ranging from 0 to 5 milliamperes and at differences of 0.5 mA each.
A POLARITY SELECTION SWITCH :
ON/OFF
JACK WITH CABLE: Jack with black and red spiral cables.
BLACK PLUG: For impression trays with electrode.
RED PLUG: For electrode to be held in hand.
ELECTRODE FOR TOUCHING: An impression tray with electrode is used over the dental arch.
ELECTRODE TO BE HELD: Usually positive, connected to the red cable. [ Fig.1]
CASE REPORT:
Mrs. Prema 36/F reported to Department of periodontics with a chief complaint of sensitivity in the right lower back teeth since three months.
On clinical examination, Abrasion involving 44 and 45. [Fig.4]
EVALUATION TEST:
Before doing the procedure, sensitive tooth was isolated by cotton rolls and subjected to tactile, air blast tests.
Tactile test:
Dentinal hypersensitivity of the surface was examined using dental explorer which was moved around the surface in sweeping motion.
Air blast test:
Air blast using air syringe was directed on the selected tooth surface for one second. [Fig.5]
VRS (Verbal Rating Scale) was used to record scores:
No discomfort
Mild discomfort
Moderate discomfort
Severe pain only during application of stimulus
Severe pain persisting after removal of stimulus.
PROCEDURE:
The sensitive tooth to be treated is isolated with cotton rolls and dried. A sponge with Thin layer of APF (Acidulated phosphate fluoride) gel is applied in a tray. [Fig.6] An autoclaved tray with disposable sponges applied with APF gel is to be kept in contact with affected teeth surfaces. The metal electrode with red spiral is held in the patient hand. Metal electrode with black spiral should contact with rectangular slot in the impression tray. [Fig.7] Resistance knob is slowly turned clock wise and increasing current 0.5 to maximum 2.5 mA to the tooth until the patient experience pain or sensitivity. [Fig.8] Procedure is repeated at a low ampere current. Current is applied for 2 minutes.
Once the treatment is over, knob is turned off and the electrodes are removed from the patient’s dental arch. Repeat the tactile and Air blast test and evaluate the response from the patient.
Before starting the treatment dentinal hypersensitivity of selected tooth surface was checked and scores were recorded, immediately after treatment the surface was checked and scores recorded. Post operatively dentinal hypersensitivity was checked and scores recorded after 1 week, 1 month, and 6 months. [ Fig.9,10 ]
Iontophoresis is repeated after one week in those patients with persistent dentinal hypersensitivity.
DISCUSSION
Several hypotheses have been proposed by which iontophoresis produces desensitization of dentin. One mechanism proposed by Lefkowitz (1963) states that the application of current to dentin leads to formation of reparative dentin which results in dead tracts in primary dentin. A second possible explanation of iontophoresis is that the electrical current produces paresthesia by altering the sensory mechanisms of pain conduction.[8]
A third alternative explanation of iontophoretic desensitization causes ionic movement by electrical current which may enhance ion uptake by the dentinal tubules results in desensitization.[8]
Parr and Brokaw remarked that the rate of penetration of the drug would be increased by giving more electrical current. It also minimize the treatment time.[2]
Carlo et al found that patients with severe hypersensitivity required a second application, while those with mild-to-moderate sensitivity experienced highly significant relief and did not require any additional therapy.[2]
Corticosteroids Iontophoresis (Methyl prednisolone) increases peritubular mineralization. Thus lumen would be decreased, resulting in less dentin tubule fluid movement, reducing dentin sensitivity.[8]
Topical sodium fluoride, strontium chloride reducing hypersensitivity by precipitating calcium fluoride, and recrystallization in form of strontium apatite complex and thus blocking dentinal tubules, reduce the diameter of open tubules respectively. Both of the techniques are short lived, because of wearing away of resin layer.[8,9]
Recently tooth pastes containing carbonated hydroxyl apatite Nano crystals are being studied. It produces biomimetic coating on enamel and thus prevents tooth decay, revitalize the teeth and seal the dentinal tubules. But these are technique sensitive.[10]
Lasers reduces dentin hyper sensitivity with formation of secondary dentin by odontoblast due to bio stimulation but long term relief was not achieved.[5]
CONCLUSION
Reduction in dentinal hypersensitivity was almost immediate with iontophoresis technique and was confirmed in this case. This Iontophoresis technique is safe and effective treatment option to treat dentinal hypersensitivity.
APF gel Iontophoresis is more effective in reducing hypersensitivity immediately and effects are comparatively long lasting by deeper penetration of fluoride ions. Iontophoretically treated teeth had a fluoride concentration twice that of topically applied fluoride. Before any other therapeutic steps like resin primers and low level laser therapy this can be used as a first line of treatment.
Further studies with larger population sample are being carried out in our college to prove the effectiveness of treating dentinal hypersensitivity with Iontophoresis.
ACKNOWLEDGEMENT
Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to author’s /editors/ publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.
Englishhttp://ijcrr.com/abstract.php?article_id=146http://ijcrr.com/article_html.php?did=146
American Academy of Periodontology. Glossary of periodontal Terms (2001).
Sandhu SP, Sharma RL, Bharti V. Comparative evaluation of different strengths of electrical current in the management of dentinal hypersensitivity. Indian Journal of Dental Research. 2010 ;21(2):207-12
Sethi R, Indurkar MS. Comparison of Fluoride Iontophoresis and Dentin Bonding Agent Application in the Treatment of Dentin Hypersensitivity: A clinical study. Indian J Dent Adv. 2015; 7(1):13-7.
Porto IC, Andrade AK, Montes MA. Diagnosis and treatment of dentinal hypersensitivity. Journal of oral science. 2009; 51(3):323-32.
Indurkar MS, Maurya AS. The Clinical Effect of Diode Laser versus Iontophoresis with Acidulated Phosphate Fluoride Gel in the Treatment of Dentin Hypersensitivity. Indian Journal of Applied Research. 2015; 5(7):33-5.
Bhasker SN. Orban's oral histology and embryology. Vol. ed. 1991;11.
Arowojolu MO. Fluoride Iontophoresis versus Topical Fluoride Application In The Treatment Of Dentine Hypersensitivity. Nigerian Journal of clinical practice. 2002 Dec; 5(2):87-90.
Kaur M. Methyl Prednisolone with Iontophoresis in the Treatment of Dentine Hypersensitivity. An In-Vitro and In-Vivo Study. IOSR Journal of Dental and Medical Sciences (IOSR-JDMS). 2016; 15(2):52-60.
Shasi kanth Hegde, Mankude S. Rajesh Kashyap, Arun kumar M.S. Comparative assessment of efficacy of iontophoresis with APF gel and a commercially available Desensitizing agent – Bi fluoride varnish. IOSR journal of dental and medical sciences, 2015;14(3): 34- 41.
Khetawat S, Lodha S. Nanotechnology (Nanohydroxyapatite Crystals): Recent Advancement in Treatment of Dentinal Hypersensitivity. JBR Journal of Interdisciplinary Medicine and Dental Science. 2015; 3(3):1-4.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241823EnglishN-0001November30HealthcareCEREBRAL ASYMMETRY: A CADAVERIC STUDY
English2125Archana RaniEnglish Rakesh Kumar VermaEnglish Arvind Kumar PankajEnglish Rakesh Kumar DiwanEnglish Anita RaniEnglish Deepshikha KoriEnglishObjectives: Anatomical brain asymmetries in cadaveric human brains are not well defined in the literature. Therefore, the present study was undertaken to observe the variations in right and left cerebral hemispheres using various parameters.
Methods: Twenty eight adult human brains irrespective of sex were taken for the present study. Various measurements of cerebral hemisphere including fronto-occipital length, cerebral width, Sylvian fissure length and distance between anterior Sylvian point to inferior Rolandic point were taken. These measurements were taken by Vernier calipers. Different configuration of the anterior ascending and anterior horizontal ramus of the Sylvian fissure were also examined as U-shaped, V-shaped and Y-shaped. The presence or absence of triangular sulcus located within pars triangularis was also observed.
Results: The fronto-occipital length was longer on right side while the cerebral width and Sylvian fissure length were more on left side. These values were significant statistically.
The configuration of the anterior ascending and anterior horizontal ramus of the Sylvian fissure was V-shaped in maximum number of hemispheres followed by U-shaped. The triangular sulcus was noted in thirty hemispheres i.e. 16 right and 14 left.
Conclusions: Hemispheres of the brain are not identical and functional asymmetries have an underlying anatomical basis.
EnglishCerebrum, Morphometry, Sylvian fissure, Triangular sulcusINTRODUCTION
Anatomical brain asymmetries are understated and still little studied in humans. The Sylvian fissure (SF) has long been shown to be an important indicator of the leftward cerebral asymmetry present in human language reception regions. The language ability and handedness are localized on the left side.
