Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241811EnglishN2016June11HealthcareRELATIONSHIP BETWEEN PERSONALITY DISORDERS AND MIGRAINE HEADACHE IN REFERRAL PATIENTS TO ARDABIL NEUROLOGY CLINIC, 2013-14
English0104Ghasem Fattahzadeh-ArdalaniEnglish Hamidreza AzizzadehEnglishBackground: Some problems related to migraine as one of the most common causes of headache is caused by psychiatric disorders associated with it. Understanding relation between personality disorders with migraine headache can be effective in the diagnosis and treatment of migraine.
Aim: The aim of this study was to investigate the association between personality characteristics and migraine incidence in neurology clinic in Ardabil during 2013-14.
Methods: In this descriptive-analytical study, 50 migraine patients were selected randomly. In order to determine personality disorders, MCMI-3 questionnaire was used. Collected data analyzed using statistical methods in SPSS.16.
Results: Of all patients,62% were female and 38% were male with the mean age of 37.02 years old (range 18-65). The personality disorders that observed among patients by the highest frequency order were histrionic personality disorder (70%) and obsessive (40%). The prevalence of depressive personality disorder among women (P = 0.020) and the prevalence of narcissistic personality disorder higher among men (P = 0.049) were significantly higher than others.
Conclusion: Results showed that people with migraines have certain personality characteristics such as anxiety, irritation, depression, anxiety, and obsession that these characteristics leads into internal frustration and cause to headache and finally lead to migraine headache.
EnglishPersonality disorders, Headache, MigraineINTRODUCTION
Migraine is one of the most common causes of headache which implicated about 15 percent of women and 6 percent of men [1]. A Migraine is an attacking headache which its occurrence not certainly clear, but it seems that patients with migraine have abnormalities in sensory information processing whose centers are in the brainstem [2-3]. To find out the underlying dysfunction in migraine, one of the methods is to assess the associated diseases. Previous studies suggest that migraine is associated with psychiatric diseases [4-10]. Investigation of the relationship between migraine and psychiatric disorders is useful in the treatment of people with migraine and select the best treatment. Many studies have been done on the effects of biological, environmental, psychological and social factors on migraine attacks among the patients. Psychological problems are prevalent among patients with migraine and most of them with chronic migraine headaches are depressed [11]. Lipton and et al in a study showed a clear and meaningful correlation between introvert personality traits, depression and hypochondriasis with chronic migraine headache. Although it is not clear yet whether psychological factors are the causes of headache or not [12]. Wilson and et al in a study showed that the prevalence of psychological symptoms among individuals with migraine is 2.5 times more than other people [13]. According to Kaplan and Sadouk idea, two-thirds of patients with migraine have obsessive-compulsive personality traits and are idealistic and aggressive and they may be suffer from headache after emotional stresses [14]. Several studies have shown stress and depression can cause starting and repeating of migraine attacks [4,15]. Given the high prevalence of migraine and psychiatric disorders and the importance of them, as well as the lack of adequate research on the prevalence of them in Iran, there is a need to do more research in this area. The aim of this study was to investigate the relationship between personality disorders and migraine headache in patients referred to neurologic clinic of Ardabil city in 2014.
MATERIALS AND METHODS
In this descriptive-analytical study, 50 patients with migraine were selected randomly from the patients referring to neurology clinic of Alavi Hospital in Ardabil city. In order to determine personality disorders in these patients, MCMI-3 personality questionnaire was used. The collected data were analyzed using statistical methods in SPSS version 16 software.
RESULTS
Of the total patients, 19 patients (38%) were male and 31 (62%) were female. The mean age of the subjects in the present study was 37.2± 13.2 (range 18-65). Most of patients were in the age group 30 to 39 years old (28%).The most observed disorder in all patients with migraine was histrionic personality disorder(35.7%) which was the most frequency in both sexes, but the order of other personality disorders was different between two sexes. The prevalence of depression among women was significantly more than men, and the prevalence of narcissistic personality disorder among men was significantly more than women. (Table 1)
DISCUSSION
In this study, the prevalence of migraine among women with 62% was higher than men (38%). According to the study of Tazikiand et al, [16] and Firoozabadi and et al, [17], the prevalence of migraine in women was more than men. The mean age of the subjects in the present study was 37.2 ± 13.2 and the patients were in the age range of 18 to 65 years old. This finding was consistent with the findings of other studies [16-17]. Three personality disorders, which have the highest frequency among migraine patients in the present study, were as follows: histrionic personality disorder (70%), obsessive-compulsive personality disorder (40%) and depressed personality disorder (36%). Patients with histrionic personality disorder need a lot of attention and praise, and they behave in an exhibitive and community friendly manner to have their peace of mind supplied. They behave in a flattering and very obediently manner. They are pretentious, pathetic and probably in search of stimulation, excitement, and attention. They simply react to stimuli around them and often highly motivated, but motivation does not last long and the pattern of arousal and relaxation are constantly repeated. These people, in spite of their values and beliefs, in order to protect their mental security, avoid conflicts and disagreements with others [18]. In terms of behavior, patients with OCD are serious, consistent, conscientious, polite, regular, accurate, punctual, often perfectionist, uptight, conservative, strongly agree and submit to the law, honest, self-made and hardworking. They have much control over their behaviors and actions. In terms of emotional, they are prevented. They suppress their anger and hate and control their emotions and they rarely present their feelings and emotions. They are socially compatible and consistent with the group and, as a result, they have commitment to a set of behaviors and rigid rules and repetitive and monotonous way of life. They fear from social disapproval and they are models of courtesy and restraint in their communities. To avoid criticism, they try not to make mistakes. Their behaviors stem from a conflict between the feeling of hostility, which they want to present, and the fear they have from social disapproval [18].Depressed patients are often grim, stern, pessimist, serious, quiet and passive and are easily distracted with negative events. They often feel bad and have a low self-esteem. They tend to have undue inconvenience and worry. However, they are usually responsible and conscientious and they are self-criticize because of the smallest errors. They are often depressed and they do not seem happy even in the most pleasant experiences. They feel that their efforts to improve their relations or any other important aspect of their lives are useless. This is due to the fact that persistent pessimism leads them to have a failure perspective.
Their soulless behavior often makes others away from them. These patients constantly feel guilty because they are very dependent on others to gain the support and acceptance. They also have difficulty in expressing their anger and often swallow their anger. These people, inspite of depressed mood and negative thoughts, do not consider themselves depressed. This personality style may occur even in the absence of clinical depression. Cold treatment and soulless behavior in combination with passivity and self-doubt may put them in occupational and marital problems. Also, if they are under stress due to the lack of loved ones, they will probably suffer from dysthymic [18].In the study conducted by Wolf and et al, it was noted that migraine patients are rigid, obsessive, idealistic and ambitious and aggressive[19]. Turain et al. study showed that migraine patients are generally uncertain, hesitant, idealistic, detail-oriented and are sensitive to criticism. According to the report, these people don’t have close and deep communications [20]. In another study [21] that has been done by the MMPI test on 80 patients with migraine showed that migraine patients had higher scores on scales of paranoia and introversion compared with the control group. Comparing the two groups of men and women participating in the study, it was found that the prevalence of depressive personality disorder among women was significantly higher and the prevalence of narcissistic personality disorder was significantly higher among men. The results of a study conducted by Rezaiand et al, showed that disruption of social relationships and physical, hysteria and obsessive-compulsive occur more in women with migraine headaches and depression and anxiety are more obvious in men with migraine headaches [22].In a study conducted by Karakoram on the personality characteristics of 35 patients with acute migraine and 50 patients with chronic migraine, the results showed that depression and anxiety may be a risk factor in the onset of migraine and depression, hysteria and hypochondriasis in patients with chronic migraine are more specific and more obvious than in acute migraine[23]. According to the studies of Kaplan and Sadouk, two-thirds of migraine patients have aggressive, oppressive and obsessive-compulsive personality traits and their headaches are aggravated by worry and anxiety [14]. The study conducted in 1990 on 80 patients with acute and chronic migraine showed that the most common psychiatric disorders observed in patients with migraine were anxiety and depression[24]. In a study conducted by Martin and et al on 652 students with migraine headaches showed a clear relationship between migraine and features of OCD, anxiety, depression and anxiety[25]. The results of a study on 70 patients with tension and migraine headaches revealed that the scales of violence and aggression in patients with migraine without aura were significantly higher than normal patients. In this study, the scales of anxiety and depression in patients with tension and migraine headaches with aura were more than the control group[26].
CONCLUSION
The results showed that histrionic, obsessive-compulsive and depression personality traits in patients with migraine headaches were more common than other patients. Given that migraine headache is a multifactorial disease, undoubtedly hereditary, psychological and social factors play roles in its occurrence and intensity. Thus, it is suggested for the researchers to evaluate the effect of cognitive-behavioral interventions in patients with migraine. Also, due to the limited population of this study to the migraine patients in Ardabil city and more generalize the results, future studies with larger sample sizes and control groups in different parts of the country are recommended.
Conflict of Interest: none-declared
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.
Englishhttp://ijcrr.com/abstract.php?article_id=243http://ijcrr.com/article_html.php?did=2431. Bresleu N. Headache and major depression. Neurology 2000; 45:308-12.
2. Gisela T. Tietjen E. Migraine, Depression may have genetic link, neurology 2010;74:288-294
3. Rezaei A, Shamsaei F, Rezaei N. Personality characteristic in patient with migraine headache. Pak J Med Sci 2006; 22(4):480- 2.
4. Goldstein J, Silberstein SD, Saper JR, Ryan RE Jr, Lipton RB. Acetaminophen, aspirin, and caffeine in combination versus ibuprofen for acute migraine: results from a multicenter, doubleblind, randomized, parallel-group, single-dose, placebo-controlled study. Headache. 2006;46(3):444-453.
5. Goadsby PJ, Zanchin G, Geraud G, et al. Early vs. non-early intervention in acute migraine-‘Act when Mild (AwM)’. A double-blind, placebo-controlled trial of almotriptan [published correction appears in Cephalalgia. 2008;28(6):679]. Cephalalgia. 2008;28(4):383-391.
6. Mathew NT, Landy S, Stark S, et al. Fixed-dose sumatriptan and naproxen in poor responders to triptans with a short half-life. Headache. 2009;49(7):971-982.
7. Singh A, Alter HJ, Zaia B. Does the addition of dexamethasone to standard therapy for acute migraine headache decrease the incidence of recurrent headache for patients treated in the emergency department? A meta-analysis and systematic review of the literature [published correction appears in AcadEmerg Med. 2009;16(5):435]. AcadEmerg Med. 2008;15(12):1223-1233.
8. Bigal ME, Serrano D, Buse D, Scher A, Stewart WF, Lipton RB. Acute migraine medications and evolution from episodic to chronic migraine: a longitudinal population-based study. Headache. 2008;48(8):1157-1168.
9. Lipton RB, Dodick DW, Silberstein SD, et al. Single-pulse transcranial magnetic stimulation for acute treatment of migraine with aura: a randomised, double-blind, parallel-group, shamcontrolled trial. Lancet Neurol. 2010;9(4):373-380.
10. Pringsheim T, Davenport WJ, Dodick D. Acute treatment and prevention of menstrually related migraine headache: evidencebased review. Neurology. 2008;70(17):1555-1563.
11. Huber D. Henrich G. Personality traits and stress sensitivity. Behave Med 2003; 29(1):4-13.
12. Lipton RB, Stewart WF, Diamond S, Diamond ML, Reed M. Prevalence and burden of migraine in the United States: data from the American Migraine Study II. Headache. 2001;41(7):646- 657.
13. Wilson JF. In the clinic. Migraine [published correction appears in Ann Intern Med. 2008;148(5):408]. Ann Intern Med. 2007;147(9):ITC11-1–ITC11-16.
14. Kaplan HI, Sadock BJ, Grebb JA. Kaplan and Sadock’s synopsis of psychiatry. 7th Ed. Baltimore: Williams and Wilkins; 1994. P. 516-712.
15. Locker TE, Thompson C, Rylance J, Mason SM. The utility of clinical features in patients presenting with nontraumatic headache: an investigation of adult patients attending an emergency department. Headache. 2006;46(6):954-961.
16. Taziki SA , Fathi D , Ramezannezhad A , Behnampour N , Salari H, Personality characteristics in migraine and tension headache in Gorgan, Iran (2007-08), Journal of Gorgan University of Medical Sciences, 2013 14(4): 65-69.
