Clinico-bacteriological study of vesical calculus.
Urinary calculi are the third most common affliction of the urinary tract exceeded only by urinary infection and pathologic condition of the prostate.  Bladder stones are the most common manifestation of lower urinary tract lithiasis, currently accounting for 5% of all urinary stone disease.  Exact aetiopathogenesis of stone is still unknown, but Metabolism, Infection, Stasis/Obstruction, Foreign bodies and Malnutrition/Vit. A deficiency has been proved to be a factor behind its formation, no matter where it forms in the body. [3, 4, 5, 6) Presence of upper urinary tract calculi is not necessarily a predisposition to the formation of bladder stones.
The incidence of primary bladder calculi in developed countries has been steadily and significantly declining since the [19.sup.th] century because of improved diet, nutrition and infection control. [7, 3] In these countries, vesical calculi affects adults. However, bladder calculi remain common in developing and less developed countries. In these populations, it remains a disease that affects children. Among children, the disease is far more common in boys than girls.  The information obtained by bacteriological study of stones and their sensitivity to various drugs not only help in establishing the aetiology, but also useful in planning the treatment and prevention of the recurrence.
Our study was conducted in order to establish the bacteriology of stone and urine in an attempt to evaluate the role of infection in the formation of stones. Associated factors like age, sex, site of infection, obstruction, diet were also evaluated.
MATERIAL AND METHODS
The present study was carried out in 94 patients of vesical calculi, who were admitted in Surgical Wards of Sanjay Gandhi Memorial Hospital associated with Shyam Shah Medical College, Rewa (M. P.) India, during the period from August 2014 to July 2015.
The patients admitted in Surgery Ward with the symptoms of pain in abdomen, burning in micturition, frequency of micturition, dribbling or weak stream of urine, retention of urine, haematuria and other symptoms of uraemia, signs such as distension of bladder or abdominal tenderness were admitted in surgical wards as provisional diagnosed cases of urinary calculus and were subjected to investigations including routine Hb%, TLC, DLC, RBS, urine analysis, renal function test, X-ray KUB region, USG and intravenous pyelography as and when required.
Following investigations were carried out.
* Routine and Microscopic.
Collection of Sample
* After preliminary cleaning of genitalia by soap and water, the concerned patients were asked to collect clean mid-stream morning samples in sterile culture tubes. Catheterization was only allowed in cases of retention of urine. The specimens of urine thus collected were examined within 1 hour and not more than 6 hours. These culture tubes were sent to pathology department.
* Routine examination: Colour, Albumin and Sugar.
* Microscopic examination: Red blood corpuscles, pus cells, epithelial cells and crystals and casts. Microscopic examination will be done after centrifugation of 1.0 mL of urine at 1500 R.P.M. Supernatant will be decanted off and one drop of the deposit will be taken on a clean glass slide cover-slip was put over it and examined under low power and higher power field for pus cells, R.B.C, cast and crystals.
It was Carried Out in Maximum Number of Cases
a. Direct Smear Examination by Gram staining method.
b. Culture Material Inoculation on MacConkey media or nutrient agar media.
Haemoglobin, Total leucocyte count, Differential leucocyte count, Blood urea, Blood sugar and Serum creatinine.
* X-ray KUB region, Ultrasonography and Intravenous pyelography as and when required.
* Surgical Treatment: The patients who were fit for surgery were treated by surgery under local, spinal or general anaesthesia according to their age and comorbid conditions.
* Majority of the cases were subjected to Suprapubic Cystolithotomy alone or in conjunction with suprapubic prostatectomy, in some cases cystoscopic litholapaxy were done, access for any complication and they discharged after suture removal and regular follow-up in surgery OPD.
* The stones removed were grossly examined for number, colour, size, shape and type. Core culture of stone was done.
The core culture technique that we employed involved washing the stone surface with sterile physiological saline, crushing the stone with a sterile hacksaw. The crushed stone core was cultured in 5 mL thioglycollate broth, which was incubated at room temperature for 18-24 hours and then subcultures were made on blood agar and MacConkey agar plate for isolation of aetiological agent.(8) The isolated organisms were identified by standard techniques.
Findings were analysed and following observations were recorded.
A highest incidence was seen in the age group of 0-10 (43.61%), out of these 25 cases from 0-5 age group and 16 cases were from 6-10 year of age. Youngest patient seen in the study was 2 years old male and the eldest was 82 years male.