The two human cerebral hemispheres are not simply mirror image of each-other. Brain asymmetry has been observed in animals and humans in terms of structure, function and behavior. The evidence of lateralization was first provided in 1861, by Broca who described a case of expressive aphasia resulting from an infarction of the left posterior inferior frontal lobe, which became to be known as Broca’s area. The later discovery of Wernicke’s area in the left posterior temporal and inferior parietal lobes also provided unequivocal evidence of another lateralized function and demonstrated an asymmetry for language comprehension as well as for speech production. Association of language impairment with left hemisphere lesions led to the more general concept of a dominant left hemisphere and a minor right hemisphere.1
Speech and language are one of the most lateralized of all cerebral functions.2 Their cortical areas are also some of the most asymmetrical in the brain.3 These areas are located within pars opercularis and pars triangularis in the frontal lobe. They are bounded anatomically by the anterior horizontal and anterior ascending ramus of the SF. Sylvian fissure is a most commonly used landmark for neurosurgical interventions. The triangular sulcus, also known as the incisura capitis, separates the pars triangularis into an anterior and a posterior part.4
The present study was undertaken to note the morphometry, asymmetry and variations of the right and left cerebrum and SF using various parameters.
MATERIAL AND METHODS
28 adult human brains with no gross deformity preserved in formalin irrespective of sexes were obtained from the Department of Anatomy, King George’s Medical University, Lucknow, Uttar Pradesh, India. The various measurements including fronto-occipital (FO) length, cerebral width, Sylvian fissure (SF) length and distance from anterior Sylvian point (ASP) to inferior Rolandic point (IRP) were taken. The sulcus (if present) located within the pars triangularis (PT) and pars opercularis (PO) was also evaluated. Identification of various sulci and gyri was done according to standard anatomy books. Confirmation was further strengthened by consensus of two authors. A tag with a specific number was attached to each specimen to avoid intermingling of brains.
Measurements were done by Vernier calipers. The fronto-occipital length was the maximum distance between the frontal and occipital poles measured from the inferior aspect of cerebrum (Figure 1). On superolateral surface of cerebral hemisphere, the lowermost point of central sulcus (Rolandic sulcus) which intersects with SF was referred as inferior Rolandic point (IRP). Lateral sulcus (Sylvian fissure) length was measured on the superolateral surface from anterior Sylvian point (ASP) i.e. most anterior point of SF where it divides into three rami (anterior horizontal, anterior ascending and posterior) to posterior Sylvian point (PSP) i.e. where the SF separates into ascending and descending posterior ramus (Figure 2). Width of cerebrum was measured by putting the two arms of Vernier calipers at the superomedial and inferolateral borders respectively at the level of IRP in midline. ASP to IRP distance and presence of the triangular sulcus (if present) located within the PT was also evaluated (Figure 3). Various pattern of division at ASP into anterior horizontal ramus (AHR) and anterior ascending ramus (AAR) of the Sylvian fissure as U, V and Y-shaped was also noted and analyzed (Figure 4).
Statistical Analysis was done using Statistical Package for Social Sciences (SPSS), version 15.0. Continuous data was compared using paired‘t’-test. Categorical data was compared using chi-square test. A 'p' value less than 0.05 (Englishhttp://ijcrr.com/abstract.php?article_id=147http://ijcrr.com/article_html.php?did=1471. Mancall EL, Brock DG.Gray’s Clinical Neuroanatomy. The Anatomical Basis for Clinical Neuroscience. Elsevier Saunders Philadelphia, 1st ed., 2011; 308-11.
2. Knaus TA, Corey DM, Bollich AM, Lemen LC, Foundas AL. Anatomical asymmetries of anterior perisylvian speech-language regions. Cortex. 2007;43:499–510.
3. Greve DN, Van der Haegen L, Cai Q, Stuffle beam S, Sabuncu MR, Fischl B, et al. A surface-based analysis of language lateralization and cortical asymmetry. J Cogn Neurosci. 2013;25:1477– 92.
4. Petrides M. Neuroanatomy of Language Regions of the Human Brain. 1sted, In: Morphological features of the core language regions: The Sulci and Gyri. Amsterdam, Boston: Elsevier, 2013: 21-22.
5. Toga AW, Thompson PM. Mapping Brain Asymmetry. Nat. Rev. Neurosci. 2003; 4: 37.
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7. Mellor C. Dermatoglyphic evidence of fluctuating asymmetry in schizophrenia. Br Jpsychiatry. 1992;160: 467–72.
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9. Burton C, Stevenson JC, Williams DC, Everson PM, Mahoney ER, Trimble JE. Attention deficit hyperactivity disorder (AD/ HD) and fluctuating asymmetry in a college sample: An exploratory study. Am. J. Hum. Biol. 2003; 15: 601–19.
10. Naugler CT, Ludman MD. Fluctuating asymmetry and disorders of developmental origins. Am. J. Med. Genet. 1996; 66: 15–20.
11. Barden HS. Fluctuating dental asymmetry: A measure of developmental instability in Down syndrome. Am. J. Phys. Anthropol. 1980; 52: 169–73.
12. Galaburda AM, Rosen GD, Sherman GF. Individual variability in cortical organization: its relationship to brain laterality and implications to function. Neuropsychologia. 1990; 28: 529– 46.
13. Chi JG, Dooling EC, Gilles FH. Gyral development of the human brain. Annals of Neurology. 1977; 1 (1): 86-93.
14. Eberstaller O, Das Stimhim. Ein Beitragzur Anatomie der oberflache des Gehirns. Urban and Schwarzenberg, Vienna, Austria, 1890.
15. Cunningham DJ. Contribution to the surface anatomy of the cerebral hemispheres. Dublin, Royal Irish Academy, 1892.
16. Rubens AB, Mahowald MW, Hutton JT. Asymmetry of the lateral (sylvian) fissures in man. Neurology. 1976; 26: 620-24.
17. Boni RC, Prodoscimi FC, Bonsi AB, Almeida TM, Ribeiro LAM. Asymmetries ofthe left and right temporal lobes. Int. J. Morphol. 2007; 25(1):117-20.
18. Foundas AL, Faulhaber JR, Kulynych JJ, Browning CA, Weinberger DR. Hemispheric and sex-linked differences in Sylvian fissure morphology: a quantitative approach using volumetric magnetic resonance imaging. Neuropsychiatry Neuropsychol Behav Neurol. 1999; 12 (1):1–10.
19. Idowu OE, Soyemi S, Atobatele K. Morphometry, asymmetry and variations of the Sylvian fissure and sulci bordering and within the pars triangularis and pars opercularis: An autopsy study. J Clin Diagn Res 2014;8(11): AC11-AC14.
20. Chakrabarti S, Vijayalakshmi S. Interhemispheric variation of Sylvian fissure: A Cadaveric brain study. Int J Anat Res. 2015;3(2):1143-48.
21. Ono M, Kubik S, Abernathy CD. Atlas of the cerebral sulci. New York: Georg Thieme Verlag. 1990; 62-74.
22. Ayberk G, Yagli OE, Comert A, Esmer AF, Canturk N, Tekdemir I, et al. Anatomic relationship between the anterior sylvian point and the pars triangularis. Clin. Anat. 2012;25:429–36.
23. Galaburda AM, LeMay M, Kemper TL, Geschwind N. Rightleft asymmetrics in the brain.Science.1978; 199(4331): 852-6.
24. Rumeau C, Tzourio N, Murayama N, Peretti-Viton P, Levrier O, Joliot M, et al. Location of hand function in the sensorimotor cortex: MR and functional correlation. Am J Neuroradiol. 1994; 15:567–72.
25. Sanes JN, Donoghue JP, Thangaraj V, Edelman RR, Warach S. Shared neural substrates controlling hand movements in human motor cortex. Science. 1995; 268:1775–77.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241823EnglishN-0001November30HealthcareKNOWLEDGE, ATTITUDE AND PRACTICE EVALUATIONOF ADVERSE DRUG REACTIONS REPORTING AMONG HEALTH CARE PROFESSIONALS IN KIMS HOSPITAL, NARKETPALLY, TELANGANA, INDIA
English2628C. Dinesh M. NaiduEnglish Anand VardhanEnglish Mangesh BankarEnglish Vinay Singh RaghuvanshiEnglish Sagar SharmaEnglishA huge number of cases of morbidity and mortality worldwide are attributed to Adverse Drug Reactions (ADRs), therefore this study was done with the primary Objective of assessing the Knowledge regarding pharmacovigilance and to evaluate the impact of an educational intervention on the same among healthcare professionals in a tertiary care centre.
Methods: A questionnaire containing 20 questions was given to all the participants before the educational session. Later, an interactive educational session on pharmacovigilance was conducted for them. After the educational session, the same questionnaire was given to all participants. The impact of effectiveness of educational session among health care professionals was evaluated by statistical analysis. 80 health care professionals were involved in the study. 37 Post Graduates of different departments, Assistant Professors 14, senior residents 10, Associate Professors 15, and 4 Professors were participants of this study.
Results: The overall response rates during pre intervention was 23.48%, which significantly increased to 25.99% during post intervention (P value EnglishPharmacovigilance, Educational Intervention, Adverse Drug Reactions, Spontaneous Reporting of ADR’sINTRODUCTION
Adverse drug reactions (ADRs) are the cause of morbidity and mortality worldwide. Recently it has been shown that ADRs are the fourth major cause of death in the United States.[1] According to World Health Organization (WHO) definition, an Adverse drug reaction is any unintended, and undesired effect of a drug, which occurs at doses used in humans for prophylaxis, diagnosis, or therapy.[2] Therefore to identify the drugs which cause number of ADRs, pharmacovigilance programs have been initiated in several countries in the recent past. There are variation in drug response among individuals, prescription pattern, drug regulatory system, drugs availability, it is recommended for every country to set up their own pharmacovigilance programme.[3]
Spontaneous reporting of ADR’s are a major source of information in pharmacovigilance. Spontaneous reporting of ADR’s are responsible for preventing the occurrence of tragedies caused by new medicines and also improving the safety labeling of pharmaceutical products. However, spontaneous reporting schemes are very less in number.[4]The success of any pharmacovigilance program in any country depends upon the involvement of the healthcare professionals who participate in reporting the ADRs.
