Two main populations of nephrons are recognizable in the kidney: those possessing a short loop of Henle and those with a long loop of Henle . The loop of Henle is composed of the straight portion of the proximal tubule (pars recta), the thin limb segments, and the straight portion of the distal tubule (thick ascending limb, or pars recta). The length of the loop of Henle is generally related to the position of its parent glomerulus in the cortex. Most nephrons originating from superficial and midcortical locations have short loops of Henle that bend within the inner stripe of the outer medulla close to the inner medulla. A few species, including humans, also possess cortical nephrons with extremely short loops that never enter the medulla but turn back within the cortex. Nephrons originating from the juxtamedullary region near the corticomedullary boundary have long loops of Henle with long descending and ascending thin limb segments that enter the inner medulla. Many variations exist, however, between the two basic types of nephrons, depending on their relative position in the cortex. The ratio between long and short loops varies among species. Humans and most rodents have a larger number of short-looped than long-looped nephrons
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TheÂ urinary systemÂ orÂ urinary tractÂ is theÂ organ systemÂ that produces, stores, and eliminatesÂ urine. In humans it includes twoÂ kidneys, twoÂ ureters, theÂ bladderÂ and theÂ urethra. The female and male urinary system are very similar, they differ only in the length of the urethra.
The kidneys are bean-shaped organs that lie in theÂ abdomen,Â retroperitonealÂ to the organs of digestion, around or just below theÂ rib cage and close to theÂ lumbar spine. The organ is about the size of a human fist and is surrounded by what is called Peri-nephric fat, and situated on the superior pole of each kidney is anÂ adrenal gland. The kidneys receive theirÂ bloodÂ supply of 1.25 L/min (25% of theÂ cardiac output) from the renal arteries which are fed by theÂ abdominal aorta. This is important because theÂ kidneys' main role is to filterÂ water solubleÂ waste products from the blood. The other attachment of the kidneys are at their functional endpoints theÂ ureters, which lies more medialÂ and runs down to theÂ trigone of urinary bladder. The kidneys perform aÂ number of tasks, such as: concentrating urine, regulating electrolytes, and maintainingÂ acid-base homeostasis. The kidney excretes and re-absorbsÂ electrolytesÂ (e.g.Â sodium,Â potassiumÂ and calcium) under the influence of local and systemicÂ hormones.Â pHÂ balance is regulated by the excretion of Â bound acidsÂ andÂ ammonium ions. In addition, they removeÂ urea, aÂ nitrogenousÂ waste product from theÂ metabolismÂ ofÂ amino acids. The end point is aÂ hyper osmolar solution carrying waste for storage in the bladder prior toÂ urination.
HumansÂ produce about 2.9 litres of urine over 24 hours, although this amount may vary according to circumstances. Because the rate of filtration at the kidney isÂ proportionalÂ to theÂ glomerular filtration rate, which is in turn related to the blood flow through the kidney, changes in body fluid status can affect kidney function. Hormones exogenous and endogenous to the kidney alter the amount ofÂ bloodÂ flowing through theÂ glomerulus.Â
Diabetes mellitus, is a group of metabolic disease characterized by highÂ blood sugar, due to either of the factors such as reduced insulin secretion, decreased glucose utilization, and increased glucose production.
This metabolic dysregulation associated with DM causes secondary pathophysiologic changes in multiple organ systems and impose a tremendous burden on the individual with diabetes and on the health care system.
DM will be a leading cause of morbidity and mortality for the foreseeable future.
Globally, as of 2012, an estimated 346Â million people have typeÂ 2 diabetes.
Globally, as of 2010, an estimated 285Â million people had diabetes, with typeÂ 2 making up about 90% of the cases. Its incidence is increasing rapidly, and by 2030, this number is estimated to almost double.Â Diabetes mellitus occurs throughout the world, but is more common in the more developed countries . Based on current trends, the International Diabetes Federation projects that 438 million individuals will have diabetes by the year 2030. Although the prevalence of both type 1 and type 2 DM is increasing worldwide, the prevalence of type 2 DM is rising much more rapidly, presumably because of increasing obesity, reduced activity levels as countries become more industrialized, and the aging of the population.The increase in incidence in developing countries due to urbanization and change in lifestyle.
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India has more diabetics than any other country in the world, according to the International Diabetes Foundation.Â The disease affects more than 50 million Indians, 7.1% of the nation's adults and kills about 1 million Indians a year. The average age of onset is 42.5 years. The high incidence is attributed to a combination of genetic susceptibility and adoption of a high-calorie, low-activity lifestyle mainly by India's growing middle class.