17. Firoozabadi M., Manshaee G., Danae Z., Sharifzadeh G., Effectiveness of cognitive behavioral stress management on depression and anxiety symptoms of patients with epilepsy and migraine, Journal of Birjand University of Medical Sciences 2015; 21 (4) : 407-415.
18. Ali G, Azadeh S, Mehrdokht M, Mehrdokht S R, Mohammad Z. Correlation between migraine headaches and obsessive-compulsive Disorder: a two year study. Tehran Univ Med J. 2012; 69 (12) :781-786.
19. Wolf HG. Personality features and reactions of subjects with migraine. Arch Neurol- psychiatr 1937; 37:895-921.
20. Tourine GA, Draper G. The migrainous patients: a constitutional study. J NervMent Dis 1934; 80: 183-204.
21. Arthur YY. MMPI manifestation of Chinese migraine syndromes: a control study. Am J Chinese Med 1999; 27(1):37-42.
22. Rezaei A, Shamsaei F, Rezaei N. Personality characteristic in patient with migraine headache. Pak J Med Sci 2006; 22(4):480- 2.
23. Kara Kurum B. Personality, depression and anxiety as risk factor for chronic migraine. Int J NeuroSci 2004; 114(11): 1391-9.
24. Brandth JD, Celentano S. Personality and emotional disorder in a community sample of migraine headache sufferers. Am J Psychiatr 1990; 147:303-8.
25. Martin Hertz SP, Smith MS, McMahan RJ. Psychosocial factors associated with headache in Junior high school students. J PediatrPsychol 1999; 24: 13-23.
26. Ming Cao, Shiyang Wang, Yehan Wang. Personality traits in migraine and tension- type headache: a five factor model study. Psychopathology 2002; 35(4): 254-8.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241811EnglishN2016June11HealthcareGENE XPERT BASED ONE YEAR ANALYSIS OF PULMONARY KOCH’S FROM NORTH MAHARASHTRA REGION
English0509Mrudula DravidEnglish Sukhada BuwaEnglish Shubhangi DangeEnglish Hitesh AdchitreEnglishIntroduction: Tuberculosis is a major public health problem in the world, especially in the developing countries like India. Also MDR-TB and HIV: TB co-infection are major hurdles to achieve the aim and objectives of our national tuberculosis programme.
Objectives:
1. To diagnose Mycobacterium tuberculosis (MTB) infection in clinically suspected cases of pulmonary tuberculosis using Gene Xpert.
2. To find out HIV: TB co-infection rate.
3. To find out prevalence of Rifampicin sensitive and resistant cases in diagnosed tuberculosis patients.
4. To study factors responsible for Rifampicin resistance (MDR-TB)
Material and Methods: This retrospective study included 278 sputum samples from Jan 2015-Dec 2015 from registered RNTCP patients. The samples were subjected to Xpert MTB/RIF Assay for use with the Cepheid Gene Xpert. The Ziehl - Neelsen smear finding was provided by the RNTCP –DOTS regional centres.
Results: A total number of 278 sputum samples were subjected to Gene Xpert analysis in the year 2015. MTB could be detected in 137 (49.28%) cases. In the rest 141 cases where MTB was not detected 14 samples reported error on Gene Xpert. Out of 278 cases, 209 (75.18%) were HIV negative, and 69 (24.82%) were HIV positive. In 69 clinically suspected HIV: TB co-infection cases, MTB could be detected in 22 (31.88%) cases. Out of these 22 cases, 4 (18.18%) were smear positive. Out of 137 MTB detected samples, 117 (85.4%) were rifampicin sensitive, 2 (1.46%) were rifampicin indeterminate resistant and 18 (13.14%) were rifampicin resistant. According to RNTCP programme criteria for suspected MDR –TB, out of 18 MDR-TB cases, 12 (66.67%) cases were - smear positive at diagnosis , retreatment case; 3 (16.67%) cases were - any follow up smear positive, 2 (11.11%) - had contact of known MDR -TB case and 1(5.56%) - was HIV: TB case.
Conclusion: Gene Xpert was a useful tool in detection of HIV-TB co-infection. In our study out of 69 clinically suspects HIV: TB co-infection cases, one third cases were confirmed by Gene Xpert. Thus, 44 patients were not put on unnecessary AKT and were kept on follow-up. Even though the association of MDR-TB and HIV co-infection was not very significant in this study, it would not be too long before witnessing a rapid increase of MDR-TB among HIV patients if adequate and immediate measures are not taken. In the present study, MDR-TB detection rate was high among re-treatment cases. Emphasis has to be given for completion of primary treatment on time and taking proper nutrition. Patients usually stop treatment once they feel better within 2 month of starting the treatment. Special counselling and education is needed at this juncture. In our study, two cases of primary MDR-TB were from household contact. Hence, health workers must generate awareness and educate patients and family members about the risk of acquiring Primary MDR-TB to prevent its spread.
EnglishGene Xpert, Pulmonary tuberculosis, MDR-TB, HIV: TB co-infectionINTRODUCTION
India has the world’s maximum burden of TB (approximately 3.4 million cases), which accounts for one fifth of global incident cases, and ranks first among the 22 TB high-burden countries.1 Drug-resistant TB (DR-TB) is commonly seen in India; although its existence has been recognized since anti-tuberculosis drugs were first introduced for the cure of TB, adequate information regarding the prevalence of drug-resistant tuberculosis (TB) has not been reported from India.2 According to WHO Reports on global TB control, Multidrug-resistant tuberculosis (MDR-TB) in India has been reported between 2.5–2.8% amongst new TB cases and 14–17% in retreated patients.3-7 MDR-TB is a growing worldwide threat, most cases arising from a combination of physician error and patient non-compliance during treatment of susceptible TB. The amount of burden of MDR-TB varies significantly from nation to nation and region to region.8 The threat of development of active tuberculosis is high in HIV infected patients. Although antiretroviral therapy for HIV reduces this risk, TB still remains 5 times more common in HIV/AIDS persons.9 The present study was undertaken with following primary objectives: 1. To diagnose Mycobacterium tuberculosis infection in clinically suspected cases of tuberculosis using Gene Xpert 2. To find out HIV: TB co-infection rate. 3. To find out prevalence of Rifampicin sensitive and resistant cases in diagnosed tuberculosis patients. 4. To study factors responsible for Rifampicin resistance(MDR-TB)
MATERIALS AND METHODS
Study Design: Retrospective study Study period: From Jan 2015 to Dec 2015 The study was approved by Institutional Ethical Committee of SBHGMC, Dhule. Clinical data was obtained from the RNTCP filled requisition form sent along with the samples. Collection of specimen: Sputum samples from registered RNTCP patients were subjected to Xpert MTB/RIF Assay for use with the Cepheid Gene Xpert system, a semi-quantitative, nested real-time PCR in-vitro diagnostic test. After proper instruction to the patient, two sputum samples were collected per patient. The Ziehl - Neelsen smear finding was provided by the RNTCP-DOTS regional centres. The specimen were stored and transported at 2 to 8°C prior to processing whenever needed. In the laboratory, specimens were examined for the presence of obvious food particles or other solid particulates and were rejected. Processing of the samples:10 Each Xpert MTB/RIF cartridge was labelled with the sample ID. The lid of the sputum collection container was opened carefully and approximately 2 times the volume of the sample reagent (SR) to the sputum (2:1 dilution; SR: sputum) was poured in the container. The lid was replaced and shaked vigorously 10 to 20 times. The samples was incubated for 10 minutes at room temperature, and then shaked vigorously 10 to 20 times. The sample was incubated at room temperature for an additional 5 minutes. The test was started within 4 hours of adding the sample to the cartridge. The cartridge lid was opened and using the provided transfer pipette, the liquefied sample was aspirated to the line on the pipette. Care was taken so that no air bubble enters the pipette. The sample was transferred into the sample chamber of the Xpert MTB/ RIF cartridge and was dispensed slowly to minimize the risk of aerosol formation. The cartridge lid was closed firmly and loaded as per the instruction given by the manufacturer.
RESULTS
A total number of 278 sputum samples were subjected to Gene Xpert analysis in the year 2015. MTB could be detected in 137 (49.28%) cases, while in 141(50.72%) cases MTB could not be detected. In the rest 141 cases where MTB was not detected 14 samples reported error on Gene Xpert. Out of 278 cases, 209 (75.18%) were HIV negative, and 69 (24.82%) were HIV positive. In 69 clinically suspected HIV: TB co-infection cases, MTB could be detected only in 22 (31.88%). Out of these 22 HIV: TB cases, only 4 (18.18%) were smear positive (Table 1). Out of 137 MTB detected samples, 117 (85.4%) were rifampicin sensitive, 2 (1.46%) were rifampicin indeterminate resistant and 18 (13.14%) were rifampicin resistant (Figure 1). According to RNTCP programme criteria for suspected MDR –TB, out of 18 MDR-TB cases, 12 (66.67%) cases were - smear positive at diagnosis , retreatment case; 3 (16.67%) cases were - any follow up smear positive, 2 (11.11%) - had contact of known MDR -TB case and 1(5.56%) - was HIV: TB case. Out of the 22 HIV: TB co-infection cases, MDRTB was detected only in 1 case.
DISCUSSION
The WHO South-East Asia Region (SEAR), having almost one fourth of the world’s inhabitants, accounts for 38% morbidity and 39% mortality of the global burden of tuberculosis, with an approximate 4.5 million prevalent and 3.4 million incident cases and 440,000 deaths in 2013. India having a population of about 1252 million is the largest country in the Region, and is ranked first among the high-burden countries having contributed approximately 24% of the global incident TB cases and about 20% of global TB-related deaths in 2013. In 2013, the prevalence and incidence rates of all forms of tuberculosis were 211 and 171 respectively per 100,000 population.11 Thus, India is the highest TB burden nation accounting for one fifth of the worldwide incidence.12 In the present study, a total number of 278 sputum samples were subjected to Gene Xpert analysis in the year 2015. All these samples were from RNTCP programme of Dhule district. MTB could be detected in 137 (49.28%) cases, while MTB was not detected in 141 (50.72%) cases. While HIV/AIDS and tuberculosis can independently be the foremost causes for apprehension as stand-alone public health threats, the blend of the two has proven to have a far greater impact on the epidemiologic progression and subsequently on the impact it has on the global health prospect. The twin infection has been termed “accursed duet”.13 Research shows that of the opportunistic infections distressing HIV-infected patients, TB is found to be the most frequent with soaring risk for mortality.14,15 Worldwide, about 14.8% of patients with TB are co-infected with HIV.16 There is extensive inconsistency in HIV seropositivity amongst TB patients in India, ranging from 9.4% in New Delhi to 30% in Mumbai.17 Out of 278 cases, 209 (75.18%) were HIV negative, and 69 (24.82%) were HIV positive. Gene Xpert was very helpful in diagnosis of HIV: TB coinfection cases as HIV positive TB cases are more likely to be smear negative. In 69 clinically suspected HIV: TB co-infection cases, MTB could be detected in 22 (31.88%) cases. Out of these 22 cases, only 4 (18.18%) were smear positive. Sethi et al18 has reported the prevalence of HIV-TB as 20.1%, while Ghiya et al 19and Noeske et al 20 have reported the prevalence of HIV:TB as 49.2% and 32% respectively. MDR–TB is a most important challenge for TB control globally and is also linked with increased mortality and development of XDR-TB. Surveillance studies for the estimation of resistance rates and detection of MDR-TB are thus vital so as to optimize empiric drug treatment and to avoid the spreading of resistant strains in the community.21 Out of 137 MTB detected samples, 117 were rifampicin sensitive, 2 were rifampicin indeterminate resistant and 18 were rifampicin resistant i.e. rifampicin was sensitive in 85.4% of our diagnosed cases of tuberculosis and was resistant in 13.14% cases. Worldwide, 5% of TB cases were estimated to have had MDR-TB in 2014.22 While rates of MDR-TB infections are comparatively low in North America and Western Europe; they are progressively new grave dilemma worldwide, especially in areas of the Russian Federation, the former Soviet Union and other parts of Asia.23 In 2008, after China (100,000 cases), India had second highest total number of estimated MDR TB cases (99,000).24 According to WHO Reports on global TB control, MDR-TB in India continues to be reported between 2.5–2.8% and 14– 17% amongst new TB and retreated patients respectively.3-7 According to RNTCP programme criteria for suspected MDR-TB, out of 18 MDR-TB cases, 12 (66.67%) cases were - smear positive at diagnosis , retreatment case; 3 (16.67%) cases were - any follow up smear positive, 2 (11.11%) - had contact of known MDR -TB case and 1(5.56%) - was HIV: TB case. HIV may not constantly be a risk factor of MDR-TB, depending on the people investigated. In the present study, out of 22 HIV: TB confection patients only 1 case was MDR-TB. The hastening and magnifying sway of HIV infection and postponement in identification and diagnosis of tuberculosis were found to add to the outbreaks of MDR-TB among HIV infected patients in USA25 and European countries.26 However, a Thailand study 27 showed that the percentage of MDR-TB among HIV seropositives was the same as in the HIV negative group. Among the high risk groups of MDR-TB, failure of retreatment regimen and contact of MDR-TB may be the highest prospect of MDR-TB and warrant special attention. Rapid testing of resistance to rifampicin might be essentially helpful in the confirmation of MDR-TB.28
CONCLUSION
Gene expert was a useful tool in detection of HIV: TB coinfection as HIV positive TB cases are more likely to be smear negative, thus early diagnosis of TB in HIV: TB patients helps in the management of these patients, reducing the morbidity and mortality. In our study out of 69 clinically suspects HIV:TB co-infection cases, one third cases were confirmed by Gene Xpert. Thus, 44 patients were not put on unnecessary AKT and were kept on follow-up. Even though the association of MDR-TB and HIV co-infection was not very significant in this study, it would not be too long before witnessing a rapid increase of MDR-TB among HIV patients if adequate and immediate measures are not taken. In the present study, MDR-TB detection rate was high among retreatment cases. Emphasis has to be given for completion of primary treatment on time and taking proper nutrition. Patients usually stop treatment once they feel better within 2 month of starting the treatment. Special counselling and education is needed at this juncture. In our study, two cases of primary MDR-TB were from household contact. Hence, health workers must generate awareness and educate patient and family members about the risk of acquiring Primary MDR-TB to prevent its spread.