Pharmacovigilance is still a young branch in India and there is very limited knowledge about this discipline. The Indian national Pharmacovigilance Programme is suffering setbacks due to lack of awareness and inadequate training about drug safety monitoring among healthcare professionals in India.[5]Different studies done in this field indicate inadequate knowledge about pharmacovigilance among healthcare professionals as well as attitudes that are associated with underreporting. Although Pharmacovigilance programs are becoming successful in improving drug use patterns, under-reporting of ADRs is still a major problem.[6]
Assessing the level of awareness of Pharmacovigilance among the healthcare professionals is extremely important, Therefore this study was conducted to assess awareness of pharmacovigilance among the healthcare professionals and to evaluate the impact of an educational session for improving awareness of pharmacovigilanceamong Physician, and post graduate residents in Kameini institute of Medical Sciences, Narketpally, Telangana.
MATERIALS AND METHODS
A Cross sectional study was conducted in Kameini institute of Medical Sciences, Narketpally, Telangana in June 2015 over a period of 1 week where All the participants were given a questionnaire as pre-test and the same question as post-test was given after one week of integrated educational session. The questionnaire consisted of information about knowledge and practice of ADR reporting and how frequently they are encountering the ADR’s, factors affecting their reporting of ADR and suggestions towards how a better ADR reporting can be achieved. The collected thus data was analysed statistically using chi-square test and p value was obtained.
RESULTS
Out of 80 health care professionals in this study, 37 participants were post Graduates, 14 were Assistant Professors,10 were senior residents,15 were Associate Professors , and 4 were Professors of different clinical departments were the enrolled. The overall response rates before the educational session(Pre Test) was 23.48%, which significantly increased to 25.99% after one week of educational session(Post Test) and the p value came out to be Englishhttp://ijcrr.com/abstract.php?article_id=148http://ijcrr.com/article_html.php?did=148
Somayeh H, Hassan T, Hayatshahi A, Gholami K, Javadi M. Knowledge, attitudes and practice of nurse regarding adverse drug reaction reporting. Iran J Nurs Midwifery Res, 17(1), 2012, 21–25.
World Health Organization. Safety of Medicines. A guide to detecting and reporting adverse drug reactions. Geneva, Switzerland: World Health Organization 2002. WHO / EDM / QSM /2002.2
Safety monitoring of medicinal products. The importance of pharmacovigilance. World Health Organization, Geneva, 2002. (http://apps.who.int/medicinedocs/pdf/s4893e/s4893e.pdf.)
Mishra H, Kumar V. Pharmacovigilance: Current Scenario in a Tertiary Care Teaching Medical College in North India. J Pharmacovigilance, 1, 2013, 108.
Rajesh R, Vidyasagar S, Varma DM An Educational Intervention to assess Knowledge Attitude Practice of pharmacovigilance among Health care professionals in an Indian tertiary care teaching hospital. Int J PharmTech Res, 3(2), 2011, 678-92.
Palaian S, Ibrahim MI, Mishra P. Health professionals' knowledge, attitude and practices towards pharmacovigilance in Nepal. Pharmacy Practice (Internet), 9(4), 2011, 228-35.
Desai CK, Iyer G, Panchal J, Shah S, Dikshith RK et al. An evaluation of knowledge, attitude, and practice of adverse drug reaction reporting among prescribers at a tertiary care hospital. PerspectClin Res, 2(4), 2011, 129–36.
Fadare JO, Enwere OO, Afolabi AO et al. Knowledge, Attitudeand Practice of Adverse Drug Reaction Reporting among Healthcare Workers in aTertiary Centre in Northern Nigeria. Trop J Pharm Res, 10 (3), 2011, 235-42.
Chatterjee S, Lyle N, Ghosh S: A survey of the knowledge, attitude and practice of adverse drug reaction reporting by clinicians in eastern India. Drug Saf, 29, 2006, 641-2.
Khalili H, Mohebbi N, Hendoiee N, Keshtkar AA, Khavidaki SD . Improvement of knowledge, attitude and perception of healthcare workers about ADR, a pre- and post-clinical pharmacists’ interventional study. BMJ Open, 2, 2012, e000367.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241823EnglishN-0001November30HealthcareRAPID DETECTION OF MULTI-DRUG RESISTANT TUBERCULOSIS USING DIRECT DRUG SUSCEPTIBILITY TESTING AND LINE PROBE ASSAY
English2938Pranali MedhekarEnglish Nilma HiraniEnglish Sarala MenonEnglish Ameeta JoshiEnglish Abhay ChowdharyEnglishIntroduction: Tuberculosis, particularly multi-drug resistant tuberculosis (MDR TB), is a leading cause of morbidity and mortality in developing countries. Conventional methods of DST have a long turnaround time of around 2-3 months. Rapid detection of MDR TB can be achieved by direct tests, liquid based indirect susceptibility tests and molecular line probe assays. This study aims to evaluate the utility of Direct DST and line probe assay (LPA) for rapid detection of MDR TB.
Methodology: A total of 510 sputum concentrates (smear 1+/ more) were subjected to Direct DST and LPA. Conventional DST was put up from the primary cultures of all 510 strains using economic variant of 1% proportion method, considered the gold standard.
Results: Direct DST showed 100% specificity and sensitivity of 98.7%, 97.3% and 97.5% respectively for detection of Isoniazid (INH), Rifampicin (RIF) resistance and for MDR strains. LPA showed specificity of 98.4%, 96.8%, 97% and sensitivity of 97.5%, 98.9% and 98.9% for detection of INH, RIF resistance and MDR strains respectively. The commonest mutation pattern detected in our region was S531L (MUT3) for rpoB gene locus signifying RIF resistance + S315T1 (MUT 1) for katG gene locus signifying high level INH resistance.
Conclusion: Direct DST can be used as a good alternative for rapid detection of MDR-TB in resource poor settings. LPA is definitely a good tool, but requires requisite laboratory infrastructure and trained man-power. From our study, rifampicin resistance can be considered as a surrogate marker of MDR TB.
EnglishMDR TB, Direct DST, LPAINTRODUCTION
Tuberculosis (TB) continues to be a leading cause of morbidity and mortality in developing countries [1]. Global efforts for TB control are being challenged by the steady increase in drug resistant TB, particularly multidrug resistant tuberculosis (MDR TB), defined as resistance to rifampicin (RIF) and isoniazid (INH) [2]. As per the WHO Global TB Report 2015, there were an estimated 480 000 (range: 360 000-600 000) new cases of MDR-TB globally in 2014 and MDR TB has been reported in 3.3% new and 20% previously treated cases, respectively. [3]
MDR-TB requires 18-24 months of treatment with expensive second line drugs, some of which are parenteral drugs. The cure rate is much lower at about 60% than for drug susceptible TB [4]. Therefore, it is crucial that MDR TB should be detected as early as possible and measures implemented to effectively control its spread.
Detection of MDR-TB could be achieved by phenotypic and genotypic methods of drug sensitivity testing. The phenotypic methods include: a) Conventional methods of solid drug susceptibility testing [5] for detection of MDR TB which include 1% proportion method, resistance ratio and absolute concentration method. These methods require growth from primary cultures of specimens on Löwenstein Jensen (LJ) medium. Hence, they have a long turnaround time of around 2-3 months. b) Direct tests for DST of mycobacteria in which decontaminated sputum specimens are directly inoculated on drug free and drug containing LJ media. This obviates the need to wait for growth on primary culture, thus, reducing the turnaround time by 4-6 weeks and can be applicable in a resource poor setting. Examples of direct tests are Direct DST [6] on drug containing media, Nitrate Reductase Assay [NRA] [7], Microscopic Observation Drug Susceptibility [MODS] [7, 8]. c) Liquid based indirect susceptibility tests such as MB/BACT, BACTEC and MGIT960 which have an improved turnaround time of about 25 -45 days, as compared to the solid conventional DST, but, these are costly and not available where the need is greatest.[7,8]
The genotypic methods for rapid detection of MDR-TB include molecular line probe assays (LPA) which have a turnaround time of 48-72hrs. But, require specific laboratory infrastructure, technical expertise and are expensive. [9]
This study aims to evaluate the utility of DST and LPA for early detection of MDR-TB and also to evaluate rifampicin resistance as a surrogate marker for MDR-TB.
.MATERIAL AND METHODS
This was a prospective study carried out in a tertiary care hospital. The study protocol was approved by institutional ethics committee.
Inclusion criteria: Clinically diagnosed pulmonary tuberculosis (PTB) cases with sputum smear 1+ and more
Exclusion criteria: 1. Sputum smear positive PTB cases less than 1+.