It is evident from the above table that the opacity in bladder was found in 91 cases (96.80) and 3 cases were diagnosed in USG KUB region.
It is evident from above table that USG KUB revealed calculus in 100% cases (n=52)
It is evident from the above table that the 23.04% patients were anaemic, blood urea was raised in 15.95% cases and serum creatinine was raised in 10.63% cases, blood sugar was raised in 3.19% cases.
It is evident from the above table that the UTI was present in 37.2% cases.
The present study "Clinicobacteriological Study of Vesical Calculus" was carried out in 94 patients of vesical calculus who were admitted in Surgical Wards of Sanjay Gandhi Memorial Hospital associated with Shyam Shah Medical College, Rewa, during the period from Aug 2014 to July 2015. Vesical calculus has been one of the most common and distressing maladies of mankind. Since the ancient times and are still with us affecting the patients of all age group, sex and socioeconomic status, although incidence varies in different groups.
Even after extensive research on various aspects no definite aetiological factor is known. Recurrence and its management is still a great problem.
Dietary analysis in cases of vesical calculus gives information about various properties of different foods. Some are calculogenic, while the others are helpful in preventing this by various ways. This information may be of use in decreasing the incidence and specially recurrence of stones.
The bacteriological analysis of vesical calculus and urine, impart the information regarding the urinary tract infection in calculus formation.
In present study, the incidence of vesical calculus in relation to total admission and total urolithiasis cases was 1.13% and 33.69% respectively
In the past 50 years the incidence of vesical calculi in developed countries has declined significantly, but underdeveloped/developing nations still suffer from endemic bladder calculi.
In the present series 43.61% cases of vesical calculus belongs to age group 0-10 years, out of these 25 (26.55% of total) cases were belong to 0-5 years' age group. The second highest incidence was seen in >50 years of age group (26.59%). The youngest patient seen in the study was 2 years of male and oldest was 82 years of male. The findings are conformity with the study of S.G. Kabra et al (1972), Shakya G.R. (1996) and Tiwari et al (2000) who reported that the maximum incidence of vesical calculus is below 10 years of age. Pathogenesis of bladder calculi is likely complex with multiple contributing lithogenic factor, in adult (mainly men over 50 years) outlet obstruction is the main aetiological factor, BPH being the most common cause. The causes of bladder stone formation in children Vit. A deficiency/malnutrition (diet low in animal proteins, which consists mainly cereals), other factors include dehydration, fever, diarrhoea, infection and metabolic abnormality.
In the present series male constituted about 93.61% cases of vesical calculus as compared to 6.38% female. Male-to-female ratio was 14.6:1. This is nearly similar to the finding of M. Singh (2003), S. Singh et al (1992) who reported male-to-female ratio 12:1 and 9:1 respectively.
The study indicates that disease is mainly prevalent in male. The higher incidence of vesical calculus in males in comparison to female may be due to increased serum testosterone level, which favours increased endogenous oxalate production by liver which in turn predisposes to oxalate stone formation. Moreover, increased urinary citrate concentration in females may help in protection against urolithiasis.
In present study 92.55% cases belonged to rural area, while only 7.45% cases were urban; 69.23% cases were pure vegetarian and only 30.77% cases were non-vegetarian (Occasional). Majority of cases (88.29%) belonged to low socioeconomic status and 11.71% cases belonged to middle class and no case belonged to upper class.
In present study, factors appearing to be responsible for vesical calculus are- Vitamin A deficient diet, imbalance diet, diet low in protein, carbohydrate rich diet, low phosphate diet, cheap and easily available food rich in oxalic acid, poor hygiene, lack of knowledge and delay in seeking medical help.
Clinical Features Symptoms
In present study, pain in lower abdomen was seen in 85.10% cases and it could have been due to associated chronic retention of urine and urinary tract infection. Pain usually due to movement of stone, irritating the trigone of bladder in vesical calculus, while ureteric and renal colic is attributed to higher intraluminal pressure due to distension. Burning micturition (92.55%) is almost always associated with urinary tract infection. Retention of urine (19.14%) was due to stone obstructing the outflow and enlarged prostate.