Abbreviations:
MDR-TB: Multidrug resistance tuberculosis
MTB: Mycobacterium tuberculosis
TB: Tuberculosis
RNTCP: Revised National Tuberculosis Programme
DOTS: Directly Observed Therapy, Short-Course
AKT: Anti Koch’s treatment XDR-
TB: Extensively drug resistant tuberculosis
WHO: World Health Organisation HIV: Human Immunodeficiency Virus AIDS: Acquired Immunodeficiency Syndrome
ACKNOWLEDGEMENT
We are thankful to Mr. Gopal Chaure for his technical support. We also extend thanks to RNTCP staff of Dhule district. Authors also 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.
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19. Ghiya R, Naik E, Casanas B, Izurieta R, Marfatia Y. Clinico-epidemiological profile of HIV/TB coinfected patients in Vadodara, Gujarat. Indian J Sex Transm Dis . 2009 Jan;30(1):10–5.
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21. Magana-Arachchi DN. Tuberculosis - Current Issues in Diagnosis and Management [Internet]. Mahboub B, editor. InTech; 2013 [cited 2016 Mar 30]. Available from: http://www. intechopen.com/books/tuberculosis-current-issues-in-diagnosis-and-management/epidemiology-of-multidrug-resistant-tuberculosis-mdr-tb22. WHO 2015. World Health Organization: MDR-TB 2015 Factsheet Source. Available from: http:// www.who.int/tb/challenges/mdr/mdr_tb_factsheet.pdf.
23. MDRTB_Indicators_map [Internet]. [cited 2016 Mar 30]. Available from: https://extranet.who.int/sree/Reports?op=vsandpath=/ WHO_HQ_Reports/G2/PROD/EXT/MDRTB_Indicators_map 24. WHO.Global Tuberculosis Control:WHO Report 2010.2010[Sept18,2011] Available from: http://www.who.int/ tb/publication/global report/2010/en/index.html 25. BrudneyK,Dobkin J. Resurgent tuberculosis in New York City. HIV virus, homeleness and the decline of tuberculosis control programme. Am Rev Respir Dis 1991;144:748-9. 26. Angarno G, Carbonara S,Costd GoriA. Drug resistant TB in HIV infected persons in Italy.Int J Tuberc Lung Dis 1998;2:301- 11. 27 Maranetra KN. Treatment of multidrug-resistant tuberculosis in Thailand. Chemotherapy1996;42:(Suppl 3):10-15. 28. Chiang C-Y, Centis R, Migliori GB. Drug-resistant tuberculosis: past, present, future. Respirology. 2010;15(3):413–32.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241811EnglishN2016June11HealthcareCOMPARISON OF DIFFERENT PHENOTYPIC METHODS FOR THE DETECTION OF EXTENDED SPECTRUM b- LACTAMASE (ESBL) IN BACTERIAL ISOLATES FROM TERTIARY CARE CENTRE
English1014Shaikh N. K.English Mundhada S. G.English Lalngaihzuali R.English Ingole K. V.EnglishBackground and objective: Extended spectrum β lactamases (ESBLs) continue to be a major problem in clinical setups. Their detection is essential for proper antibiotic therapy, to limit the spread of resistance mechanisms and for epidemiological purposes. The objective of our study was to compare different phenotypic methods for detection of ESBL to know the best suitable one in our setup.
Material and method: A total of 127 Gram negative isolates were identified by standard protocol and Antibiotic susceptibility testing (AST) was done. Isolates which were resistant to one of the third generation cephalosporins were selected provisionally as ESBL producers and then subjected for confirmation by Phenotypic confirmatory disc diffusion test (PCDDT), Double disk synergy tests, Modified double disk synergy test (MDDST), Indirect modified three dimensional test (IMTDT) to evaluate their ability to detect ESBLs. Minimal inhibitory concentration (MIC) by the agar dilution method was used as the standard reference method.
Result: MIC detection by agar dilution method had confirmed all 63(49.60%) screening positive isolates as ESBL producers. E.coli which constitute 39(61.90%) of the isolates was found to be highest ESBL producer among them. PCDDT by CLSI detected ESBL in 55(87.30%) isolates. DDST using amoxicillin-clavulanic acid detected the same in 49 (77.78%) cases. MDDST using cefepime and piperacillin-tazobactam detected ESBL in 60(95.23%) cases. IMTDT detected 62(98.41%) ESBL producing isolates.
Conclusion: IMTDT was found to be superior method than MDDST, PCDDT and DDST for detection of production of ESBL alone or in presence of other β- lactamases like Amp C.
EnglishAmp C, DDST, ESBL, IMTDT, MDDST, PCDDTINTRODUCTION
The most common cause of bacterial resistance to β-lactam antibiotics is the production of enzyme β-lactamases. Over the last 20 years, many new β-lactam antibiotics have been developed which were specifically designed to resist to the hydrolytic action of β-lactamases. However, with each new class that has been used to treat patients, new β-lactamases emerged that caused resistance to that class of drug.1 The latest in the series of these enzymes is the evolution of Extended Spectrum Beta Lactamases (ESBLs) due to extensive use of 3rd generation cephalosporins. These ESBLs efficiently hydrolyze oxyimino cephalosporins conferring resistance to third generation cephalosporins and to monobactams.2 Plasmids coding for ESBLs may also carry additional β-lactamase genes as well as genes conferring resistance to other antimicrobial such as quinolones, aminoglycosides and sulphonamides. This can limit the chemotherapeutic options for ESBL-producing pathogens and facilitate the inter and intraspecies dissemination of ESBLs.3
Considering the increasing rate of infections caused by ESBL producing organism, their detection is essential for prescribing appropriate antibiotic therapy, to limit the spread of resistance mechanisms and for epidemiological purposes. Various methods like phenotypic (disk diffusion, disk potentiation, double disk synergy procedure, three dimensional test and E- test ESBL strip), semi-automated [Phoenix Automated Microbiology System (BD Diagnostic Systems, Sparks, MD), the VITEK 2 System (bioMe´rieux, Marcy l’Etoile, France), and the MicroScan Walk Away-96 System (Dade Behring, Inc., West Sacramento, CA)] and molecular methods (PCR, PCR-RFLP and nucleotide sequencing) have been described for ESBL detection; however, each method has its own limitations.4 In the present study, we compared different phenotypic methods for detection of ESBL to know the best suitable one in our setup.
MATERIAL AND METHOD
The present study was carried out at a tertiary care centre in the Microbiology department from September 2015 to February 2016. Total 127gram negative bacteria were isolated from various samples including blood, pus, surgical site wounds, burn wounds, tracheal aspirates, central venous pressure tips and urine. Identification of isolates was done based on cultural characteristics and reaction in standard biochemical tests.5, 6 Antimicrobial sensitivity testing was done by the Kirby-Bauer disc diffusion method.7 ESBL detection was done in those Gram negative isolates which revealed resistance/decrease susceptibility to 3rd generation Cephalosporin i.e. Ceftazidime (30ug) or Cefotaxime (30ug) or both according to CLSI guideline (Screening test).Detection of minimum inhibitory concentration (MIC) by the agar dilution technique for 3rd generation cephalosporins with and without clavulanic acid in accordance to CLSI guidelines- 7 was taken as standard reference method for comparison purpose with different phenotypic method for detection of ESBL as given below. 1. Phenotypic confirmatory disc diffusion test (PCDDT)7 – It is performed according to CLSI guideline. Ceftazidime (30 µg) discs alone and in combination with clavulanic acid (ceftazidime + clavulanic acid, 30/10 µg discs) were placed on a Muller Hinton agar (MHA)plate which was inoculated with the test strain. An increase of ≥ 5mm in the zone of inhibition of the combination discs in comparison to the ceftazidime disc alone was considered to be an ESBL producer. 2. Double disk synergy test (disk approximation test)8 - MHA plate was inoculated with test strain. Augmentin (20μg amoxicillin + 10μg clavulanic acid) disc was place in centre of plate. Disc of cefpodoxime (30μg), ceftazidime (30μg) and cefotaxime (30μg) were placed 15-20 mm (centre to centre) around augmentin disc. Enhancement in zone of inhibition of any cephalosporin toward augmentin indicated presence of ESBL producing organism. 3. Modified double disk synergy test (MDDST)8 - The original double disk synergy test was modified for detecting ESBL in AmpC producing clinical isolates. A disc of augmentin was placed in the centre of MHA; then discs of cefpodoxime (30μg), ceftazidime (30μg), cefotaxime (30μg), aztreonam (30ug) and cefepime (30μg) were kept 16 to 20 mm (centre to centre) around augmentin disc. From the cefepime disc at a distance of 22 to 25 mm centre to centre disc of piperacillin-tazobactam (100/10μg) was placed. The organisms were considered to be ESBL producer when the zone of inhibition around cefepime or any of the extended-spectrum cephalosporin discs showed a clear-cut increase towards the piperacillin-tazobactam (PIT) disc or augmentin disc. 4. Indirect modified three dimensional test (IMTDT)9 - MHAplate was inoculated with standard sensitive strain (E. coli ATCC 25922) matching 0.5 McFarland turbidity standards. A disc of 3rd generation cephalosporin (ceftazidime/ceftriaxone/cefotaxime) or aztreonam was placed in the centre of the plate. At a distance of 2 mm from the antibiotic disc a well of 4 mm (diameter) was punched and inoculum (30µL) of the test strain preadjusted to 5.0 McFarland standards was seeded into the well. Heart shaped distortion of zone of inhibition around the β -lactam disc was indicative of an ESBL production. Quality control -Klebsiella pneumonia ATCC 700603 and Escherichia coli ATCC 25922 were used as ESBL positive and negative controls, respectively. RESULT Among the 127 gram negative bacteria isolated, 63(49.60%) were resistant or revealed decreased susceptibility to 3rd generation cephalosporin on screening giving provisional diagnosis of ESBL. All screening positive 63 isolates were confirmed as ESBL producers by standard reference method. (MIC detection by the agar dilution technique)ESBL production was seen highest in E.coli isolates 39(61.90%) followed by Klebsiella pneumonia, Pseudomonas aeruginosa, Proteus species, Citrobacter species and Acinetobacter species. (Table I) Antibiotic Resistance pattern of ESBL producing isolates is as shown in Table II. Imipenem, Pipracillin-Tazobactam and Amikacin were found to be most sensitive antibiotic while maximum resistance was observed against cefotaxime, ceftazidime, Trimethoprim-sulfamethoxazole and Tetracycline. Prevalence of ESBL production in different isolates in present study was detected by various phenotypic methods. Table III shows evaluation of these phenotypic methods in comparison to reference standard method (MIC detection by Agar dilution). Int J Cur Res Rev | Vol 8 • Issue 11 • June 2016 12 Shaikh et.al.: Comparison of different phenotypic methods for the detection of extended spectrum b- lactamase (ESBL)... DISCUSSION The prevalence of ESBL among gram negative bacteria constitutes a serious threat to current β lactam therapy leading to treatment failure and consequent escalation of costs of hospital stay. Many clinical laboratories are not fully aware of the importance of the ESBL producers and of methods to detect them. This lack of understanding is responsible for a continuing failure to respond appropriately to prevent the rapid, worldwide dissemination of the pathogens which possess these β-lactamases. Hence, ESBL detection must be efficient to aid in formulating an antibiotic policy and containment measures to solve the issue of antibiotic resistance.10 Previous studies from India have reported the prevalence of the ESBL producers to be 6.6 to 68%.11, 12, 13, 14 In the present study 49.60 % of gram negative bacteria were ESBL producers. E.coli was the predominant ESBL producer in our study. Similar finding were reported by Kulkarni et al and Ananthakrishanan et al.15, 16We observed that a majority of the isolates in our study were susceptible to imipenem (100%),Amikacin (77.77%) and piperacillin-tazobactum (73.02%). Studies by Dalela et al 17 and Baby et al18 also observed similar result. We observed that imipenem is the most active drug for the treatment of infections caused by ESBL producers, followed by Amikacin and piperacillin/tazobactam. These drug should be used as reserved one, their indiscriminate use should be minimize to prevent emergence of resistance. Different methods were used in present study for detection of ESBL production. PCDDT, theconfirmatory method recommended by CLSI had detected ESBL production in 87. 30% of isolates. While DDST, using Amoxiclav as inhibitor of ESBL showed positive result in 77.78% of isolates. In present study between these two tests, PCDDT was more sensitive than DDST. Similar finding were observed in other studies by Khan et al and Modi et al.8, 9 Sensitivity of both PCDDT and DDST were less compared to standard reference test because Clavulanic acid which was used as ESBL inhibitor in PCDDT and DDST act as inducers of high level AmpC production and it led to resistance to 3rd generation cephalosporins as well 3rd generation cephalosporins + clavulanic acid. So even if ESBL was present, it would not be detected and resulted in false negative test.9 For MDDST, few modifications were done in DDST like addition of piperacillin- tazobactam as ESBL inhibitor, use of 4th generation cephalosporins(Cefepime) and optimum spacing of drugs for detection of synergy. If AmpC was present, cefepime would be sensitive so synergy could be seen with 4th generation cephalosporins and inhibitor antibiotic.8 Tazobactam and sulbactam were less likely to induce AmpC B-lactamase compared to clavulanic acid. So, simultaneous Amp C induction did not occur. In present study, MDDST had detected 60(95.23%) isolates which is 11 isolates more than detected by DDST. MDDST found to be more sensitive than DDST and PCDDT in our study. These finding can be correlated with observation made by Modi et al9 in their study. In present study, IMTDT identified 62(98.41%) ESBL positive isolates. Thus it was the most sensitive test among all other tests. It was superior in detecting ESBL than PCDDT, DDST and MDDST in our study. Other studies carried out by Menon et al19 and Modi et al9 also reported similar observations. Thus, PCDDT and DDST should be used in the isolates which produce only ESBL but are not useful for detection of ESBL in isolates who also produces other β-lactamases like AmpC enzyme. While MDDST due to modification in it and IMTDT had showed good sensitivity when compared to standard reference method.