2. Smear negative PTB cases.
3. Extrapulmonary (+/-) specimens.
METHODOLOGY
Each patient was asked to submit two sputum samples (one early morning and one spot sample) as per RNTCP Guidelines [11]. These were then subjected to direct smears by Ziehl Neelsen staining [12] and graded according to RNTCP guidelines [11]. The sputum samples were decontaminated and concentrated using the N-acetyl- L- Cysteine (NALC) – NaOH Concentration Method.[12]
Direct Drug Susceptibility Test (Direct DST) [6]
The sputum deposits obtained by NALC –NaOH method were inoculated with a 5 mm loop (27 SWG) onto a pair of plain LJ and drug containing LJ media (INH 0.2 μg /ml; R-40 μg/ml) along with a Para nitro benzoic acid (PNB) slant to confirm the growth of M.tuberculosis. The slopes were incubated at 37oC and examined weekly for 8 weeks and the growth was recorded weekly.
The definition of resistance was based on the amount of growth seen on the drug-free medium. Thus, when the growth on the drug-free medium was 2+ or more, growth of 1+ or more on the drug containing medium was defined as resistance to the drug. When growth on the drug-free medium is 1+ or less (i.e. < 100 colonies), any growth on the drug-containing medium was considered to be an indication of resistance to that particular drug. For this purpose, the higher growth observed on the paired slopes were considered for interpretation.
Conventional DST by 1% proportion method (economic variant) [5]
Inoculum preparation : With a 3 mm loop, 4-5 mg ( loopful) of primary culture was taken and inoculum was prepared in a sterile Bijou bottle containing 2 ml sterile distilled water and 6 sterile glass beads of diameter 3mm. Turbidity of the bacterial suspension was matched with McFarland standard no. 1 (10 7 to 108 cfu/ml ) against a black background. This was the neat bacterial suspension standardized at 1 mg/ml. Further’ 2 log dilutions were prepared. A loopful (using a 3 mm calibrated loop) of each dilution was inoculated on drug free and drug containing media [Isoniazid: 0.2μg/ml, Ethambutol: 2μg/ml, Streptomycin: 4μg/ml and Rifampicin: 40μg/ml.]
The slopes were incubated at 37°C. The growth was read at 28 days and again at 42 days.
(Growth recorded as - Confluent growth = 3+; More than 100 colonies = 2+; Record actual number of colonies = 1-100 cols.)
Interpretation
First reading was taken at 28th day after inoculation. The actual number of colonies (up to 100 colonies on the slope) on drug containing and drug-free slopes were counted.
Proportion of resistant bacilli existing in the strain was obtained by dividing the number of colonies obtained on drug containing slopes by that on drug free slope. Below a certain value – the critical proportion (1%)– the strain is classified as sensitive; above that value, it is classified as resistant. The proportions were reported as percentages. [5]
Internal Quality Control
Along with each batch of samples to be tested, a culture of H37Rv (a standard strain of M.Tuberculosis) or a known all sensitive strain was also incorporated and the test was validated.
Line Probe Assay (LPA) [13]:
Line probe assay is a DNA strip technology using a cellulose acetate membrane strip for the detection of resistance to RIF and/or INH. The kit used at our center for LPA was GenoType MTBDR Plus version 1.0 manufactured by Hain Lifescience, Germany which detects resistance to RIF and/or INH in sputum samples (smear 1+ / more) and culture isolates.. The steps involved are DNA Extraction from sputum concentrates, Multiplex Amplification, reverse hybridization and interpretation of results.
DNA Extraction:
A 500 µl of the smear positive (1+ or more) decontaminated sputum specimen was used for the DNA extraction which involved centrifugation of the specimen at 10,000 g for 15 minutes and suspension of the pellet in 100 µl of molecular grade water. It was then subjected to heat killing at 95oC for 20 minutes followed by sonication for 15 minutes and finally centrifugation at 10,000g for 5 minutes. 70-80 µl of the supernatant was taken and deep frozen at -20 0C of which 5 µl was used for PCR.
Multiplex amplification:
The master mix was prepared in a DNA-free room and the DNA extract was added to the master mix in a PCR hood. Both these procedures were done in separate rooms following the restricted access and unidirectional work flow protocol. A master mix negative control was also included with every batch of master mix prepared.
The amplification was done using a thermo cycler, with a set programme for amplification i.e. different for direct smear positive sputum specimens and culture isolates
Reverse hybridization:
It involved the chemical denaturation of the amplicons, hybridization of the biotin-labeled amplicons to the single stranded membrane-bound probes on the cellulose acetate membrane strip. It was followed by stringent washing, addition of streptavidine-alkaline phosphate conjugate to the wells and an enzymatic reaction mediated by alkaline phosphatase was observed in the form of a coloured band following addition of substrate in those regions where the amplicon and probe had hybridized. After the final washing, the strips were removed from the wells, allowed to air dry and placed onto the sheet provided with the kit and interpreted.
Each run incorporated one positive control and two negative controls (1 extraction negative control + 1 master mix negative control).
Interpretation of Results
Valid results can be interpreted by the presence of CC (conjugate control) and AC (amplification control) bands for each sample run. The presence of TUB band indicates that the organism belongs to M.tuberculosis complex.
A mutation in the relevant gene (and resistance to the relevant drug) is signified by either an absence of wild type band and/or the presence of a mutant band for each gene. The rpoB, katG and inhA gene loci each have a control band which needs to be present in order to interpret the results.
The presence of mutations in the rpoB gene locus predicts RIF resistance, in katG predicts high level INH resistance while in the inhA gene locus predicts low level INH resistance.
For interpretation of results, the intensity of all the bands (except CC band) should be equal to or greater than the intensity of the AC band.
In order for a batch of results to be valid, the negative control strip has to have a CC and AC band present, but no other bands.
Presence of bands other than CC and AC bands in the NC indicates contamination and the run need to be repeated.
The product insert was referred to for interpretation of banding patterns.
Statistical analysis:
Sensitivity, specificity, positive predictive value, negative predictive value of Direct DST and LPA was calculated considering 1% proportion method the gold standard.
RESULTS
A total of 531 smear positive (1+ and more) sputum specimens were collected from the patients attending the outpatient as well as inpatient departments of tertiary care hospital. Out of these 531 specimens, 9 specimens showed no growth and12 were contaminated on Direct DST, thus could not be processed further by 1% Proportion method. Hence these 21 specimens were excluded from data analysis, since the results of Direct DST and 1% Proportion method (gold standard test for comparison) were not available for them. Primary cultures of the remaining 510 specimens were then subjected to economic variant of 1% proportion method. Thus total 510 specimens (n=510) all of which were subjected to all 3 tests were included for analysis.
Table: 1 shows the comparative evaluation of LPA, Direct DST % 1% Proportion method in terms of the number of MDR, RIF and INH monoresistant and pansensitive strains.
Table: 2 shows the turn-around time by Direct DST. 74 out of 176 specimens with smear grading of 3+ gave results at 4th week, whereas for 172 out of 183 specimens with smear grading of 1+, results were obtained around 8th week; most of these being sensitive strains. Thus it was observed that higher the smear grading, we obtained the results earlier.
Tables: 3A and 3B- Comparison of LPA with proportion method for detection of MDR strains, RIF and INH resistance. LPA has shown a sensitivity of 98.9%, 98.9 % and 97.5 % and specificity of 97 %, 96.8 % and 98.4 % for detection of MDR, rifampicin and isoniazid resistance, respectively.
Tables: 4A and 4B- Comparison of Direct DST with 1% Proportion method for detection of MDR strains, RIF and INH resistance. DST has shown a sensitivity of 97.5%, 97.3 % and 98.7 % and specificity of 100% for detection of MDR, rifampicin and isoniazid resistance, respectively.
Table: 5 shows the banding patterns amongst resistant strains by LPA. There were 45 different patterns observed amongst the resistant strains. Commonest pattern (57.1%) seen was absence of wild type 8 band and simultaneous presence of mutation 3 band in the rpoB gene region coding for the S531L codon signifying RIF resistance + absence of wild type band and simultaneous presence of mutation 1 band in the katG gene region coding for S315T1 codon signifying high level INH resistance. 14 (2.7%) strains showed mixed infections and one strain showed complete inhA gene deletion. Low level INH resistance was seen in 13 (2.5%) of the 510 strains, of which 8 (1.5%) were MDR strains and 5 (0.9%) were INH mono-resistant strains.
Table 6 shows the distribution of mutation in various gene loci. In the rpoB gene locus, the most common mutation was found to be the presence of MUT3 band coding for S531L codon in 265(89.8%) of the 295 strains. The katG gene locus showed maximum mutations in the MUT1 band region coding for S315T1 codon amounting to 289(96.3%) of the 300 strains, while in the inhA gene locus maximum mutations were seen in the MUT1 band region coding for C15T codon amounting to 66(91.7%) of the 72 strains.
DISCUSSION
In the present study, we have performed the comparative evaluation of the results of drug susceptibility testing of total 510 sputum specimens by Direct DST, Line probe assay (LPA), and 1% Proportion method, considered the gold standard.
The performance of the Direct DST and the Genotype MTBDR plus used for LPA test directly from smear positive (1+/ more) sputum specimens correlated very highly with conventional DST by 1% proportion method. (Table: 1)
The Direct DST described here would serve the purpose well in countries with limited resources. In our study, DST has shown a sensitivity of 97.5%, 97.3 % and 98.7 % and specificity of 100% for detection of MDR, rifampicin and isoniazid resistance, respectively. The concordance of the results of the 1% proportion method and Direct DST was found to be 97.6%. Thus, its performance characteristics suggest that the technique is equivalent to conventional 1% proportion method. The turn-around-time of the results obtained by Direct DST are shown in Table 2. Maximum results with smear grading of 3+ were obtained around 4th week, whereas for most of the specimens with smear grading of 1+ results were obtained around 8th week; most of these being sensitive strains. Thus it was observed that higher the smear grading, the results were obtained earlier.