Screaming (pulling of prepuce in male or labia in female) was present in 78.72% cases. Screaming is because of irritation of trigone that stimulate the second and third sacral nerves to the scrotum and third and fourth sacral nerve to perineum. Vitamin A deficiency was present in 3 Cases, one had history of night blindness and Bigot's spot was seen during ophthalmic examination in 2 cases.
In the present series main physical signs were stone palpable in bladder (22.34%), enlarge prostate (19.14%) in per rectal examination.
Suprapubic distension (10.63%)--distended bladder was seen in either impacted urethral or impacted vesical calculus or enlarged prostate with associated vesical calculus.
Stricture urethra was present in 4 cases and phimosis was seen in 2 cases.
BPH, PEM and Hypertension were common associated illness with vesical calculus.
In present study, plain X-ray KUB region revealed calculus in bladder region in 91(96.80%) cases. In 3 cases ROS was not seen either obscured by overlying bowel gas shadows or not radiopaque. In 84 cases radio opacity was seen in bladder region alone. Radio-opacity associated with kidney in 5 cases, one case in urethra and one case in ureter. In one case radio-opacity was seen on DJ stent in situ in bladder. In 10 cases, multiple stones were seen in UB region. Present study indicates that the X-ray KUB region is the main diagnostic tool, non-radiopaque urinary bladder stone can be detected by USG KUB region; 18 cases of BPH, 5 cases of cystitis and one case of bilateral hydronephrosis diagnosed in USG KUB region.
In present study, 23.04% patients were anaemic. Blood urea was raised in 15.95% cases and serum creatinine was raised in 10.63% cases. Blood sugar was raised in 3.19% cases. In 45-year-old female diagnosed as vesicle calculus with B/L hydronephrosis with uterine prolapsed had blood urea 89 mg/dL and serum creatinine was 5.14 mg/dL. The above investigations were carried as and when required. These abnormalities were corrected prior to surgical management.
In the present series, urine in the majority of cases (86.17%) was found to be acidic. Alkaline pH is because of infection with urease producing bacteria (Proteus, strepto, staph). Phosphate calculi mainly develop in alkaline urine and oxalate, both in acidic and alkaline.
Urine Microscopic and Culture
In the present series, urinary tract infection was present in 37.2% cases. The culture was positive in 26 cases out of 84 cases (30.95%). In 23 cases single organism was identified and in 3 cases mixed organism were found.
Surgical Procedures for Bladder Stones
In present study, all cases were managed surgically. Suprapubic cystolithotomy was done and associated disease as enlarged prostate, phimosis, stricture urethra was also operated in same settings.
Bladder calculi were mainly treated by suprapubic cystolithotomy (97.8%). In 14 cases, SPCL with prostatectomy (14.89%) and in 3 case SPCL with bladder neck dilation was done. In one case where stone was present in urethra, it was first pushed into the bladder followed by suprapubic cystolithotomy. In 2 cases cystoscopic litholapaxy was done. In 2 cases, circumcision and in 4 cases urethral dilatation was done along with SPCL.
Number of Stones
Out of the 91 cases operated in the present series, most of them 81 cases (89.01%) had single stone. In 10 cases (10.98%), multiple stones were present.
Gross Examination of Stone
In present study, size (maximum length) of maximum stones was 3-4 cms (42.69%). In 22.2% cases size of stone was 4 to 5 cm, 5.61% cases it was 5-7 cm. The explanation for large stone may be due to ignorance, late presentation of symptom, lack of knowledge because of low socioeconomic status and type of calculus as phosphate calculi remains asymptomatic.
Maximum size of stone removed was 9 x 7 cm weighing 460 gms. Maximum number of stone had rough surface (76.4%), dirty white colour (84.26%) and oval in shape (80.89).
In the present series, most of the patients were free of complications. The common postoperative complications were suprapubic urinary leakage (4.39%), wound infection (3.29%), wound gapping (2.19%), catheter blockade (2.19%) and haematuria (1.09%).
Suprapubic urinary leakage was associated with suprapubic cystolithotomy; possible contributing factors were the presence of urinary tract infection, wound infection, catheter blockade and accidental early removal of urethral catheter. All the cases of urinary leakage were treated conservatively. Wound infection was associated with comorbid condition like anaemia, diabetes and treated by dressing and proper antibiotic.
In present study, there was no mortality.