CONCLUSION
Infections caused by ESBL producers often limit therapeutic options and cause treatment failures. Thus, detection of ESBL production should be perform routinely in microbiology laboratories so that the appropriate antimicrobial therapy can be instituted and the dissemination of ESBL producers may be prevented by employing appropriate infection control measures. It is important to increase awareness of among physician about ESBL producers and different methods available to detect them. Molecular assays may provide accurate results in the identification of ESBL genes, but their accessibility is often limited and they are expensive. Phenotypic methods are easy to perform and interpret. In our study, IMTDT was found to be superior method than MDDST, PCDDT and DDST for detection of production of ESBL alone or in presence of other β- lactamases like AmpC.
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.
Source of Funding – Nil
Conflict of interest- Nil
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2. Mundhada S, Waghmare P, Ingole k, Rathod P. Prevalence of various β-Lactamases production among gram negative isolates in burn care unit in a tertiary care hospital. International Journal of Applied Research2015; 1(12): 474-8.
3. Poulou A, Grivakou E, Vrioni G, Koumaki V, Pittaras T, Pournaras S, Tsakrisa A. Modified CLSI Extended-Spectrum -Lactamase (ESBL) Confirmatory Test for Phenotypic Detection of ESBLs among Enterobacteriaceae Producing Various β-Lactamases. Journal of Clinical Microbiology 2014;52(5):1483–9.
4. Goyal A, Prasad A, Ghoshal U, Prasad K. Comparison of disk diffusion, disk potentiation and double disk synergy methods for detection of extended spectrum beta lactamases in Enterobacteriaceae.Indian J Med Res 2008:209-211 5. Forbes BA, Sahm DF, Weissfeld AS. Overview of bacterial identification methods and strategies. Bailey and Scott’s Diagnostic Microbiology. 12th ed. Missouri: Mosby Elsevier; 2007. p. 218- 47.
6. Collee JG, Miles RS, Watt B. Tests for identification of bacteria. In: Colle JG, Fraser AG, Marimon BP, Simmons A, editors. Mackie and McCartney Practical Medical Microbiology. 14th ed. Edinburg: Elsevier Churchill Livingstone; 2006. p. 131-149.
7. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-fifth Informational Supplement. CLSI document M100- S25. Wayne, PA: Clinical and Laboratory Standards Institute; 2015.
8. Khan M, Thukral S, Gaind R. Evaluation of a Modified Double-Disc Synergy Test for Detection of Extended Spectrum Β-Lactamases in Ampc Β-Lactamase-Producing Proteus mirabilis. Indian Journal of Medical Microbiology, (2008). Pg. 58- 61
9. Modi D, Patel D, Patel S, Jain M, Bhatt S, Vegad M. Comparison of various methods for the detection of extended spectrum beta lactamase in klebsiella pneumoniae isolated from neonatal intensive care unit, Ahmedabad. National journal of medical research 2012;2(3):348-53.
10. Krishnamurthy V, Vijay Kumar G, Sudeepa Kumar M, Prashanth H, Prakash R, Nagaraj E. Phenotypic and Genotypic Methods for Detection of Extended Spectrum β Lactamase Producing Escherichia coli and Klebsiella pneumoniae Isolated from Ventilator Associated Pneumonia.Journal of Clinical and Diagnostic Research 2013;7(9): 1975-78.
11. Subha A, Ananthan S. Extended-spectrum β-lactamase (ESBL) mediated resistance to the third generation cephalosporins among Klebsiella pneumoniae in Chennai. Indian J Med Microbiol 2002; 20:92-95.
12. Mathur P, Kapil A, Das B, Dhawan B. Prevalence of extended spectrum β-lactamase producing gram negative bacteria in a tertiary care hospital. Indian J Med Res 2002; 115:153-57.
13. Rodrigues C, Joshi P, Jani SH, Alphonse M, Radhakrishnan R, Mehta A. Detection of β-lactamases in nosocomial gram negative clinical isolates. Indian J Med Microbiol 2004; 22(4):247- 50.
14. Singhal S, Mathur T, Khan S, Upadhyay DJ, Chugh S, Gaind R,Rattan A. Evaluation of the methods for AmpC β-lactamase in gram negative clinical isolates from tertiary care hospitals. Indian J Med Microbiol 2005; 23(2):120-24.
15. Kulkarni R ,Dohe V, Ghadge D, Bhore A. A study of extended spectrum β- lactamases (ESBL) producers in clinical isolates. Medical journal of western India.2013; 41(1).
16. Anathakrishanan AN, Kanungo R. Detection of extended spectrum β-lactamase produces among surgical wound infections and burns patients in JIPER. J Lab Physicians 2009;1(1):7-10.
17. Dalela G. Prevalence of extended spectrum beta-lactamase (ESBL) producers among gram-negative bacilli from various clinical isolates in a tertiary care hospital at Jhalawar, Rajasthan, India. J ClinDiag Research. 2012;6(2):182-187.
18. Baby PS, Appala RB, Mani KR. Detection of Enterobacteriaceae producing CTX-M extended spectrum beta-lactamases from a tertiary care hospital in south India. Indian J Med Microbiol 2008; 26:163-66.
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Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241811EnglishN2016June11HealthcareROLE OF BIOFILMS IN STAPHYLOCOCCUS COLONISING INTRAVENOUS CATHETERS
English1522Aruna JadhavEnglish Vaishali DoheEnglish Anju KagalEnglish Renu BharadwajEnglishIntroduction: Staphylococci are major nosocomial pathogen associated with indwelling medical devices, especially with intravascular catheter (I V) related infections. Major virulence factor of Staphylococcus is its ability to form biofilm on polymeric surfaces.
Objective:
• To isolate and identify the pathogens colonising IV catheters.
• To select the Staphylococcus species and to detect their ability to produce biofilms along with antimicrobial susceptibility testing.
Material and methods: A total of 373 IV catheter tips from 373 patients were collected and processed for isolation of bacterial pathogen. Total 119(31.9%) IV catheter tips were culture positive. 100 Staphylococcus strains were tested for biofilm detection by Tissue culture plate (TCP) method and Tube method (TM). Antimicrobial susceptibility testing was done by Kirby-Bauer disc diffusion method as per CLSI guidelines. The time frame since insertion and removal of catheter was also noted.
Results: 125 organisms were isolated from 119(31.9%) culture positive IV catheter tips. Staphylococcus spp.(80%) was the most common organism followed by Enterococcus spp(9% ). 93(24.4%) catheters were colonised within one week of insertion.56% of the Staphylococci were Coagulase negative staphylococci (CoNS) and 44% were S.aureus. Of 100 Staphylococcus spp. 84% were biofilm producer by TCP method and 75% by Tube method. Sensitivity and specificity of TM method vis-a-vis TCP method was 89.3%and 100% respectively.
Conclusion: Majority of Staphylococcus isolated from IV catheters were biofilm producers. TCP method is standard method for detection of strong, moderate and weak biofilm producing strains, but TM method is technically simple. Antimicrobial resistance was significantly higher in biofilm producing Staphylococcal species.