The advantages of the Direct DST over the conventional 1% proportion method are that it gives sensitivity results at the same time as that of primary culture. This process not only reduces the turn-around-time by 4 weeks, but also contamination by eliminating the step of doing a subculture. Most importantly, the results of the Direct DST are more closely the representative of the bacterial population in the given sample unlike in the conventional test, which can suffer from errors of selection when drug susceptibility test is set up from a primary culture. In the Direct DST, resistance can be reported if adequate growth is seen on the drug slopes even when the plain medium is contaminated. Mathew et al also have reported the utility of Direct DST in MDR detection. Their results also indicate that resistance could be detected with growth as low as 10 colonies and in total agreement with the results of conventional test.[6] Hence, it is recommended that such results be accepted provisionally and confirmed with the conventional test on subculture.
The performance of Genotype MTBDRplus assay has co-related very well with the conventional 1% proportion method for DST with a sensitivity of 98.9%, 98.9 % and 97.5 % and specificity of 97 %, 96.8 % and 98.4 % for detection of MDR, rifampicin and isoniazid resistance, respectively. This test has the advantage of performing well and giving good results even in those specimens which are finally lost on culture.
Rifampicin resistance due to mutation in the rpoB gene was detected in 288 out of 291 phenotypically resistant RIF isolates. The resultant high sensitivity (98.9 %) for detection of rifampicin resistance found in our study could be attributed to all these mutations being present in the 81 bp- hotspot region of the rpoB gene (Table 6). This is in accordance with various other studies conducted across the world, such as S. Africa, Pakistan, Mongolia, India and Vietnam which have proven the mutations in the 81-bp region alone have got 98-100% sensitivity in detecting RIF resistance.[14-18]
Among all the gene mutations conferring to Rifampicin resistance, codon S531L was found to be the most frequently encountered mutation in 265 out of 295 (89.8%) which is comparable to the Indian study by Raveendran et al in 2012 (84.6%) [17], but, higher than that reported in various other studies across the world [14,16,18].
Mutations at codon 526 in our study were less common (2.7%) than reported from the studies conducted in various other studies such as S. Africa (8.6%)[14], Europe (15 %)[19], India (19 %)[17], Pakistan (22.5%)[15] and Iran (45. 6%)[20], but higher than the study conducted in Vietnam ( 1.8%)[18].
Mutations in the rpoB gene at the D516V codon were at 1.4 % in our study which is very low compared to the studies from S. Africa (9.6%)[14] and Europe (44%)[19].
18 isolates (6.1%) showed unknown mutations with the absence of both wild type and mutation band. It could be attributed to a rare mutation in the corresponding genomic region which is not probed on the strip (e.g mutations in the 530-533 region of the rpoB gene other than S531L mutation leading to absence of both, wild type 8 and MUT 3 which codes S531L codon specifically) or it could be a silent mutation since this test only screens the nucleic acid sequence and not the amino acid sequence. Therefore it is possible that mutations that do not cause an amino acid change (silent mutations) will still produce the absence of one of the wild type probes. In such cases phenotypic resistance determination should be considered[13]. If deletion of wild type band is the only indication of RIF resistance and there is no mutation probe, it would be advisable to continue the patient on RIF therapy till phenotypic sensitivity report is available.[17]
Mutations in the katG gene locus were detected in 300 out of 313 (95.8%) INH resistant strains. These were found to be maximum in the S315T1 codon region of the katG gene amounting to 96.3%. This is in accordance with Huyen et al’s study from Vietnam [18] and Van Rie et al’s study from S, Africa [21]. A high prevalence of katG mutations has been reported in high TB prevalent countries which is attributed to INH resistance and much lower prevalence in low TB-prevalent countries. This could be due to the on-going transmission of the strains in the high- burden settings.[22]
The presence of mutations in the inhA region signifying low level INH resistance accounts for 72 (23%) of the 313 INH resistant strains, with maximum mutations in the C15T (MUT1) region amounting to 66 (91.7%) of the 72 strains. Mutations in the inhA gene region accounting for 23.5% (66/281 strains) in MDRTB cases and 18.8% (6/32 strains) in INH mono-resistant cases were seen in our study, which is similar for MDR strains but lower for INH resistant strains when compared to the study by Barnard et al at 27% of MDR strains and 54% of INH mono-resistant strains.[14] The presence of mutations in inhA gene region vary in different geographical locations from 2% isolates in Western Cape Province of South Africa [21], 5.4% in the study by R. Raveendran et al[17] from India, 18% in the study from Vietnam by Huyen et al [18], 24% from KwaZulu-Natal [23] to 67.6% in Mongolia[16].
Out of the 327 strains showing mutations one strain exhibited complete inhA gene deletion, which is a very rare finding and the probable explanation for the absence of all inhA bands including the inhA locus control band, is a mutation in the primer annealing sequence, which results in missing of the specific amplification product. Since this strain was MDRTB with katG mutations as well, this presentation would not have any implication on patient treatment.
In our study, low level INH resistance alone was seen in 13 (2.5%) of the 510 strains, of which 8 (1.5%) were MDRTB strains and 5 (0.9%) were INH mono-resistant strains. These strains would not have been detected as MDR by the previous version of the assay (GenotypeMTBDR) which only tested for mutations in the katG gene.[24-26] The GenotypeMTBDRplus assay, with incorporation of additional probes to detect mutations in the inhA gene as well has a better sensitivity for detecting INH resistance.
In our study, 14 cases (2.7%) of mixed infections were detected by this assay showing presence of both the wild type as well as mutant band in the corresponding gene region. This is similar to the findings by Huyen et al[18] (3.6%). Of the 14 cases, mixed infections were seen in both, RIF and INH genomic loci in 5 (35.7%), while 9 (64.3%) out of 14 cases showed mixed infections either in the RIF or INH genomic loci.
In our study 21 discrepant results were obtained with LPA when compared to conventional DST, thus giving concordance of 95.9% (489/510 strains). 7/16 strains detected as RIF resistant by LPA were found to be sensitive on conventional DST. 5 out of these 7 false positive RIF resistant strains showed the presence of silent/unknown mutation which was not phenotypically expressed. In this situation results of phenotypic DST are confirmatory. For the rest of the false positive RIF resistant strains, performing phenotypic DST using minimum inhibitory concentration (MIC) would be confirmatory to check whether these isolates are showing resistance on a lower drug concentration. Rifampicin resistance was not detected in 3 strains by LPA which were RIF resistant on conventional DST. This could be attributed to other mutations outside the 81 bp-hotspot region which are not probed on the strip, hence LPA could not detect them. INH resistance was detected in 3 strains by LPA which were INH sensitive by conventional DST; 2 of which showed an unknown/silent mutation which was not phenotypically expressed and the remaining 1 strain showed low level INH resistance on LPA. LPA could not detect INH resistance in 8 strains which were resistant by conventional DST. This could be because of the mutation being present in other genomic loci such as kasA, intergenic region of oxyR-ahpC complex which are not probed on the DNA strip.[27,28]
This study also evaluated detection of monoresistance to INH and RIF. This approach was adopted because these 2 drugs are the key elements in short course TB chemotherapy and provide the most robust drug susceptibility results. Moreover, the only susceptibility data that are likely to be translated into clinical action (in terms of a change in drug regimen) in a programmatic setting would be identification of MDR TB. This is also seen with other well standardized methods, as resistance results for ETH and STR are less reliable than for INH and RIF. We found a higher incidence of INH monoresistance as compared to RIF monoresistance which is in accordance with the other studies worldwide. [14,16,18]
Because RIF mono resistance is rare, resistance to RIF is considered a surrogate marker of MDR TB. [29] Our results (Table 1) show that RIF resistance can be considered as a surrogate marker for MDR by all the three tests. Patients infected with RIF resistant strain of MTB generally have a poor prognosis, particularly because RIF resistance is often associated with resistance to other first line drugs.
Hence to summarize, successful treatment of MDR TB relies on prompt laboratory detection of drug resistance. This study found that Direct DST is a good rapid technique in resource-constraint settings. LPA is also a good rapid technique for rapid detection of MDR TB, but, it needs specific laboratory infrastructure, trained personnel, is expensive and needs an external quality assurance system. Both the techniques were well comparable with the gold standard 1% proportion method. Therefore, development of efficient laboratory strategies for rapid and reliable drug susceptibility testing of M tuberculosis is of importance for proper management of patients, particularly those with multi-drug resistant tuberculosis.
CONCLUSIONS
The results of this study have led us to following conclusions.
Direct DST can be used as a good method, extremely feasible for rapid drug susceptibility testing of M tuberculosis in resource poor settings. Similarly, Direct DST can also compete with LPA in terms of turnaround time (as cheaper technique and less turnaround time compared to1% proportion method).
Line probe assay is definitely a good tool for rapid detection of MDR provided one has the requisite laboratory infrastructure and technical expertise.
1% Proportion method still continues to remain the gold standard, when it comes to smear negative cases as well as to identify extensively drug resistant (XDR) cases of tuberculosis.
Rifampicin resistance can be considered as a surrogate marker for MDR-TB in settings with high prevalence of drug resistant tuberculosis.