Type 1 DM is the result of interactions of genetic, environmental, and immunologic factors that ultimately lead to the destruction of the pancreatic beta cells and insulin deficiency. Type 1 DM results from autoimmune beta cell destruction, and most, but not all, individuals have evidence of islet-directed autoimmunity. Some individuals who have the clinical phenotype of type 1 DM lack immunologic markers indicative of an autoimmune process involving the beta cells and the genetic markers of type 1 diabetes. These individuals are thought to develop insulin deficiency by unknown, nonimmune mechanisms. Individuals with a genetic susceptibility have normal beta cell mass at birth but begin to lose beta cells secondary to autoimmune destruction that occurs over months to years. This autoimmune process is thought to be triggered by an infectious or environmental stimulus and to be sustained by a beta cell-specific molecule. In the majority, immunologic markers appear after the triggering event but before diabetes becomes clinically overt. Beta cell mass then begins to decrease, and insulin secretion progressively declines, although normal glucose tolerance is maintained. The rate of decline in beta cell mass varies widely among individuals, with some patients progressing rapidly to clinical diabetes and others evolving more slowly. Features of diabetes do not become evident until a majority of beta cells are destroyed (70-80%). At this point, residual functional beta cells exist but are insufficient in number to maintain glucose tolerance. The events that trigger the transition from glucose intolerance to frank diabetes are often associated with increased insulin requirements, as might occur during infections or puberty. After the initial clinical presentation of type 1 DM, a "honeymoon" phase may ensue during which time glycemic control is achieved with modest doses of insulin or, rarely, insulin is not needed. However, this fleeting phase of endogenous insulin production from residual beta cells disappears as the autoimmune process destroys remaining beta cells, and the individual becomes insulin deficient. Some individuals with long-standing type 1 diabetes produce a small amount of insulin (as reflected by C-peptide production) and some individuals have insulin-positive cells in the pancreas at autopsy.
Insulin resistance and abnormal insulin secretion are central to the development of type 2 DM. Although the primary defect is controversial, most studies support the view that insulin resistance precedes an insulin secretory defect but that diabetes develops only when insulin secretion becomes inadequate. Type 2 DM likely encompasses a range of disorders with common phenotype of hyperglycemia. Most of our current understanding of the pathophysiology and genetics is based on studies of individuals of European descent. It is becoming increasing apparent that DM in other ethnic groups (Asian, African, and Latin American) has a different, but yet undefined, pathophysiology. In these groups, DM that is ketosisprone (often obese) or ketosis-resistant (often lean) is commonly seen.
The cause of diabetes depends on the type.
TypeÂ 1 diabetes is partly inherited, and then triggered by certain infections, with some evidence pointing atÂ Coxsackie B4 virus. However, even in those who have inherited the susceptibility, typeÂ 1 DM seems to require an environmental trigger. The onset of typeÂ 1 diabetes is unrelated to lifestyle.
TypeÂ 2 diabetes is due primarily to lifestyle factors and genetics.
The following is a comprehensive list of other causes of diabetes:
I. Type 1 diabetes (beta cell destruction, usually leading to absolute insulin deficiency)
II. Type 2 diabetes (may range from predominantly insulin resistance with relative insulin deficiency to a predominantly insulin secretory defect with insulin resistance)
III. Other specific types of diabetes
A. Genetic defects of beta cell function characterized by mutations in:
1. Hepatocyte nuclear transcription factor (HNF) 4 (MODY 1)
2. Glucokinase (MODY 2)
3. HNF-1 (MODY 3)
4. Insulin promoter factor-1 (IPF-1; MODY 4)
5. HNF-1 (MODY 5)
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6. NeuroD1 (MODY 6)
7. Mitochondrial DNA
8. Subunits of ATP-sensitive potassium channel
9. Proinsulin or insulin
B. Genetic defects in insulin action
1. Type A insulin resistance
3. Rabson-Mendenhall syndrome
4. Lipodystrophy syndromes
C. Diseases of the exocrine pancreas-pancreatitis, pancreatectomy, neoplasia, cystic fibrosis, hemochromatosis, fibrocalculous pancreatopathy, mutations in carboxyl ester lipase
D. Endocrinopathies-acromegaly, Cushing's syndrome, glucagonoma, pheochromocytoma, hyperthyroidism, somatostatinoma, aldosteronoma
E. Drug- or chemical-induced-glucocorticoids, vacor (a rodenticide), pentamidine, nicotinic acid, diazoxide, adrenergic agonists, thiazides, hydantoins, asparaginase, interferon, protease inhibitors, antipsychotics (atypicals and others), epinephrine
F. Infections-congenital rubella, cytomegalovirus, coxsackievirus
G. Uncommon forms of immune-mediated diabetes- "stiff-person" syndrome, anti-insulin receptor antibodies
H. Other genetic syndromes sometimes associated with diabetes- Wolfram's syndrome, Down's syndrome, Klinefelter's syndrome, Turner's syndrome, Friedreich's ataxia, Huntington's chorea, Laurence-Moon-Biedl syndrome, myotonic dystrophy, porphyria, Prader-Willi syndrome
SIGNS AND SYMPTOMS
The classical symptoms of untreated diabetes are loss of weight,Â polyuriaÂ (frequent urination),Â polydipsiaÂ (increased thirst) andÂ polyphagia(increased hunger).Â Symptoms may develop rapidly (weeks or months) in typeÂ 1 diabetes, while they usually develop much more slowly and may be subtle or absent in typeÂ 2 diabetes.