EnglishTissue culture plate (TCP), Tube method (TM), Biofilm, Intravascular catheter (I V).INTRODUCTION
The use of indwelling medical devices is important in treatment of critically and chronically ill patients.1 These indwelling medical devices become the focus for infections, like intravascular catheter related infections. Use of vascular catheters has become an indispensable part of modern medicine practice. The predominant organisms isolated in these infections are Staphylococcus epidermidis and Staphylococcus aureus. 2 The major virulence factor for these organisms is their ability to form biofilm.2 Biofilm consist of slime which is a complex extracellular polymeric substance produced by most of the Staphylococcus. 3 In biofilm, specific initial adherence to device surface is mediated by polysaccharide adhesion (PSA). Following initial adhesion, polysaccharide intercellular adhesion (PIA) is involved in cell-cell adhesion. The synthesis of PIA is mediated by the chromosomal ica gene (intercellular adhesion), which is an operon structure contains the icaADBC genes.4,5 Biofilm formation is a major concern in nosocomial infections because it protects microorganisms from host immune response along with antimicrobial agents. Such infections are resistant to systemic antibiotic therapy and removal of infected device becomes necessary. Therefore, once biofilmassociated Staphylococcal infections occur, they are difficult to eradicate.6 Biofilm formation in Staphylococcus can be studied by various methods such as-microscopic examination by using epifluroscence, scanning electron microscope (SEM), confocal laser scanning microscope (CLSM). Molecular techniques such as Polymerase chain reaction (PCR) which amplifies the gene (ica ABCD).7 The present study was carried out to assess the incidence of Staphylococcus spp. colonising intravenous catheters. An effort was also made to study the presence of biofilm in these Staphylococcus spp and their antimicrobial sensitivity pattern. MATERIAL AND METHODS: Collection and processing of IV catheter tips: A total of 373 IV catheter tips (336 peripheral venous catheter tips, and 37 central venous catheter tips) were collected from 373 patients, admitted in Medicine, Surgery, Paediatrics wards, and Intensive care units. The distal 5 cm of catheter was cut off and placed in a sterile screw caped container with 1ml brain heart infusion (BHI) broth.10 [fig.1] All tips were processed for quantitative culture technique. For this, catheter tip in BHI broth was vortexes for 1min and 0.1 ml of suspension was spread over Blood agar and MacConkey agar 11,12. The plates were incubated aerobically overnight at 370 C and were observed for growth. Catheter tip colonization was said to be present if more than 100 cfu /catheter segment by this vortexing techniques. 11 A total of 100 Staphylococcus spp. were isolated from 373 catheter tips. Speciation was done by Standard microbiological techniques. 11 All Staphylococcus isolates were subjected to antimicrobial susceptibility testing on Muller-Hinton agar by Kirby-Bauer disc diffusion method14. Methicillin resistance was detected by using Cefoxitin-30μg disc as per CLSI.14Inducible Clindamycin resistance were detected by D test as per CLSI guidelines. 14A record was kept of the time since insertion of all catheters removed and cultured in the present study. Biofilm detection methods: Biofilm formation in Staphylococcus spp. was done by tube method(TM) and tissue culture method (TCP).This was done to evaluate the usefulness of the tube method for biofilm detection. Tube method(TM):1,3 10 ml Trypticase soya broth (TSB) with 1% glucose was taken in a new glass test tube which was inoculated with loopful of Staphylococcus growth from overnight culture plate. It was further incubated at 37°C for 24 hours. Tube was decanted and washed with PBS (PH 7.3) and then further it was air dried. A dried tube was stained with 0.1% crystal violet. Excess stain was removed by distilled water. Tubes were dried in inverted position and observed visually for biofilm formation. Each test was performed in triplicate to minimise errors. S. epidermidis A TCC 35984 (strong biofilm producer) used as positive control while, uninoculated TSB broth with 1% glucose as negative Control. Biofilm formation is said to be positive when a visible thin film lined the wall and bottom of the test tube. Ring formation at the liquid interface is not considered as an indication of biofilm formation. Biofilm formation was scored asStrong, moderate, weak, and negative. [fig.3] Tissue culture plate method (TCP): 1,9,15 A. Biofilm Cultivation - All Staphylococcal isolates from fresh agar plates were inoculated in TSB with 1% glucose and incubated for 18 hr at 37o C in stationary condition and then diluted 1 in100 with fresh medium. Individual wells of sterile, polystyrene, 96 well-flat bottom tissue culture plates were filled with 200µL aliquots of the diluted cultures. The inoculated plate was covered with a lid and incubated aerobically for 24 hr at 370 C under static condition. B. Washing - After incubation content of each well was gently removed by tapping the plates. Wells were washed three times with 300 µL of sterile phosphate-buffered saline (PBS; PH 7.2). C. Fixation - After washing remaining attached bacteria were fixed by air drying. D. Staining - Adherent biofilm layer formed in each microtiter plate well was stained with 150 µL of 0.1% Crystal violet for 15 min at room temperature. After staining, washing was done until washing are free of stain. Then microtiter plate was air dried at room temperature. S. epidermidis ATCC 35984 (strong biofilm producer) used as a positive control while uninoculated TSB broth with 1% glucose as negative control. E. Measurement and interpretation of results15 Uniformly Stained adherent Staphylococcal cells on well of microtiter plate were considered as biofilm producer [fig.3].
Optical density (OD) of the stained adhesive Staphylococcus bacteria was determined with a micro-ELISA reader at a wavelength of 570 nm (OD570 nm ). The result of the test is recorded and these OD values are considered as a true indication of bacteria adhering to the surface and biofilm formation. Tests were performed in triplicate to minimise errors and for analysis of data. Interpretation of obtained results requires definition of the cut –off value that separates biofilm producing from nonbiofilm producing strain. i. Average OD values were calculated for all tested strain (ODt) and negative controls (since all tests are performed in three times). ii. Cut-off value (ODc) was calculated. It is defined “as three standard deviations (SD) above the mean OD of negative value”. [ODc = average OD of negative control + (3xSD of negative control)]. iii. ODc value was calculated for each microtiter plate separately. For easier interpretation of results, strains were divided in to the following categories. • No biofilm producer = ODt ≤ ODc • Weak biofilm producer = ODc < ODt ≤ 2xODc • Moderate biofilm producer = 2xODc < ODt ≤ 4x ODc • Strong biofilm producer = 4x ODc < ODt Statistical Methods: Statistical evaluation of the TM method for detection of biofilm formationResults and observations were analysed by using In Silico statistical software. Chi-square test and P-value was calculated by using this software for comparison and to determine the statistical significance Results and observations: Total 373 IV catheter tips were processed out of which 119 (31.9%) were culture positive, which include 95(28.3%) peripheral and 24(64.9%) central IV catheter tips.[Table1] Common indication for peripheral IV line removal [85 i.e.89.5%] was when no longer required. In central IV line it was removed in majority of patients [20 i.e.83.4%] due to local sign and symptoms of catheter related infection. Peripheral IV lines were colonised with bacteria earlier than central IV line.55 (57.9%) peripheral IV catheter colonization was seen after 2-4 days of catheter use while100% colonisation with CVC when duration of catheter use was > 7days [Table 2] Total 125 organisms were isolated from 119 culture positive IV catheter tips. Staphylococcus spp.100 (80%) was the most common organism followed by Enterococcus spp 9%.[Table 3] 6 catheter tips showed mix growth i.e. two organism from each. Out of 100 Staphylococcus species, 44% were S.aureus and 56% CoNS. Amongst the all CoNS, S.epidermidis 22 (39.28%) was the commonest isolate followed by S.haemolyticus 13 (23.1%). [Table 4] In present study 84% of Staphylococci were biofilm producer by tissue culture plate method and 75% by tube method. TCP method is considered standard test for biofilm detection. The tube method is relatively and technically simple method. Out of these 84% of Staphylococci 39(46.4%) were S.aureus and 45(53.3%) were CoNS. However in CoNS 21(52.5%) were S.epidermidis and 10(25 %) S.haemolyticus 3] 6 catheter tips showed mix growth i.e. two organism from each. Out of 100 Staphylococcus species, 44% were S.aureus and 56% CoNS. Amongst the all CoNS, S.epidermidis 22 (39.28%) was the commonest isolate followed by S.haemolyticus 13 (23.1%). [Table 4] In present study 84% of Staphylococci were biofilm producer by tissue culture plate method and 75% by tube method. TCP method is considered standard test for biofilm detection. The tube method is relatively and technically simple method. Out of these 84% of Staphylococci 39(46.4%) were S.aureus and 45(53.3%) were CoNS. However in CoNS 21(52.5%) were S.epidermidis and 10(25 %) S.haemolyticus. Sensitivity and specificity of TM method vis-a-vis TCP method was 89.3%and 100% respectively with 100% Positive predictive value (PPV), and 64% negative predictive value (NPV). Biofilm formation by tube method was found to be equivalent in specificity to the tissue culture plate method for detecting biofilms. [Table 5] TM method was comparable with TCP method to differentiate strong and moderate biofilm producing strains but not the weak one. The TCP method was definitely better in detecting weak biofilm producer. [Table 6] In this study >50% Staphylococcal spp. were multi drug resistant (MDR) [Table-7]. Overall 75% of S. aureus and 60.7% CoNS were resistant to Penicillin and Methicillin resistance was 63.6% in S. aureus and 42.8% in CoNS [Table7]. However, inducible Clindamycin resistance (MLSBi) was 21% amongst all Staphylococcus species and constitutive Clindamycin resistance (MLSBc) was 35%. Antimicrobial resistance was higher in biofilm producing Staphyloccus spp. than non biofilm forming one [Table 8].
DISCUSSION
The extensive use of intravenous catheters in hospitalized patient has led to increased incidence of catheter-related infection (CRI), especially blood stream infections. These infections originate from the microbial colonisation of the intravascular catheters. 16 In the present study out of 373 IV catheter tips processed, 119 (31.9%) were culture positive [Table 1]. Rao SD et al (2005) reported 74(54.8%) culture positive out of total 135 IV catheter tips. This was higher than present study. This could be probably due to the fact that IV catheter tips were collected from paediatric intensive care unit (PICU) only in theirstudy.17
Peripheral IV catheter tip culture positivity rate, in our study was 95 (28.3%). In Nahirya P et al (2008) showed 20.7%
culture positivity of peripheral IV catheter tips collected from paediatric wards.18However, Rao SD et al (2005) detected 54(52.4%) were culture positive IV tips out of 103 peripheral IV catheters.17 Central IV catheter (CVC) tip culture positivity rate in present study was 24 (64.9%) This was comparable with 62.5% each given by Subba Rao SD et al in (2005)17 and Gahlot R et al in (2013).19While Chopdekar K et al(2011) found that culture positivity rate was 57.6% in their study. 20 Peripheral IV catheters were removed in 89.5% of patients when they were no longer required. However, Central IV catheter removal in 83.4% of patients was due to the presence of clinical sign and symptoms of infections like local signs of inflammation .Majority of CVC catheter tips were collected from adult ICUs with co-morbid conditions like hypertension, diabetes, malignancy or receiving chemotherapy. The duration of IV catheterisation is a significant factor which determined the development of catheter related infections. In our study peripheral IV catheter colonisation was seen, in 57.9% of patients, after 2-4 days of catheter use [Table2]. These findings were comparable with Rao SD et al17 (2005). In present study central IV catheters culture positivity rate was 100% when duration of use was more than7days [Table2]. Rao SD et al (2005) showed 100% culture positive rate, after duration more than 11days. 17 Total 125 organisms were isolated from 119 cultures positive catheter tips in our study. Out of which CoNS 44(44.8%) and S.aureus 56(35.2%) were predominant ones, followed by Enterococcus spp.(9%)[Table 3]. Nahirya et al (2008) found S.aureus (60.5%) as the predominant pathogen followed by S.epidermidis (23.4%).18 Rao SD et al (2005)17 detected CoNS (32.4%) followed by Pseudomonas spp.(31%).While in Chopdekar et al, (2011) study revealed maximum colonisation with non-albicans Candida spp.(22.6%).20The microbial profiles of catheter colonisation vary in different settings or areas due to the impact of environmental contaminants in the pathogenesis of device related infections. CoNS is frequently responsible for catheter colonization due to its capacity to adhere to polymer surfaces and consequent biofilm production. Out of 100 Staphylococcus spp. isolated in our study, 56% were CoNS and 44% were S.aureus. Two recent studies Prasad S et al21 (2012) and Patil HV et al (2011) isolated 57.1% and 65% of CoNS respectively from indwelling IV catheter tips, which were comparable with our study.22 Amongst the 56% CoNS in present study 39.28% were S.epidermidis followed by S.hemolyticus 23.1% [Table 4]. Patil HV et al (2011)22 showed 45% of S.epidermidis and 15% of S.hemolyticus in their study. S.aureus is a known pathogen in hospital infections. It was the second common organism in our study which was (35.2%). Rate of S.aureus was much lower in Khanna et al (2013) 23 who got 13.25% as compared to our study The higher rate of S.aureus in present study could be due to the lack of dedicated IV catheter insertion team, as well as lack of standardized protocol for insertion and replacement of IV catheters. In the present study 100 Staphylococcus spp. were screened for biofilm detection by modified TCP method and TM method, along with antimicrobial susceptibility pattern. 84% and 75% of Staphylococcus spp. showed biofilm production by TCP and TM method respectively [Table 5].Bose et al (2009) reported 54.2% and 42.4% of Staphylococcus spp. were biofilm producer by TCP and TM method respectively.24 While Mathur et al (2006) showed 53.9% and 41.4% of Staphylococcus spp. as biofilm producer by TCP and TM method respectively.1 Biofilm production in present study was higher than these studies. These could be because the isolates from above two studies were obtained from all clinical samples and not only catheter tips like the current study. In this study 84% Staphylococcus species were true biofilm producer, i.e. positive by standard TCP method [Table-5]. However, 9 strains were positive by TCP but negative by TM method. This is because it was difficult to differentiate between weak and non-biofilm producers by TM method. Otherwise TM method correlated well with TCP method for Strong and moderate biofilm production.[Table-6]. TCP method was definitely better test to detect weak biofilm producing strains. This variability in results of weak biofilm production was also observed by other similar studies.1,24 Sensitivity and specificity of TM method vis-a-vis TCP method was 89.3% and 100% respectively with 100% Positive predictive value (PPV), and 64% negative predictive value (NPV) [Table-5]. This finding is supported by two similar studies. 1,24 Multidrug resistance (MDR) was significantly higher in all biofilm producing strains than non biofilm producing strains in present study [Table-8]. MDR strain was taken as a strain resistant to > 2 classes of antibiotic. Thus making it difficult to treat intravenous catheter related infections. There are some highly accurate methods like PCR to detect icaADBC operon. These encode polysaccharide intercellular adhesion (PIA) which mediates biofilm formation. These are expensive methods. In a developing country like India, low cost method like TCP and TM methods are useful for screening purpose of biofilm producing strains.