Core Culture of Stone
Bacteriological study of stone removed from bladder (Core culture of stone) was performed in 70 cases in present study. Culture was positive in 18 cases (25.71%). E. coli were seen in majority of the cases (18.57%); while Staphylococcus aureus (2.85%), Klebsiella aeruginosa (2.85%) and Proteus (2.5%) In 52 cases core culture was sterile.
Similar observation has been noted by R. Kumar (1980), Shigetta M (1993) and M. Singh (2003).
The explanation for the presence of bacteria within the calculi may be due to recurrent urinary tract infection, insignificant intermittent bacteraemia from where the bacteria are excreted in bladder and may act as a nidus for deposition of crystals either by damaging the mucous coat or perhaps also by acting as a nidus for crystallization of salts  Thus, a vicious cycle starts, the infection leading to stone formation and then the stone causing infection [8, 10] Most of the current literature on the subject focuses on pathogenesis of infectious urinary stones. Griffith et al showed that bacterial urease is a primary cause of infection stones. The bacteriological study of urine and stone samples revealed that commonest pathogens were E. coli, Klebsiella aerogenes, Staphylococcus and Proteus spp. E. coli is not a urease producing organism and is not considered to be a stone producing micro-organism. However, the present study revealed that E. coli was predominant microorganism recovered from core of stones.
In majority of cases hospital stay was up to 10 days, average hospital stay was 11.67 days. Average hospital stay in SPCL alone was 10.30 days, in SPCL with prostatectomy was 15.57 days, in SPCL with bladder neck dilatation was 11.3 days and in cystoscopic litholapaxy was 3.5 days.
Postoperative longer stay was because of complication like wound infection, wound gap, abnormal investigation and poor general condition.
SUMMARY AND CONCLUSION
The present study "Clinicobacteriological Study of Vesical Calculus" was carried out in 94 patients of vesical calculus who were admitted in Surgical Wards of Sanjay Gandhi Memorial Hospital associated with Shyam Shah Medical College, Rewa, during the period from Aug 2014 to July 2015.
After admitting the patients, detailed history was recorded systematically including presenting complaints, history of urinary tract infection and history of night blindness. Detailed general and systemic examination was carried out with special emphasis on genitourinary system. Routine investigations were done, special investigation like urine culture and core culture of stone was done to find out aetiological factors. Treatment was planned and patients were operated accordingly. Patients were followed up for any complications during the hospital stay.
Relevant Literature was Reviewed, Observations were Systematically Recorded, Critically Analysed and Following Conclusions Were Made
1. Incidence of vesical calculus was 1.13% out of total surgical admissions and 33.69% out of total urolithiasis cases.
2. Maximum number of patients (43.61%) was in the age group 0-10 year. The second highest incidence (26.59%) was seen in >50 years of age group. Youngest patient was 2 years of male and eldest patient was 82 years of male.
3. Males predominated the females with male-to-female ratio was 14.6:1.
4. Majority of cases were from low socio-economic income group (88.29%).
5. Majority of cases were from rural areas 92.55%.
6. Majority of cases were vegetarian (69.23%).
7. Most common symptom was burning in micturition (92.55%) followed by increased frequency of micturition (88.29%). Pain in lower abdomen (85.10%) and screaming (78.72%).
8. BPH, PEM and Hypertension were common associated illness with vesical calculus.
9. Plain X-ray KUB revealed calculus in urinary bladder region in 91 (96.80%) cases. In 3 cases, ROS was not seen either obscured by overlying bowel gas shadows or not radiopaque. USG KUB revealed calculus in 100% cases (n=52).
10. Anaemia was present in 23.04% cases, blood urea was raised in 15.95% cases and serum creatinine was raised in 10.63% cases and 3 patients were Diabetic.
11. Majority of cases reaction of urine was acidic (86.17%) and in (13.82%) it was alkaline.
12. Urinary tract infection was present in 37.2% cases. Majority of cases urine culture was positive (30.95%). E. coli was the commonest organism (19.04%) followed by Klebsiella aerogenes (3.57%), proteus (2.38%), Staphylococcus aureus (1.19%), pseudomonas (1.19%) and mixed organism was found in 3.3% cases.
13. Bladder calculi were mainly treated by suprapubic cystolithotomy (97.8%). In 14 cases SPCL with prostatectomy (14.89%) and in 3 cases SPCL with bladder neck dilation was done. In one case where stone was present in urethra; it was first pushed into the bladder followed by suprapubic cystolithotomy. In 2 cases cystoscopic litholapaxy was done. In 2 cases circumcision and in 4 cases urethral dilatation was done along with SPCL.