ACKNOWLEDGEMENT
We acknowledge the support from technical staff of TB culture and DST laboratory, Sir JJ Group of Hospitals Mumbai. We are also grateful to authors, editors and publishers of all those articles, journals and books included in the references of this manuscript.
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4. Blumberg HM, Burman WJ. Chaisson RE , Dailey CL, Etkind SC ,Fujivara P, et al: American Thoracic Society/ Centers For Disease Control and Prevention/ Infectious Diseases Society of America; treatment of Tuberculosis . Am. J Resp Crit Care Med 2003, 167(4) :603-662.
5. Manual of Standard Operating Procedures (SOPs). Culture of Mycobacterium tuberculosis and Drug Susceptibility Testing on solid medium. 1-142(2009) available at www.tbcindia.org
6. Mathew S, Nalini SM, Rahman F, Sundaram V and Paramasivan CN. Simple Direct Susceptibility Tests on sputum samples for early detection of resistance in tubercle bacilli: Indian J Tuber 2007; 54: 184-189.
7. Angeby KA , Klintz L, Hoffner SE :Rapid And Inexpensive Drug Susceptibility Testing of Mycobacterium tuberculosis with a nitrate reductase assay, J Clin Microbiol 2008 ,40(2) :553-555
8. Woods GL Susceptibility Testing for Mycobacteria Clin inf Dis 2000, 31 (5);209-1215.
9. Miotto P, PianaF, Cinllo DM, Miglori GB; Genotype MTBDRplus: a further step Toward rapid identification of drug resistant Mycobacterium tuberculosis J Clin Microbiol 2008, 46(1); 393-394.
10. Sharma M, Sethi S, Mishra B, Sengupta C, Sharma SK; Rapid detection of mutations in rpoB gene of rifampicin resistant Mycobacterium tuberculosis strains by line probe assay;Indian J Med Res 2003;pp 76-80.
11. Revised National Tuberculosis Control Programme: Guidelines on Programmatic Management of Drug Resistant TB (PMDT) in India May 2012.
12. Koneman’s Colour Atlas and Textbook of Diagnostic Microbiology. Washington Winn, Jr. Stephen Allen, William Janda, Elmer Koneman Gary Procop, Paul Schreckenberger, Gail Woods. 2006; Sixth Edition.
13. GenoType MTBDRplus,version 1.0 (product insert). (Internet) Feb 2007 (updated 2007 Apr 3; accessed 2007 May 9). Nehren, Germany; Hain Lifescience, GmbH. Available from: http:// www.hain-lifescience.com/pdf/304xx_pbl.pdf
14. Bernard N, Albert H, Coetzee G Rapid Molecular screening of multi drug resistant Tuberculosis in A high volume public health laboratory in South Africa; Am J Resp Cri Care Med, 2008,pp 787-792. 35 Int J Cur Res Rev | Vol 8 • Issue 23 • December 2016 Medhekar et.al.: Rapid detection of multi-drug resistant tuberculosis using direct drug susceptibility testing and line probe assay
15. Farooqui J. Q. Erun Khan, Syed Muhammed Zaheer Alam, Asho Ali, Zahra Hasan, Rumina Hasan, Line Probe Assay for detection of rifampicin and isoniazid resistant tuberculosis in Pakistan; Journal of Pakistan Medical Association; vol 62 No. 8, August 2012.
16. Buyankhishig B, T. Oyuntuya, B. Tserelmaa, J. Sarantuya, Marilla G. Lucero, S. Mitarai. Rapid molecular testing for multiresistant tuberculosis in Mongolia: A diagnostic study; International Journal of Mycobacteriology , vol 1, Issue 1, March 2012i
17. Raveendran R, Wattal C, Oberoi JK, Goel N, Datta S, Prasad KJ. Utility of Geno Type MTBDRplus assay in rapid diagnosis of multidrug resistant tuberculosis at a tertiary care centre in India; Indian Journal of Medical Microbiology vol 30, Issue 1, 2012.
18. Mai NT Huyen, Edine W Tiemersma, Nguyen TN Lan, Frank GJ Cobelens, Nguyen H Dung, Dinh N Sy, et al: Validation of the GenoType® MTBDRplus assay for diagnosis of multidrug resistant tuberculosis in South Vietnam; BMC Infectious Diseases 2010, 10:149.
19. Miotto P, Saleri N, Dembele M, Ouedraogo M, Baooum G, Pinsi G, et al. Molecular detection of rifampicin and isoniazid resistance to guide chronic TB patient management in Burkina Faso. BMC Infect Dis 2009;9:142.
20. Bahrmand AR, Titov LP, Tasbiti AH, Yari S, Graviss EA,. Highlevel rifampin resistance correlates with multiple mutations in the rpo B gene of pulmonary tuberculosis isolates from Afganistan border of Iran. J Clin Microbiol 2009; 47: 2744-50.
21. Van Rie A, Warren R, Mshanga I,Jordaan A, van der Spuy GD, Richardson M, Simpson J, Gie RP, Enarson DA, Beyer’s N et al. Analysis for a limited number of gene codons can predict drug resistance of Mycobacteriun tuberculosis in a high incidence community. J Clin Microbiol 2001;39-636-641.
22. Mokrousov I, Narvaskaya O, Otten T, Limenschenko E, Steklova L, Vyshnevskiy B. High prevalence of katG Ser315Thr substitution among isoniazid resistant Mycobacterium Tuberculosis clinical isolates from Northwestern Russia, 1996-2001. Antimicrob Agents Chemother 2002;46:1417-1424.
23. Kiepiela P, Bishop KS, Smith AN, Roux L, York DF. Genomic mutations in the katG, inhA and ahpC are useful for the prediction of isoniazid resistance in M. Tuberculosis isolates from KWA Zulu Natal, South Africa. Tuber Lung Dis 2000;80:87-56
24. Miotto P, Piana F, Penati V, Canducci F, Migliori GB, Cirillo DM. Use of Genotype MTBDR assay for molecular detection of rifampicin and isonoazid resistance in Mycobacterium tuberculosis in clinical strains isolated in Italy. J Clin Micobiol 2006;44:2485-2491.
25. Hilleman D, Rusch-Gerdes S, Richter E. Application of Genotype MTBDR assay directly on sputum samples. Int J Tuberc Lung Dis 2006;10:1057-1059.
26. Somoskovi A, Dormandy J, Mitsani D, Rivenberg J, Salfinger M. Use of smear-positive samples to assess the PCR-based genotype MTBDR assay for rapid, direct detection of the Mycobacterium tuberculosis complex as well as its resistance to isoniazid and rifampicin. J Clin Microbiol 2006;44:4459-4463.
27. Somoskovi A, Parsons LM, Salfinger M, The molecular basis of resistance to isoniazid, rifampin and pyrizinamide in Mycobacterium tuberculosis. Resspir Res 2001; 2:164-8.
28. Ramaswamy S, Musser JM. Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update. Tuber Lung Dis 1998; 79:3-29.
29. Molecular Line Probe Assays for Rapid Screening of Patients at Risk of Multi-Drug Resistant Tuberculosis (MDR-TB): WHO Expert group report.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241823EnglishN-0001November30HealthcarePRESCRIPTION AUDITING AND DRUG UTILIZATION PATTERN IN INDOOR PATIENTS OF PEDIATRICS DEPARTMENT
English3946Patel NehaEnglish Shah ShreyaEnglish Asari PratikEnglish Amin AnandEnglishObjectives: To analyze the rationality status of prescriptions and drug utilization pattern in indoor patients of pediatrics department.
Method: The present observational prospective study was undertaken in pediatrics indoor patient department for a period of six months during which data of 100 patients was collected. The prescriptions were analyzed for rationality score and rationality status (rational, semi-rational, irrational) using Phadke’s criteria; a 30 point score system in which choice of drugs and their dose, unnecessary drugs, irrational drugs/combinations and use of hazardous drugs were taken into consideration. Data was also analyzed for drug utilization pattern using WHO- prescribing indicators.
Result: Data of total 100 patients was analyzed, of which 69 were male and 31 were female. Rationality scores of 100 prescriptions were in range of 30 to 19, with mean rationality score of 28.4%. Of 100 prescriptions, 91 were rational and 9 were semi-rational. Main reason for getting less score were improper dose and use of second choice drug or wrong selection of drug. Average number of drugs used was 6.01 per patientsitus judi slot online deposit pulsa tanpa potongan. 54.76% drugs were prescribed by generic situs slot, 33.33% by brand name and 11.90% by both. Drugs prescribed from WHO-EML for children were 48.81%. Antimicrobials were prescribed for 93 patients, of which most common were amoxicillin+ clavulanic acid, ceftriaxone and cefotaxime.