CONCLUSION
To conclude, present study showed biofilm formation in 84% of the Staphylococcus species isolated from IV catheters tips.
Since this colonisation will result in blood stream infections unless the catheter is removed or changed early. Simple preventive measures, such as aseptic precaution during catheter insertion, daily catheter care, monitoring of catheterised patients, could help to reduce risk of colonisation and subsequent catheter related infections. Since these infections are difficult to treat, it is better to prevent such infections than attempt to treat, once they are established.
ACKNOWLEDGEMENT
I (Author) take this opportunity to 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- Not Applicable Informed consent- Not Applicable Source of funding- Not Applicable Conflict of interest- Not Applicable
Englishhttp://ijcrr.com/abstract.php?article_id=246http://ijcrr.com/article_html.php?did=2461. Mathur T, Singhal S, Khan S, Upadhyay DJ, Fatma T, Rattan A. Detection of biofilm formation among the clinical isolates of Staphylococci: An evaluation of three different screening methods. Indian J. Med. Microbiol. 2006; 24:25-9.
2. Harald S, Bernd J, Barry M.F, Catheter-Related Infections.2nd edition, Taylor and Francis e-Library, 2006
3. Christensen GD, Simpson WA, Bisno AL, Beachey EH. Adherence of slime producing strains of staphylococcus epidermidis to smooth surfaces. Infect Immun 1982; 37(1):318-26.
4. Yazdani R, Oshaghi M, Havayi A, Pishva E, Salehi R, Sadeghizadeh M, Foroohesh H. Detection of icaAD Gene and Biofilm Formation in Staphylococcus aureus Isolates from Wound Infections. Iranian J Publ Health. 2006; 35( 2):25-28.
5. Vuong C, Voyich JM, Fischer ER, Braughton KR, Whitney AR, DeLeo FR et al Polysaccharide intercellular adhesin (PIA) protects Staphylococcus epidermidis against major components of the human innate immune system. Cell Microbiol.2004; 6(3):269–75.
6. Donlan RM. Biofilms: microbial life on surfaces: Emerg Infect Dis.2002; 8(9): 881-90.
7. Lawrence JR, Neu TR. Confocal Laser scanning microscopy for analysis of microbial biofilms. Meth Enzymol 1999, 310: 131- 44
8. Freeman DJ, Falkiner FR, Keane CT.New method for detecting slime production by coagulase-negative Staphylococci. J Clin Pathol 1989; 42(8): 872-74.
9. Christensen GD, Simpson WA, Younger JA, Baddour LM, Barrett FF, Melton DM et al. Adherence of cogulase negative Staphylococci to plastic tissue cultures:a quantitative model for the adherence of Staphylococci to medical devices. J Clin Microbiol 1985;22(6):996-06.
10. Catheter tip culture updated on 2007 (www.asm.org), laboratory service manual micro/viro. Available from: (www.childrens mn.org).
11. Bouza E, Alvarado N, Alcala L, Sa´nchez-Conde M et al. A Prospective, Randomized, and Comparative Study of 3 Different Methods for the Diagnosis of Intravascular Catheter Colonization. Clinical Infectious Diseases 2005; 40:1096–100.
12. Brun-Buisson C, Abrouk F, Legrand P, Huet Y, Larabi S, Rapin M.Diagnosis of central venous catheter–related sepsis: critical level of quantitative tip cultures. Arch Intern Med 1987; 147:873–77.
13. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC. Color Atlas and Textbook of Diagnostic Microbiology. 6th edition, San Francisco Lippincott. 2006.
14. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; twenty two informational supplements. M100-S22; vol. 32.No.1: January 2012.
15. Stepanovi? S, Vukovi? D, Hola V, Di Bonaventura G, Djuki? S, Cirkovi? I et al .Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by Staphylococci. APMIS. 2007;115(8):891-99
16. Singhai M, Malik A, Shahid M, Malik MA, Goyal R. A Study on Device-Related Infections with Special Reference to Biofilm Production and Antibiotic Resistance. J Glob Infect Dis. 2012; 4(4): 193–98.
17. Rao SD, Joseph MP, Lavi R, Macaden R. infections related to vascular catheters in a pediatric intensive care unit. Indian Pediatrics 2005; 42:667-72.
18. Nahirya P, Byarugaba J, Kiguli S, Kaddu-Mulindwa D. Intravascular catheter related infections in children admitted on the paediatric wards of Mulago Hospital, Uganda. Afr Health Sci. 2008; 8(4):206-16.
19. Gahlot R, Nigam C, Kumar V, Gupta M. Catheter Related Blood Stream Infections in ICU: A study from North India. Int J Infect Control. 2013; 9:1-4.
20. Chopdekar K, Chande C, Chavan S, Veer P, Wabale V, Vishwakarma K et al. Central venous catheter-related blood stream infection rate in critical care units in a tertiary care, teaching hospital in Mumbai. Indian J Med Microbiol. 2011; 29(2):169- 71.
21. Prasad S, Nayak N, Satpathy G, Nag HL, Venkatesh P, Ramakrishnan S. Molecular and phenotypic characterization of Staphylococcus epidermidis in implant related infections. Indian J Med Res.2012; 136(3):483-90.
22. Patil HV, Patil VC, Ramteerthkar MN, Kulkarni RD. Central venous catheter-related bloodstream infections in intensive care unit. Indian J Crit Care Med. 2011;15(4): 213–23.
23. Khanna V, Mukhopadhayay C, K E V, Verma M, Dabke P. Evaluation of Central Venous Catheter Associated Blood Stream Infections: A Microbiological Observational Study. J Pathog.2013; 2013: 936864.
24. Bose S , Khodke M, Basak S , Mallick S K, et al. Detection Of Biofilm Producing Staphylococci: Need Of The Hour. Journal of Clinical and Diagnostic Research. 2009; (3):1915-20.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241811EnglishN2016June11HealthcareEFFECT OF ISOPROTURON TOXICITY ON TESTIS OF ALBINO RATS
English2327Archana RaniEnglish Anita RaniEnglish Rakesh Kumar VermaEnglish Arvind Kumar PankajEnglishObjectives: As pesticides hold a unique position among environmental contaminants; the objective of the present study was to evaluate the toxic effects of Isoproturon, a substituted phenylurea herbicide on testis of albino rats.
Methods: The compound, suspended in groundnut oil, was administered orally to rats for 60 days at the doses of 400 mg/kg and 800 mg/kg body weight for two groups respectively. Body weight, testis weight and seminiferous tubular diameter were measured. Histological study was done on haematoxylin- eosin stained 5μm thick paraffin sections.
Results: Decrease in weight gain and seminiferous tubular diameter was observed in experimental rats which was highly significant. The difference in testis weight was not significant statistically in experimental group as compared to control group. Light microscopic study showed degenerative changes in the testis.
Conclusions: Degenerative changes in the gametogenic cells indicate adverse effects on the functional ability of these cells and are highly conclusive of reproductive toxicity caused by isoproturon.
EnglishIsoproturon, Testis, Seminiferous tubules, Leydig cellsINTRODUCTION
Exposure to pesticides can range from mild skin irritation to birth defects, tumors, genetic changes, blood and nerve disorders, endocrine disruption, and even coma or death.1 Developmental effects have been associated with pesticides. Recent increases in childhood cancers in throughout North America, such as leukemia, may be a result of genotoxic and non-genotoxic pesticides due to somatic cell mutations.2 Insecticides targeted to disrupt insects can have harmful effects on the nervous systems of mammals, due to basic similarities in system structure. Both chronic and acute alterations have been observed in those who are exposed. Pesticides can act in the promotion and proliferation of cancer while causing hormone imbalance. DDT and its breakdown product DDE, with levels still present in the environment, despite its ban, are known to disturb estrogenic activity and possibly lead to breast cancer. Exposure to pesticides, for example DDT, in fetal stages has been proven to alter male penis size in animals to that much smaller than average as well as develop undescended testicles. Exposure to pesticides may occur in postnatal early stages of development, in utero, and even if either parent was exposed before conception took place. Reproductive disruption has the potential to occur by chemical reactivity and through structural changes to a system.3 Of the potential health risks associated with exposure to chemical or physical agents, a prominent concern is that these agents may interfere with the ability of individuals to produce normal and healthy children. Concern about the susceptibility of the male reproductive system to drugs or environmental agents has assumed an increasing extent. A large number of toxic chemicals in the environment are known to interfere with the endocrine system. Sexual development during the prenatal and neonatal period is under hormonal control and is therefore sensitive to exogenous substances with an endocrine effect. The present work primarily focuses on toxic effects that involve testicular and spermatogenic processes that are essential for reproductive success. Occupational or experimental exposure to herbicides alters the testicular steroidogenic function resulting in spermatogenic failure and decreased male fertility. Isoproturon is a substituted phenylurea [3-(4-isopropylphenyl)-1,1-dimethylurea], a herbicide possessing broad spectrum activity was taken to see its toxic effects on testis of albino rats. Literature suggests that very few studies have been done on testicular toxicity of isoproturon while recently it is widely used because of its broad spectrum activity. Therefore, the present study is a step to fill this lacuna. MATERIAL AND METHODS The present study was conducted in the Department of Anatomy, King George’s Medical University, Lucknow, Uttar Pradesh, India. Twenty four male albino rats weighing 50-80 g were used for the present experiment. Animals were obtained from animal house of Indian Institute of Toxicology and Research, Lucknow. The rats were maintained under standard laboratory conditions in an air conditioned room and housed in stainless steel cages at temperature 22±3°C and relative humidity 30–70%.4 Light dark cycle (photoperiod) was controlled between 8.00 a.m. to 5.00 p.m. They were fed on the standard pellet diet and tap water ad libitum. Animal care was as per Indian National Science Academy (INSA) guidelines for Care and Use of Animals in Scientific Research.5 After acclimatization for 2 weeks in laboratory conditions, animals were divided into 3 groups of 8 rats each. Group-1 served as control. Group 2 and 3 received 400 mg and 800 mg isoproturon/ Kg body weight respectively in 0.2 ml of groundnut oil orally, 6 days/week for 60 days. Body weight of all the rats i.e. control and treated were taken on day zero, at every 10 days interval and at the end of experiment (day 60) with the help of weighing machine. All treated rats along with their controls were anaesthetized by intraperitoneal administration of Nembutol (30 mg/Kg body weight). Fixation of testis was achieved by in- vivo perfusion with Bouin’s fixative. After completion of the perfusion, the scrotum was skinned and an incision was given with the scalpel and forceps. The testis was isolated from the scrotum and weighed. Testis was cut into small pieces and kept in Bouin’s fluid for 24-48 hours. Processing of tissue was done for making paraffin blocks. 5µm thick sections were cut with a sharpen knife on a rotary microtome. The sections were adhered on clean glass slides smeared with a thin layer of Mayer’s egg albumin. The slides were then dried in an incubator and staining was done with haematoxylin and eosin (H and E). Quantitative histopathology was done with the help of micrometer. It was done to know the alterations in the diameter of seminiferous tubules. Total number of tubular cross-sections was counted in 3-5 fields/testis at a magnification of 10x40 and testicular damage was assessed by determining the percentage of damaged tubules against the total tubules. Approximately, 20 circular cross-sectioned seminiferous tubules were randomly selected and two transverse diameters of each tubule was measured in order to calculate the mean tubular diameter. Statistical analysis was done by Student's t test. The difference was considered statistically significant, if the P value was < 0.05.
OBSERVATIONS AND RESULTS
Appearance
Rats treated with isoproturon appeared to be dull, drowsy, emaciated and weak.
Body weight
The mean body weight (g) of 8 rats in control group was found to be increased from 59.38±6.41 at zero day to 73.50±7.25 after 60 days. Rats treated with low dose of isoproturon exhibited less weight gain as compared to control and the value was highly significant. It was 70.50±7.09 at day zero while 78.28±6.93 after 60 days. The mean body weight of rats in high dose group increased from 71.13±5.14 to 72.26±5.34 at day zero and after 60 days respectively. Weight gain was very less and was highly significant as compared to control and low dose group (Table 1, Figure 1).