14. In majority of cases single stone was found (89.01%). In 10 cases multiple stones were removed. Size (maximum length) of maximum stones was 3-4 cms. Maximum number of stones had rough surface(76.4%), dirty white colour (84.26%) and oval in shape (80.89)
15. Maximum size of stone removed was 9 x 7 cm weighing 460 gms.
16. Commonest complication in the postoperative period was suprapubic urinary leakage (4.39%), wound infection (3.29%), wound gap (2.19%), catheter blockade (2.19%) and haematuria (1.09%)
17. Core culture of stone was positive in 18 cases (25.71%). E. coli was the predominant organism (18.57%) followed by Staphylococcus aureus (2.85%), Klebsiella (2.85%) and proteus (2.5%).
18. In majority of cases hospital stay was up to 10 days, average hospital stay was 11.67 days.
It is concluded from above study that vesical calculus are common in children of low socioeconomic status. Metabolism, infection, stasis/outlet obstruction, foreign body and malnutrition/vitamin A deficiency remained the important factors in its formation. They are commonly diagnosed by X-ray KUB. However, in places where cystoscopy is available it is the choice both for diagnostic and therapeutic purposes.
Suprapubic cystolithotomy is the most common intervention in present scenario. E. coli is the predominant organism found both in urine and core culture of stone. There is significant association regarding the presence of vesicle calculi and the development of urinary infections. Incidence of vesical calculus is progressively decreasing in urban region because of improved diet, nutrition, infection control and modification of lifestyle.
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[7.] Sarica K, Baltaci S, Kilic S, et al. 371 bladder calculi in a benign prostatic hyperplasia patient. Int Urol Nephro 1994;26(1):23-25.
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Pushpendra Singh (1), Brijesh Singh (2), Vinod Yedaiwar (3), A. P. S. Gaharwar (4)
(1) 3rd Year Junior Resident, Department of General Surgery, Shyam Shah Medical College, Rewa, Madhya Pradesh.
(2) Assistant Professor, Department of General Surgery, Shyam Shah Medical College, Rewa, Madhya Pradesh.
(3) Associate Professor, Department of General Surgery, Shyam Shah Medical College, Rewa, Madhya Pradesh.
(4) Professor and HOD, Department of General Surgery, Shyam Shah Medical College, Rewa, Madhya Pradesh.
Financial or Other, Competing Interest: None.
Submission 22-03-2016, Peer Review 15-04-2016,
Acceptance 21-04-2016, Published 05-05-2016.
Dr. Pushpendra Singh, 21, PG Boys Hostel, Shyam Shah Medical College, Rewa, Madhya Pradesh.
Table 1: Incidence of Vesical Calculus Sl. No. Month Total No. Cases of Total of Admission Urolithiasis No. 1 Aug-14 723 23 6 2 Sep-14 741 31 17 3 Oct-14 730 20 8 4 Nov-14 673 28 11 5 Dec-14 731 32 14 6 Jan-15 590 19 7 7 Feb-15 679 22 5 8 Mar-15 608 16 3 9 Apr-15 624 22 5 10 May-15 638 22 8 11 Jun-15 716 17 5 12 Jul-15 836 21 5 Total 8289 279 94 Cases of Vesical Calculi Sl. No. % Out of Total % Out of Total Admission Urolithiasis Cases 1 0.8 26.0 2 2.3 54.8 3 1.09 40.0 4 1.6 39.2 5 1.9 43.7 6 1.1 36.8 7 0.7 26.0 8 0.4 18.75 9 0.8 22.7 10 1.2 36.3 11 0.7 29.4 12 0.5 23.8 Total 1.13 33.69 Table 2: Distribution of Cases According to Age and Sex Sl. No. Age Group (For) Male % Female % 1 0-10 39 44.31 2 33.33 2 11-20 10 11.36 1 16.67 3 21-30 5 5.68 1 16.67 4 