Conclusion: Though the results reflect rational prescribing in pediatrics department of our set up, there is still scope of improvement in areas of dosage calculation, proper documentation, prescribing drugs by generic name and from WHO-EML for children as far as possible
EnglishPrescription audit, Drug utilization pattern, Rational drug utilizationINTRODUCTION
Drugs play an important role in disease prevention and in health care delivery. The availability and affordability of good quality drugs along with their rational use is required for effective health care.1 The prescription of a drug with proven efficacy at an optimal dose together with the correct information at an affordable price is very necessary to ensure the effective health care.2 The quality of life can be improved by increasing the standards of the medical treatment at all levels of the health care delivery system.3 A medical audit oversees the observance of these standards, which is defined as ‘the review and evaluation of the health care procedures and documentation for the purpose of comparing the quality of care which is provided, with the accepted standards situs slot’.4
Prescription auditing is a type of vigilance activity, which is beneficial in clinical practice in terms of reducing the burden of disease because of medication errors, i.e. because of irrational prescribing.5
In developing countries like India, a substantial proportion of medicines in the market are irrational fixed-dose-combinations and some of them are even hazardous. Analysis of a properly selected sample of prescriptions would reveal the extent of use of such irrational and hazardous drugs by doctors as well as irrational use of rational drugs. This will help in assessing the extent of wastage (health-wise and money-wise) due to irrational prescribing and in developing ways to overcome this wastage.6
Drug utilization research is defined by WHO as “the marketing, distribution, prescription, and use of drug in a society, with special emphasis on the resulting medical, social and economic consequences”.7
The principle aim of the drug utilization research is to facilitate rationale use of drugs in population. For the individual patient, rationale use of a drug implies the prescription of a well-documented drug in an optimal dose for a right indication, with the correct information and at an affordable price. Without knowledge on how drug are being prescribed and used, it is difficult to initiate discussion on rationale drug use and to suggest measure to change prescribing habits for the better.8
Infants and children constitute a large proportion of the population in developing countries. They are especially vulnerable to contract illnesses and to the harmful effect of drugs due to differences in pharmacodynamics and pharmacokinetics.9 They suffer from frequent but usually nonserious illnesses. Most of these are self limiting and often treated not only inappropriately but also resorting to polypharmacy.10 Epidemiological evaluation of medicine use in elderly is now a highly visible topic, but drug prescribing studies in pediatric patients have been limited. The need for the safe and effective drugs for use in sick neonates, infants, children and adolescents requires the establishment of thoughtful drug therapy strategies.11
Considering all these facts, the present study was designed to situs judi slot online deposit pulsa tanpa potongan check the rationality status and drug utilization pattern in indoor patients of pediatrics department.
MATERIAL AND METHODS
This observational prospective study was conducted in pediatrics in-patient department of a tertiary care teaching hospital attached to a Medical College, in western India for 6 months duration.
Pilot study was done to validate the designed proforma and feasibility of method, following which it was finalized for further work. Institutional ethics committee’s approval was obtained and verbal consent was taken from patients’ guardians before enrolling them into the study. All patients below the age of 18 years of either sex, admitted at pediatrics inpatient ward for any condition were included in the study. The patients referred to or from other specialties with conditions which can influence physician’s prescription or the patients admitted to/ transferred from NICU and PICU were excluded. Data of total 100 pediatrics in-patients were collected and were recorded in case record form. Relevant data of patients were taken from hospital records while they were admitted in the hospital. An attempt was made to include patients of different conditions or diseases admitted in pediatrics inpatient ward as far as possible.
After collecting data of all prescriptions, data were analyzed for rationality and drug utilization pattern by following criteria.
Prescription analysis
All prescriptions were analyzed by using Phadke’s criteria.12, 13 While analyzing the prescriptions, to decide for the correctness of the drug, standard textbook of pediatrics (Essential Pediatrics; OP Ghai; 7th edition) and pharmacology (Principles of Pharmacology; HL Sharma and KK Sharma; 2nd edition) were referred. Prescriptions were discussed with two consultants suggested by the Head of pediatrics department for more clarification in case of some query regarding prescription. Prescription were analyzed for diagnosis mentioned or not, number of drugs prescribed per prescription, number of drugs prescribed by brand name or generic name, drug wise analysis of prescriptions, rationality score, rationality status of prescriptions and number of prescriptions showing use of unnecessary drugs, unnecessary injections, irrational drugs or combinations. For study of rationality status of prescriptions, a maximum of 30 points score system was assigned as follows:
? Main drug - 20 points
? Complementary drug – 10 points
Out of these total points, half of the points of each category of drugs were to be allotted for the correctness of the choice of drug according to condition and half for the correctness of the dose, route, frequency of drug administration and the duration of the treatment. If more than two drugs were needed to be given in a condition. The points allocated were subdivided accordingly. From total score obtained, points were deducted if prescription include Unnecessary drug (-5 points), Unnecessary injection (-5 points), Irrational drug / combination (-5 points) or Hazardous drug (-10 points). Based on the above mentioned criteria for analysis, net score was calculated and prescription were categorized as; 0 to 14 points –Irrational, 15 to 24 points – Semirational, 25 to 30 points – Rational .
B. Drug utilization pattern
By using the prescribing indicators according to the standard WHO guidelines, the data were analyzed to study number of drugs used by trade name and by generic names, average number of drugs per prescription, percentage of prescription with an antibiotic prescribed, number of fixed drug combinations used and number of drugs prescribed from essential medicine list [WHO- EML for children, April-2013].
RESULTS
Note: As our sample size N was 100, results are mentioned in numbers only where denominator is 100.
Out of 100 patients, 69 were male and 31 were female. Amongst males, most of the patients belonged to the age group of 1 to 4 years (46.38%) followed by 5 to 8 years (27.54%), while in females most of the patients belonged to 1 to 4 years (41.94%) followed by 9 to 12 years (22.58%) of age. Male: female ratio was 2.22:1.
Of total 100 prescriptions, 91 and 9 prescriptions were rational and semi-rational respectively. There was no prescription with score less than 15, i.e. there was no irrational prescription (Table 1, Figure 1). When rationality score was calculated, 60 prescriptions had 30 score (maximum) followed by 31 and 9 prescriptions in range of 29-25 score and 15-24 score respectively. Minimum score of 19 was observed in one prescription (Table 1). Mean rationality score was 28.4. For total 40 prescriptions with rationality score less than 30, different reasons for less score were - improper dose in 25 (62.5%) prescriptions, second or wrong choice of drugs in 4 (10%) prescriptions, unnecessary drug or injection in 4 (10%) prescriptions. Irrational drug was prescribed in 2 (5%) prescriptions only. In 5 (12.5%) prescriptions, there was more than one reason for getting less scoring (Table 2).
In 81 prescriptions, only provisional diagnosis was mentioned; while in 19 prescriptions, both provisional and final diagnosis were mentioned (Figure 2).
54.76% drugs were prescribed by generic name, 33.33% by brand name and 11.90% by both (Table 3). Drugs prescribed from WHO-EML for children (April-2013) were 48.81% (Table 4). In present study, number of drugs prescribed in any given patient ranged from 1 to 13. In about half of the patients ( i.e 48), 3-5 drugs were prescribed (Figure 3). Average number of drugs per patient was 6.01.
Out of 100 patients, antibacterial agent /agents was/were prescribed in 93, among which in majority of patients, one (42 patients ) or two (35 patients) antibacterials were prescribed. Maximum number of antibacterials prescribed was 5 in this study. The most frequently prescribed group of antibacterials were cephalosporins followed by penicillins while most common prescribing antibacterials were amoxicillin+ clavulanic acid, ceftriaxone and cefotaxime ( Table 5).
Drugs used for treatment of different conditions in pediatric cases were antibacterials (22.63%) vitamins and minerals (21.46%), NSAIDs (12.65%), antihistaminics (6.66%) and antiemetics (6.16%). Apart from these, β2 agonist, antiepileptics/anticonvulsants, corticosteroids, hematinics, H2 blockers, antitussive agents, diuretics, anticholinergics and proton pump inhibitors were also prescribed (Figure 4).
In present study, total 16 FDCs were prescribed out of which 6 FDCs were rational and 10 were irrational. Of 6 rational FDCs, 4 were present in WHO-EML for children, April-2013. 10 irrational FDCs included ibuprofen + paracetamol combination, hematinics and multivitamins.
DISCUSSION
Rational prescribing is an essential part of patient care. WHO has developed Essential Medicine List to promote rational prescribing. Irrational prescribing is common worldwide with different prevalence rate at different set up. This type of study helps in assessing the extent to which rational prescribing is practiced by clinician in government as well as private set up. Pharmacological management is the most common and important form of treatment in the care of pediatric patients and irrational prescribing may lead to drug-drug interactions, development of resistance, adverse effects of drugs etc.
When we looked at the rationality of the prescriptions, we found 91 prescriptions as rational and 9 semirational. Rima Shah et al. (2011) in their study found 39.5%, 32.3%, and 28.3% rational, semirational and irrational prescriptions respectively while Shah AM et al. (2010) in their study found 53%, 30% and 17% prescriptions to be rational, semirational and irrational respectively.13, 14 Sneha Patel and Bharat Gajjar (2012) in their study found more rational prescriptions from public sector (82) compared to private sector (42). 6 Out of 100 prescriptions, 60 prescriptions had 30 score (maximum) followed by 31 and 9 prescriptions in range of 29-25 score and 15-24 score respectively. Minimum score of 19 was observed in one prescription. Mean rationality score was 28.4. In another study done by Shah AM et al. (2010), in which prescriptions of 100 OPD patients of tertiary care teaching hospital were analyzed, the mean rationality score found was 20.56. 14 In a similar study for geriatric patients (age≥ 65 years) carried out by Rima Shah et al. (2011) at tertiary care teaching hospital, the mean rationality score was 18.47.13 Sneha Patel and Bharat Gajjar (2012) had done a study of prescription audit collecting prescriptions from general practitioners from public and private sectors, using same Phadke’s criteria. In their results, they found that the mean rationality score was 25.83 for public sector and 20.45 for private sector.6 Gajjar BM (1999) in his work reported average rationality score of 19.23 and 20.83 for prescription obtained from physician of teaching institute and private sector respectively. 12
Differences found in rationality score and rationality status of prescriptions in different studies could be because of following reasons, operating one or more at a time.