Testis weight
One testis of each rat was weighed and the mean testis weight (g) of 8 rats after 60 days in control group was 1.15±0.06, in isoproturon low dose group 1.10±0.04 and in high dose group 1.11±0.05. The difference in testis weight was not significant statistically in experimental group as compared to control group (Table 2).
Histopathology Light microscopic study of testis of control rats exhibited seminiferous tubules cut in various planes of sections. The seminiferous tubules were lined by stratified cuboidal epithelium which consists of cells in various stages of spermatogenesis collectively referred to as cells of spermatogenic series. Next to basement membrane lie the spermatogonia, having spherical nuclei. Non-spermatogenic cells, called Sertoli cells having characteristically ovoid shaped nucleus was seen towards the basement membrane. In the interstitial spaces, Leydig cells having endocrine function and connective tissue cells were present (Figure 2). Testis of isoproturon treated animals revealed distorted seminiferous tubules, decreased diameter of seminiferous tubules, depressed spermatogenesis, loss of sperms, degeneration and desquamation of gametogenic cells from the basement membrane, accumulation of cellular mass in the lumen of tubules, oedematous exudation in the interstitial spaces which was more marked in the central region, vacuolation in the interstitium was also evident, some of the Leydig cells were showing signs of degeneration (Figures 3-5). All these findings were much significant with high dose of isoproturon.
DISCUSSION
Rats treated with Isoproturon appeared to be dull and drowsy. Dikshith et al (1990) noted mild to moderate toxic effects which was more pronounced in male rats.6 Sarkar and Gupta (1993) studied the effect of isoproturon on pregnant rats and their offspring. Dose-related depression and drowsiness of pregnant rats along with decreased maternal body weight, litter size, foetal weight, crown-rump and trans-umbilical lengths were observed.7 In the present study also, the rats appeared to be emaciated which runs parallel with the finding of Sarkar et al (1995). Emaciation of rats may be due to direct cumulative toxicity of the pesticide.8 None of the rat was found to be dead in the present experiment, thereby showing that these doses were not lethal. Isoproturon significantly decreased the body weight of rats at both low and high dose in the present study. Sarkar et al (1995) also noticed significant decrease in weekly body weight of rats at high dose (800 mg/kg).8 The cause for this loss in weight gain may be due to their organ directed toxicities. Loss of appetite during experimental period also explains the loss in body weight. Isoproturon did not alter the testicular weight even with the highest dose as revealed by the study of Sarkar et al (1995).8 The difference in testis weight was not significant statistically in experimental group as compared to control group in the present study also. No alteration in weight of testis can be related to the damage to the testis may be detected only at doses higher than those required to produce significant effects in other measures of gonadal status. In his study, reduction in weight of epididymis was noticed which was explained that it was due to decrease in sperm reserve. Seminiferous tubular diameter was decreased in experimental groups as compared to control and was highly significant statistically in the present study. Sarkar et al (1997) also observed that the mean diameter of seminiferous tubules was diminished by 17 and 37% with medium (400 mg/kg) and the highest doses (800 mg/kg), respectively, over the mean values of control rats, but this diminution was significant only at the highest dose level. Consequently, the number of tubular cross-sections per microscopic field was increased. Concomitant and significant increase in the percentage of damaged tubules was also detected. There was evidence of significantly decreased number of tubules with proper spermatogenesis.9 The possible cause for decrease diameter of tubules may be attributed to oedema in the interstitial spaces which compresses the nearby tubules and atrophy of the tubules. Marked histopathological changes in the present study was noticed. Distorted seminiferous tubules, depressed spermatogenesis, loss of sperms, degeneration and desquamation of gametogenic cells from the basement membrane, accumulation of cellular mass in the lumen of tubules, oedematous exudation in the interstitial spaces which was more marked in the central region and vacuolation in the interstitium was evident. Some of the Leydig cells were showing signs of degeneration. All these findings were much significant with high dose of isoproturon. These findings run parallel with those noted by Sarkar et al (1995).8 The exact mechanism through which isoproturon induced these alterations in spermatogenesis is not clear. There is a possibility that all these effects of isoproturon on both testicular and epididymal components are the result of appearance of the factor (s) from the peripheral circulation due to hepatic and/or renal toxicity of the agent. Hepatic malfunction and renal failure are known to influence testicular functions.10 Lox (1984)11 and Kiran et al (1985)12 stated that malfunctioning of liver and kidney causes general systemic toxicity which alter testicular functions. According to Sarkar et al (1997), high dose of isoproturon cause impairment of androgen biosynthetic process which affects spermatogenesis and induces maturational anomalies of sperm cells. It decreased epididymal sperm count and percent of motile sperms. The seminiferous tubular diameter was also reduced. Leydig cells maintain high concentration of testosterone in the testicular fluid. Therefore, degenerative changes in them indicate some adverse effects on the functional ability of these cells to synthesize testosterone.
CONCLUSION
The present study indicates the potential of isoproturon to affect the function, fertilizing capability and survival of spermatozoa. Therefore, correlative ultrastructural, histochemical and quantitative biochemical studies are likely to enable further insight into this interesting area of research.
ACKNOWLEDGEMENTS
I express my gratitude to the staff of the Department of Anatomy for assistance in providing infrastructure facilities and necessary help. A special appreciation to my head of the Department for his suggestions and constant encouragement in fulfilling this task. 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. Source of Funding: Nil Conflict of interest: All authors have none to declare.
ABBREVIATIONS USED: DDT-
Dichlorodiphenyltrichloroethane, DDE- Dichloro-diphenyldichloro-ethylene, INSA- Indian National Science Academy, IAEC- Institutional Animal Ethical Committee, H and E- Haematoxylin and eosin, ST- Seminiferous tubule, SC- Sertoli cell, LC- Leydig cells
Englishhttp://ijcrr.com/abstract.php?article_id=247http://ijcrr.com/article_html.php?did=2471. Lorenz, Eric S. Potential Health Effects of Pesticides. Ag Communications and Marketing. 2009; 1-8.
2. Daniels JL, Andrew FO, David AS. Pesticides and Childhood Cancers. Department of Epidemiology, School of Public Health, University of North Carolina-Chapel Hill, Chapel Hill, NC. Web of Science. 1997; 105.
3. Hodgson, Ernest, Levi, Patricia E. Pesticides: An important but underused model for the environmental health sciences. Environmental Health Perspectives Supplements. Academic Search Premier. 1996; 104 Suppl 1.
4. Rabeh NM. Effect of red beetroot (Beta vulgaris L.) and its fresh juice against carbon tetrachloride induced hepatotoxicity in rats. World Applied Sciences Journal. 2015; 33 (6): 931-938.
5. Guidelines for care and use of animals in scientific research. Indian National Science Academy New Delhi. S. K. Sahni, Executive Secretary, Indian National Science Academy, New Delhi; 2000.
6. Dikshith TS, Raizada RB, Srivastava MK. Dermal toxicity to rats of isoproturon technical and formulation. Vet Hum Toxicol. 1990; 32(5):432-434.
7. Sarkar SN, Gupta PK. Fetotoxic and teratogenic potential of substituted phenylurea herbicide, isoproturon, in rats. Indian J Exp Biol. 1993; 31 (3):280-282.
8. Sarkar SN, Chattopadhyay SK, Majumdar AC. Subacute toxicity of urea herbicide, isoproturon, in male rats. Indian J Exp Biol. 1995; 33:851-856.
9. Sarkar SN, Majumdar AC, Chattopadhyay SK. Effect of isoproturon on male reproductive system: clinical, histological and histoenzyonological studies in rats. Indian J Exp Biol. 1997; 35(2):133-138.
10. Griffin JE, Wilson JD. Harrison’s principles of internal medicine. Wilson JD, Braunwald E, Isselbacher KJ, Petersdrof RG, Martin JB, Fauci AS, Root KK. Mcgraw Hill, New York; 1991.
11. Lox CD. The effects of acute carbaryl exposure on clotting factor activity in the rat. Ecotoxicol Environ Saf. 1984; 8:280-283.
12. Kiran R, Sharma M, Bansal RC. In vivo effect of carbaryl on some enzymes of rat liver, kidney and brain. Pesticides. 1985; 19:42-43.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241811EnglishN2016June11HealthcareLONG TERM CLINICAL OUTCOME OF POST PARTUM INTRA UTERINE CONTRACEPTIVE DEVICE (PPIUCD) INSERTION
English2833Vilvapriya S.English Veeraragavan K.EnglishBackground: Majority of women are in need of effective contraceptive method for spacing. The unmet need for contraception results in unintended pregnancies, with increased maternal and neonatal complications. PPIUCD insertion is an effective contraceptive method, reduces unintended pregnancy and decreases health care cost expenditure.
Objective: This study aims to evaluate long term complications like bleeding, pain, expulsion rate, perforation, infection, missing strings and continuation rate.
Method: This prospective observational study was carried out in a tertiary care hospital in Chennai, between Jan 2012 - June 2012. CuT 380A was inserted immediately following placental delivery in caesarean section or in normal delivery or within 48 hours of normal delivery. They were followed up at the time of discharge, at 6 weeks, then at 6 monthly intervals till 30 weeks.
Results: A total of 300 PPIUCD acceptors were followed up for 30 months. Most of our acceptors are primipara group (n=199, 66.4%). Most of the insertions are in caesarean deliveries. (n=235, 78.3%), followed by post placental insertions (n=42, 13%) and immediate post partum (n=23, 7.7%). Continuation rate is high 90.7 %( n=272) at the end of 1 year and 84.3% at the end of 30 months. Expulsion rate was 4.7%, bleeding in 8.7%, pain in 8.4% and infection in 1% of acceptors. Removal rate at 30 months was 11%, majority of the removal was for opting to go for permanent method of sterilization (n=19, 6.33%), followed by planning for 2nd child (n=7, 2.3%). Pain was the cause for removal in only 0.67%, bleeding in 1.67%.
Conclusion: PPIUCD is safe and effective method of contraception with high continuation rate, low expulsion and complication rates.
EnglishPost partum, Contraception, PPIUCDINTRODUCTION
Post partum contraception can reduce one third of maternal deaths and 10% of neonatal mortality when pregnancies are spaced two years apart (1). Studies show that pregnancies within 24 months of previous birth have high risk of abortions, preterm births, low birth weight babies, post partum haemorrhage, maternal mortality and neonatal mortality. In India 61% of births were spaced less than three years. Unmet need is greater in first year of post partum (2). Only 3-5% of post partum women want another child within 2 years. So provision of IUCD in the immediate post partum period offers effective and safe method of contraception (3). The copper T 380 A is approved for 10 years. It is a cost effective, non-hormonal method. According to WHO Medical Eligibility Criteria IUCD be inserted within 48 hours of postpartum which is here referred as postpartum IUCD (PPIUCD). Insertion of copper T380A in postpartum period (PPIUCD) saves time and additional visits. There is reduced risk of perforation due to thick walled post partum uterus, reduced perception of bleeding and pain. Breast feeding does not get affected by PPIUCD. Women have been provided an effective method of contraception before discharge from the hospital. Women are highly motivated and more receptive to accept family planning in the immediate post partum period (4). By 2013, 19 states in India have started implementing PPIUCD insertion under National post partum family planning programme. Govt. of India is promoting institutional deliveries through programmes like Janani Suraksha Yojna (JSY). This conditional cash transfer scheme has expanded the accessibility of women to seek facility delivery and post partum care. The increased institutional deliveries give the health care providers an opportunity to effectively counsel and motivate for PPIUCD insertion (2). While follow up data on complications with PPIUCD insertion are available from international sources, given the scale of PPIUCD insertion in India, it is important to generate data on long term complications and continuation rate. Objective: This study aims to evaluate long term complications and continuation rate in women, who accepted PPIUCD insertion in a tertiary care centre. METHODS AND MATERIALS It is a prospective observational study carried out in a tertiary care center for a total number of 300 willing patients who accepted this contraceptive (PPIUCD) under national post partum family planning programme between Jan 2012 – June 2012 and these patients were followed up to two and a half years. Women were enrolled in the study after standardized written consent in the local language. Participants were interviewed prior to discharge after receiving a PPIUCD, at six weeks and then at 6 monthly intervals till two and a half years post insertion. Inclusion criteria: 1. Age >18 years, < 40 years 2. Delivery at term( both labour natural and caesarean deliveries) 3. Within 48 hours of delivery( immediate post placental, immediate post partum) Exclusion criteria: 1. PROM> 18 hours 2. Preterm labour 3. Uterine anomalies, fibroid uterus 4. Ante partum haemorrhage 5. Post partum haemorrhage 6. Immediate ante partum, intrapartum and post partum fever
RESULTS
(i) Age group: In our study most (n=176,58.6%) of the PPIUCD acceptors are in the age group of 20-24 years, followed by 25-29 years age group(n=80,26.6%), who are the active reproductive age women. 7.3% of women in the age group of 20-24 years and 6.3% in the age group of 25-29 years discontinued PPIUCD during our follow up period.