31-40 2 2.27 0 0 5 41-50 8 9.09 1 16.67 6 >50 24 27.27 1 16.67 Total 88 (93.61%) 100 6 (6.39%) 100 Sl. No. Total % 1 41 43.61 2 11 11.70 3 6 6.38 4 2 2.12 5 9 9.57 6 25 26.59 94 100% Table 3: Distribution of Cases According to Residence Sl. No. Residence No. of Cases Percentage 1 Rural 87 92.55 2 Urban 7 7.44 Total 94 Table 4: Distribution of Cases According to Socioeconomic Status Sl. No. Socioeconomic Status No. of Cases Percentage 1 Lower 83 88.29 2 Middle 11 11.70 3 Upper 0 0 Table 5: Distribution of Cases According to the Diet Sl. No. Diet No. of Cases Percentage 1 Vegetarian 65 69.23 2 Non-Vegetarian (Occasional) 28 29.82 Total 94 Table 6: Distribution of Cases According to the Symptoms Sl. No. Symptoms No. of Percentage Cases 1 Burning micturition 87 92.55 2 Increase frequency of micturition 83 88.29 3 Pain in lower abdomen 80 85.10 4 Screaming/Strangury 74 78.72 5 Dysuria 54 57.44 6 Retention (Acute+Chronic) (10+8) 18 19.14 7 Haematuria 12 12.76 8 Fever 7 7.44 9 H/O night blindness 1 1.06 Table 7: Vesical Calculus with Associated Illness Sl. No. Associated Illness No. of Cases Percentage 1 BPH 18 19.14 2 PEM 10 10.63 3 Hypertension 08 8.51 4 Renal calculus 05 5.31 5 Stricture Urethra 04 4.25 6 Vit. A deficiency 03 3.19 7 Rectal prolapsed 02 2.12 8 Bladder diverticulae 02 2.12 9 Phimosis 02 2.12 10 CAD 02 2.12 11 Ureteric Calculus 01 1.06 12 Hydronephrosis 01 1.06 13 Uterine Prolapse 01 1.06 14 RPD 01 1.06 Table 8: Distribution of Cases According to Various Clinical Finding Sl. No. Clinical Finding No. of Cases Percentage 1 Suprapubic distension 10 10.63 2 External genitalia (a) Phimosis 2 2.12 (b) Stricture 4 4.25 3 Rectal Examination (a) Stone palpable in bladder 21 22.34 (b) Enlarged prostate 18 19.14 4 Bitot's spot 2 2.12 Table 9: Distribution of Cases According to Radiological Finding (n=94) Sl. No. Finding on X-ray KUB Region No. of Percentage Cases 1 ROS in Bladder region alone 83 88.29 2 ROS in Bladder and Kidney 5 5.31 3 ROS in Bladder and urethra 1 1.06 4 ROS in Lower Ureter and Bladder 1 1.06 5 ROS on DJ stent in situ 1 1.06 Table 10: Distribution of Cases According to Blood Investigation Sl. No. Investigation No. of Percentage Cases 1 Hb% <10 72 76.56 >10 22 23.04 2 Blood Urea 20-40 79 84.04 41-60 14 14.89 >60 01 1.06 3 Serum Creatinine 0.5-1.2 84 89.36 1.3-2.0 09 9.57 >2.0 01 1.06 4 Random Blood Sugar <140 91 96.81 >140 03 3.19 Table 11: Distribution of Cases According to Microscopic Examination of Urine (n=86) Sl. No. PUS Cell/HPF No. of Cases Percentage 1 < 6 54 62.79 2 7 - 10 24 27.90 3 Loaded 8 9.30 Table 12: Distribution of Cases According to Urine Culture Report (n=84) Sl. No. Bacteria Grown No. of Cases Percentage 1 E. Coli 16 19.04 2 Klebsiella aerogenes 3 3.57 3 Mixed 3 3.57 4 Proteus 2 2.38 5 Staphylococcus aureus 1 1.19 6 Pseudomonas 1 1.19 7 Sterile 58 69.04 Table 13: Distribution of Cases According to Core Culture of Stone (n=70) Sl. No. Bacteria No. of Cases Percentage 1 E. Coli 13 18.57 2 Klebsiella aerogenes 2 2.85 3 Staphylococcus aureus 2 2.85 4 Proteus 1 1.42 5 Sterile 52 74.28 Total 70 100.00
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|Title Annotation:||Original Article|
|Author:||Singh, Pushpendra; Singh, Brijesh; Yedaiwar, Vinod; Gaharwar, A.P.S.|
|Publication:||Journal of Evolution of Medical and Dental Sciences|
|Date:||May 5, 2016|
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