Some studies were done in tertiary care teaching hospital while some studies were done both in public and private hospitals. In one study, prescriptions from private sector and primary health care centre were compared.
In these different studies, patients included were either from inpatient department or outpatient department, or inpatient as well as outpatient departments.
Prescribing doctors were either MBBS or MD/MS in different studies.
For some conditions, standard treatment guidelines are available making prescription analysis easy. For majority of conditions, standard treatment guidelines are not available and researchers have to refer some standard reference for deciding rationality of treatment. Referring different sources may lead to variations in rationality score.
In present study, total 40 prescriptions obtained score less than 30 for which major reasons were improper dose in 25(62.5%) prescriptions, second or wrong choice of drugs in 4 (10%) prescriptions, unnecessary drug or injection in 4 (10%) prescriptions. In a study done by Phadke et al. (1995), there were 47.4%, 23.8%, 10.5% and 19% prescriptions containing unnecessary drugs, unnecessary injections, hazardous drugs and irrational drug respectively.6
In present study, in 81 prescriptions only provisional diagnosis was mentioned; while in 19 prescriptions, both provisional and final diagnosis were mentioned. Sneha Patel and Bharat Gajjar (2012), found 81% (public sector) and 84% (private sector) prescriptions in which diagnosis was mentioned while Gajjar BM (1999) found 60% (teaching institute) and 89% (private sector) prescriptions with diagnosis mentioned.6, 12
Aggressive promotional strategies by pharmaceutical companies may lead to prescribing by brand name may lead to increased cost of therapy. Prescribing drugs by their generic name, prescribing from essential medicine list and rational prescribing are recommended measures which reduce the cost of drugs in patients and to health care system in government setups. In present study, total 84 different drugs were prescribed. 54.76% drugs were prescribed by generic name while 33.33% drugs were prescribed by brand name. There were 11.91% drugs which were prescribed by generic names in some patients and by brand names in others. When we compared results of present study with that of other studies, it was found that drugs prescribed by generic name were as high as 62.3% [Vishwanath et al. (2014)] and 67.25% [Manoj Kumar Saurabh et al. (2010)] as compared to present study while Rajesh et al. (2014) in their study, found only 15% of drugs prescribed by generic name which is low as compared to present study.15, 16, 17 In a study by Akhtar et al. (2012), percentage of drugs prescribed by generic name was only 2.63%.18 All these results suggest that there is a need to promote drug prescribing by generic name.
Out of total 84 different drugs prescribed in 100 patients in present study, 48.81% drugs were prescribed from the WHO List of Essential Medicines for children, April-2013. When results of present study were compared with other studies, percentage of drugs prescribed from WHO Essential Medicine list in other studies were 90.23% [Akhtar et al. (2012)], 86.42% [Vishwanath et al. (2014)] and 35.19% [Girija Sachdeo et al. (2013)]. 18, 15, 19
Prescribing minimum required number of drugs per patient carries less chances of drug - drug interactions and adverse effects of drugs, decreased cost of therapy and increased patient‘s compliance. In present study, number of drugs prescribed in any patient ranged from 1 to 13 with an average of 6.01% drugs per patient. In other studies, average number of drugs per encounter were 5.69 (inpatients) [Vishwanath et al. (2014)], 5.61 (outpatients + inpatients) [Akhtar et al. (2012)], 2.7 (outpatients + inpatients) [Rajesh et al. (2014)], 2.35 (outpatients) [Girija Sachdeo et al. (2013)].15, 18, 17, 19 Manoj Kumar Saurabh et al. (2010), in their study found that average number of drugs prescribed was 2.79 for government doctors and 3.12 for private practitioners.16 In the similar study, done by Rewa Shinde et al. (2013), average number of drugs per prescription was 2.11 and 2.22 for tertiary care teaching hospital and private hospitals respectively. 20
Improper use of antibacterials - overuse or not using when required - is one of the important reasons of irrational prescribing and development of antimicrobial resistance. In 93 patients in whom antibacterial was/were prescribed, in majority of them, one (42 patients) or two (35 patients) antibacterials were prescribed. Maximum number of antibacterials prescribed was 5 in single patient. Akhtar et al. (2014) in their study found use of anti-infectives in 81.12% patients while Rajesh et al. (2014) found use of antibiotics in 32% patients.18, 17 In a study done by N. Venkateswaramurthy et al. (2013), it was found that antibiotics were prescribed in 218 out of 286 prescriptions. Of this, 124 (43.4%) had a single antibiotic, while 70 (24.5%), 21 (7.3%) and 3(1%) had 2, 3 and 4 antibiotics per prescription respectively. 21
In this study, total 136 antibacterial agents were prescribed. The most frequently prescribed antibacterials were Cephalosporins followed by penicillins. Among cephalosporins, only third generation cephalosporins were prescribed which included ceftriaxone (26.47%) in majority of instances; while among penicillins, most frequently prescribed was amoxicillin+clavulanic acid (30.15%). Other antibacterials prescribed were amikacin, ofloxacin, sulfamethoxazole + trimethoprim, vancomycin, clindamycin, linezolid, meropenam and erythromycin. In the study by Vishwanath M. et al. (2014), among antimicrobials, penicillins (28.75%) were most commonly prescribed, followed by aminoglycosides (23.33%) and cephalosporins (17.5%).15 N. Venkateswaramurthy et al. (2013) in their study found that among antimicrobials, most commonly prescribed were beta-lactams followed by quinolones and aminoglycosides, while Rajesh et al. (2014) found that cefixime was most commonly prescribed antibiotic followed by ceftriaxone and amoxicillin.21, 17
Total drugs prescribed in the present study were 601. Drugs used for treatment of different conditions in pediatric patients were antibacterials (22.63%) vitamins and minerals (21.46%), NSAIDs (12.65%), antihistaminics (6.66%) and antiemetics (6.16%). Apart from these, β2 agonist, antiepileptics/anticonvulsants, corticosteroids, hematinics, H2 blockers, antitussive agents, diuretics, anticholinergics and proton pump inhibitors were also prescribed. Akhtar et al. (2011) in their study found that most commonly prescribed pharmacological group was antipyretics (100%) followed by cold and cough preparations (88.81%) and anti-infectives (81.12%).18 In the study by Vishwanath et al. (2014), it was found that most commonly prescribed pharmacological group was antimicrobial (28.10%) followed by drugs acting on respiratory system (12.18%) and NSAIDs (7.50%).15 In the study by Rewa Shinde et al. (2013), most commonly prescribed drug groups were antimicrobials (37.81% and 37.99%) followed by vitamins/minerals (22.74% and 18.47%) and analgesics (13.46% and 11.87%) in tertiary care teaching hospital and private hospitals respectively, while in similar Study by Manoj Kumar Saurabh et al. (2010) most commonly prescribed pharmacological groups were antimicrobials (25.44% and 25.96%) followed by NSAIDs (19.80% and 21.66%) in prescriptions of government doctors and private practitioners respectively.20, 16 N. Venkateswaramurthy et al. (2013) found that most commonly prescribed group was anti-infectives (24.8%) followed by anti inflammatory (20.6%) and drugs of gastro intestinal system (14.7%).21
In present study, total 16 FDCs were prescribed out of which 6 FDCs were rational and 10 were irrational. Of 6 rational FDCs, 4 were present in WHO-EML for children, April-2013. 10 irrational FDCs included Ibuprofen + Paracetamol combination, hematinics and multivitamins. In the study by Girija Sachdeo et al. (2013), 43 (39.81%) FDCs were prescribed in which 12 (27.91%) were rational and 9 (20.93%) were from WHO-EML for children [March-2011]. 19 In the study by Rewa shinde et al. (2013), total 139 and 97 FDCs were prescribed in public and private sector respectively, out of which 55 and 35 were rational and 45 and 23 were present in WHO-EML for children [March-2011] for public and private sector respectively. 20
CONCLUSION
This type of study helps to evaluate, monitor and if necessary, suggest changes or modifications in prescribing practices of clinicians which will ultimately make patient care more rational and cost- effective.
Though the results reflect rational prescribing in pediatrics department of our hospital set up, there is still scope of improvement in areas of dosage calculation, proper documentation, prescribing drugs by generic name and from WHO-EML for children as far as possible.
Development and implementation of Standard Treatment Guidelines based on essential drug concept and promoting rational drug therapy will lead to more and more rational prescribing. Periodic prescriptions analysis and effective feedback to clinician should be done based on results to ensure rational prescribing and effective health care management.
LIMITATIONS OF THE STUDY
Limitations of this study were small sample size, non randomized selection of patients, small duration of study and limitations related to Phadke’s criteria.
ACKNOWLEDGMENT
Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors/ editors/ publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed. Authors would like to acknowledge all the faculty of pediatrics department for their valuable guidance, help and support in the present study.
FUNDING AND SUPPORT: None.
CONFLICT OF INTEREST: None.
Englishhttp://ijcrr.com/abstract.php?article_id=150http://ijcrr.com/article_html.php?did=150
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