DISCUSSION
PPIUCD are the only post partum family planning method for couples requesting a highly effective, reversible, yet long term contraceptive method that can be initiated during immediate post partum period in lactating women. WHO medical eligibility criteria states that it is generally safe for postpartum lactating women to use PPIUCD with the advantages overweighing disadvantages. We can reduce the unmet need of family planning with this contraceptive. PPIUCD is more convenient for health care providers and for acceptors- using opportunity of child birth when both the mother and provider are at hospital. Another family planning visit and hospitalization is not necessary which is advantageous for socio-economically weaker section of women, who depend on Government hospitals for health care. Fewer instruments and staff are necessary for PPIUCD than for interval IUCD. Govt. of India promotes institutional deliveries and it provides increased opportunity for immediate post partum insertion of CuT. Advantages of immediate post partum insertion are high motivation, assurance that she is not pregnant and convenience. Most of the willing acceptors in our study were primipara (n=199, 66.4%), who are the ideal candidates for this non hormonal reversible spacing method of contraception. Most of our PPIUCD insertion is in intra-caesarean category (n=235, 78.3%), followed by post placental insertions (n=42, 13%) and immediate post partum (n=23, 7.7%). Comparison of our study with similar PPIUCD studies:
In our follow up study, continuation rate is high 90.7 %( n=272) at the end of 1 year and 84.3% at the end of 30 months comparable to other studies by MCHIP, Anjumafshan et al, Rajani Gowtham et al and Sahejakuttur et al. Spontaneous expulsion rate at 30 months was 4.7%, 13 out of 14 expulsion (4.3%) following first year of insertion. Expulsion rate in our study (4.3%) is less than in interval IUCD (5%) at the end of first year of insertion (9). Missing string were observed in 12.4 %( n=37), of whom nearly 2/3rd i.e. 7.7% (n=23) it was in the uterine cavity confirmed by sonography. 8.7 %( n=26) of the acceptors had excessive menstrual bleeding, in which only 5(1.67%) acceptors needed removal of IUCD. In none of the PPIUCD insertions, perforation of uterus occurred because of thick myometrium in the immediate post partum period (10, 11). Infection rate was 1% in our study (n=3) and no one needed removal of IUCD for that complication. They continued IUCD with medical management. This less incidence of infection could be due to antibiotic use in all our caesarean deliveries and careful selection of cases for PPIUCD insertion. Dysmenorrhea in our acceptor were noted in 8.3 %( mild n= 22, moderate n=3), among whom only 0.67% (n=2) needed removal of IUCD. Removal of IUCD for pain (0.67%) and excessive bleeding (1.67%) is less in our PPIUCD users (2.34%) compared to 5-15% in interval IUCD users. Most of the CuT removal in our users were for opting to go for either permanent sterilization 6.33 %( n=19) or for planning for second child 2.3 %( n=2.3%). 10 users (3.3%) needed intravenous sedation in OT for CuT removal. CONCLUSION PPIUCD is safe and effective method of contraception with low expulsion rate and high continuation rate. Women who received PPIUCD showed high level of satisfaction with their choice of contraception. It is not associated with increased risk of infection, perforation, post partum bleeding, sub involution, excessive menstrual bleeding and pain. Hence it can be highly recommended as an effective method of postpartum contraception in developing countries.
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. Conflict of interest: The authors declare that they have no conflicts of interest. Source of funding: No funding received from any source for this study.
Englishhttp://ijcrr.com/abstract.php?article_id=248http://ijcrr.com/article_html.php?did=2481. Somesh Kumar, Reena Sethi, Sudharsanam Balasubramaniam, Elaine Charurat, Kamlesh Lalchandani, Richard Semba, and Bulbul Sood Women’s experience with postpartum intrauterine contraceptive device use in India, Reprod Health. 2014; 11: 32.
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7. Rajni Gautam, K. N. Arya, S. Kharakwal, Sudhir Singh, Monika Trivedi. “Overview of Immediate PPIUCD application in Bundelkhand Region”. Journal of Evolution of Medical and Dental Sciences 2014; Vol. 3, Issue 36, August 18; Page: 9518-9526
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Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241811EnglishN2016June11HealthcareEVALUATION OF NITRIC OXIDE LEVELS AND ARGINASE ACTIVITY IN ORAL CANCER PATIENTS
English3437Ms. Shelgikar PrachiEnglish Abhang Subodhini A.English Iyer C.M.EnglishBackground: Cancer of Oral cavity is an important cancer globally. It remains a major cause of mortality and morbidity throughout the world. In India, because of cultural, geographic factors and the popularity of addictive habits, the frequency of oral cancer is high. Patients with malignant tumors induce various degrees of metabolic derangements. Substantial information has been obtained in the past decades on the role of arginine in tumor growth. Arginine has several important benefits including promotion of protein/collagen synthesis, wound healing and support of the immune system. Disturbances in arginine metabolism possibly contribute to events that lead to cancer cachexia. The net beneficial or negative effect depends on how arginine is metabolized and the strength of the activities of each of these arginine-catabolizing enzymes: Nitric Oxide Synthase and Arginase. Relative changes in these enzymatic activities serve as major determinants of Nitric Oxide (NO•) production. In view of this, the present study was planned to estimate the serum levels of NO• and arginase activity in oral cancer patients and to compare them with age, sex matched healthy controls.
Material and Methods: Fifty histopathologically proved cases of oral cancer from any stage (Stage I to stage IV) in the age group of 40-75 years admitted in Sassoon Hospital, Pune and fifty age, sex matched healthy controls were recruited for this study. Intravenous blood sample was obtained to evaluate study parameters. Arginase activity was estimated by Roman and Ray method while NO• levels were measured by Cortas and Wakid method.
Results: The results of this study showed significant increase in the activity of arginase when compared with controls and activity was found to be significantly increased in stages III+IV when compared with stages I+II (p < 0.01). Nitric oxide level was found to be significantly increased in oral cancer condition when compared to normal individuals (pEnglishArginase, Nitric Oxide, Oral cancerINTRODUCTION
Oral cavity cancer is an important cancer and is one of the ten most frequent cancers worldwide. In India, because of cultural, ethnic, geographic factors and the popularity of addictive habits, the frequency of oral cancer is high (1). It ranks number one in terms of incidence among men and third among women. Several factors like tobacco and tobacco related products, alcohol, genetic predisposition and hormonal factors are suspected as possible causative factors (1, 2). Chronic inflammation is a risk factor for cancer. Cancer-related alterations in metabolism of the host are an important factor in determining mortality. Evidence is accumulating that the amino acid arginine is of importance in cancer. Arginine is one of the 20 amino acids found in protein with numerous roles in cellular metabolism. Arginine has been shown to have several important benefits, including promotion of protein/collagen synthesis, wound healing and support of the immune system through nitric oxide production. Thus, both arginine and its product nitric oxide (NO• ) are important mediators in the defense against tumor cells, because both influence T cell–mediated immunity (3, 4), cytokine induction, and macrophage-mediated tumor toxicity (5).
Nitric oxide is a pleiotropic ancestral molecule, which elicits beneficial effect in many physiological settings but is also tenaciously expressed in numerous pathological conditions. NO• plays multiple roles in both intracellular and extracellular signaling mechanisms (6). This highly reactive molecule is produced in the body by the three isoenzymes of nitric oxide synthase (NOS) using L-arginine as a substrate. NO• is either cytostatic or cytotoxic, interacting with a number of molecular targets within cells. Cells within different tissues display varying responses to NO• . Chronic inflammation causes overexpression of NOS leading to genotoxicity. NO• may mediate DNA damage through the formation of carcinogenic nitrosamines, generation of RNS and inhibition of DNA damage repair mechanism. It can thus be considered as a tumor initiating agent. However, NO• may also have an impact on other stages of cancer development ranging from cellular transformation and formation of neoplastic lesions to the regulation of various other aspects of tumor biology (7). NO• plays an important role in host defense and homeostasis when generated at a low level for a brief period of time, but becomes genotoxic and mutagenic when generated at higher concentrations for prolonged periods of time. Thus, the biological outcome of the NO• mediated effects is complex and depends on the internal and external environment of the target and generation sites of the cells as well as the concentration of NO• generated (8). Besides, various malignant tumor tissues contain considerable amount of the enzyme arginase (9), which converts arginine to ornithine and urea. L-ornithine is the precursor of polyamines, which are essential components of cell proliferation. It was recently shown that the high arginase activity of tumors is a mechanism of tumor-induced immunosuppression through depletion of arginine concentrations in the microenvironment of the tumor (10). Arginase reflects the type of inflammatory response in a specific disease process. Arginine being the common substrate for both arginase and nitric oxide, the prevailing route determines the fate of arginine in tumor promotion or regression. With this background the present work was undertaken to estimate serum levels of arginase and nitric oxide in oral cancer patients and to compare them with age, sex matched healthy controls. MATERIAL AND METHODS The study was carried out at the department of Biochemistry, B. J. Medical College after the approval from institutional ethical committee. Study group: Included total of 50 subjects with oral cancer in the age group of 40-75 years. These patients were divided as stage I, II, III, IV. Stages were grouped as initial stage = I+II and final stage III+IV. All the cases were clinically diagnosed and histopathologically proven for oral cancer. Control group: Fifty age, sex matched healthy adults without oral cancer were included. Exclusion criteria: Subjects with other systemic diseases, taking chemotherapy, radiotherapy any medications/antioxidant supplementation will not be included in the study. A detailed case history of the patient was taken, with an informed consent which was duly signed by each patient. Collection of serum: 5ml of intravenous blood samples of the subjects was collected, centrifuged to separate the serum and stored at -80ºC till the analysis is done. Estimation of serum arginase activity by Roman and Ray method (11)
Ninhydrin reacts with ornithine formed by arginase action in the presence of MnCl2 giving a pink coloured ornithineninhydrin complex which is read spectrophotometrically at 530nm. Concentration was determined using standard graph. Estimation of serum nitric oxide (NO2 +NO3 ) levels by Cortas and Wakid method (12) Nitric oxide concentration was measured as total nitrates and nitrites (NO2 +NO3 ) by the Cortas and Wakid method. Absorbance was read at 545nm. Concentration was determined using standard graph. Statistical analysis: Results are presented as mean ± standard deviation value and statistically analyzed by ANOVA, Dunnett t (2-sided) Post Hoc tests and Student’s unpaired ’t’ test. A ‘p’ value of 0.05 or less was considered significant.
RESULT The present study involved the estimation of arginase activity and levels of nitric oxide in oral squamous cell carcinoma patients (initial stage I + II= 25, final stage III + IV = 25) and their comparison with normal individuals (N=50). We even compared stage III+IV results with stage I+II. The results are expressed in Table-I. In this study serum arginase activity was found to be significantly increased (pEnglishhttp://ijcrr.com/abstract.php?article_id=249http://ijcrr.com/article_html.php?did=2491. Byakodi R, Byakodi S, Hiremath S, Byakodi J, Adaki S, Marathe K. Oral cancer in India: an epidemiologic and clinical review. J Community Health 2012 Apr; 37(2): 316-9.
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15. Choudhury B Srivastava S , Choudhury H H et al. Arginase and C-reactive protein as potential serum-based biomarker of head and neck squamous cell carcinoma patients of north east India. Tumor Biology 2014; 35(7): 6739- 6748.
16. Rodriguez PC, Quiceno DG, Ochoa AC. L-arginine availability regulates T-lymphocyte cell-cycle progression. Blood 2007; 109: 1568-73.
17. KAPLAN ?brahim, AYDIN Yener, B?LEN Yusuf et al. The evaluation of plasma arginine, arginase, and nitric oxide levels in patients with esophageal cancer. Turk J Med Sci 2012; 42 (3): 403-409.
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