Other symptoms are
- blurred vision
- peripheral neuropathy
- loss of libido
- hyperpigmentation and skin rashes
Impaired fasting glycaemia
â‰¥ 6.1(â‰¥110) & <7.0(<126)
Impaired glucose tolerance
Diabetes mellitus is characterized by recurrent or persistent hyperglycemia, and is diagnosed by demonstrating any one of the following:
Fasting plasma glucose level â‰¥Â 7.0Â mmol/l (126Â mg/dl)
Plasma glucoseÂ â‰¥Â 11.1Â mmol/l (200Â mg/dL) two hours after 75Â g oral glucose load as in aÂ glucose tolerance test
Symptoms of hyperglycemia and casual plasma glucose â‰¥Â 11.1Â mmol/l (200Â mg/dl)
Glycated hemoglobinÂ (Hb A1C) â‰¥Â 6.5%
A positive result, in the absence of unequivocal hyperglycemia, should be confirmed by a repeat of any of the above methods on a different day.Â According to the current definition, two fasting glucose measurements above 126Â mg/dl (7.0Â mmol/l) is considered diagnostic for diabetes mellitus.
Glycated hemoglobinÂ is better thanÂ fasting glucoseÂ for determining risks of cardiovascular disease and death from any cause.
All forms of diabetes increase the risk of long-term complications. These typically develop after many years (10-20), but may be the first symptom in those who have otherwise not received a diagnosis before that time.
DKA[ diabetic ketoacidosis]
HHS[ hyperglycemic hyperosmolar state]
The chronic complications of DM affect many organ systems and are responsible for the majority of morbidity and mortality associated with the disease. Chronic complications can be divided into vascular and nonvascular complications.The vascular complications of DM are further subdivided into microvascular (retinopathy, neuropathy, nephropathy) and macrovascular complications [coronary heart disease (CHD), peripheral arterial disease (PAD), cerebrovascular disease]. Nonvascular complications include problems such as gastroparesis, infections, and skin changes. Long-standing diabetes may be associated with hearing loss. Whether type 2 DM in elderly individuals is associated with impaired mental function is not clear.
Sensory and motor (mono- and polyneuropathy)
Coronary heart disease
Peripheral arterial disease
Gastrointestinal (gastroparesis, diarrhea)
Genitourinary (uropathy/sexual dysfunction)
DKA results from relative or absolute insulin deficiency combined with counterregulatory hormone excess (glucagon, catecholamines, cortisol, and growth hormone). Both insulin deficiency and glucagon excess, in particular, are necessary for DKA to develop
Hyperglycemic hyperosmolar state
Relative insulin deficiency and inadequate fluid intake are the underlying causes of HHS. Insulin deficiency increases hepatic glucose production (through glycogenolysis and gluconeogenesis) and impairs glucose utilization in skeletal muscle (see above discussion of DKA). Hyperglycemia induces an osmotic diuresis that leads to intravascular volume depletion, which is exacerbated by inadequate fluid replacement. The absence of ketosis in HHS is not understood
The goals of therapy for type 1 or type 2 DM are to
(1) eliminate symptoms related to hyperglycemia,
(2) reduce or eliminate the long-term microvascular and macrovascular complications of DM, and
(3) allow the patient to achieve as normal a lifestyle as possible.
To reach these goals, the physician should identify a target level of glycemic control for each patient, provide the patient with the educational and pharmacologic resources necessary to reach this level, and monitor/treat DM-related complications.
- oral hypoglycemic agents
URINARY TRACT INFECTIONS
Epidemiologically, urinary tract infections are sub-divided into catheter associated or nosocomial infection or non catheter associated community acquired infections. Infections in either category may be symptomatic or Asymptomatic. Acute community infections are very common and account for more than 7 million Hospital visits annually in the united states. There infections occur in 1-3% of school girls and then increased markedly in incidence with the onset of sexual activity in adolescence. The vast majority of acute infections involve women. Acute symptomatic Urinary Tract Infection's are unusual in men under the young age group of 50 yrs. The development of asymptomatic bacteriuria parallels that of symptomatic infections and in rare among men under 50 years but common among women between 20 and 50 years. Asymptomatic bacteriuria is more among elderly men and women with rates as high as 10-50% in some studies.
Causative organisms of Urinary Tract Infection's are the most common organism are Gram Negative Bacilli
Escherichia coli - 70-80%
Klebsiella - 2-3%
Proteus - 2-4%
Enterococcus - 1-2%
Gram positive cocci
Staphylococcus saprophyticus - 10-15%
More commonly serratia and pseudomonas assure increasing important in recurrent infections and in infections associated with urological manipulation, calculi or obstruction.
Proteus species and klebsiella species by virtue of urease production and through the production of extracellular slime and polysaccharides, predispose to stone formation and are isolated more frequently from patients with calculi.
Chlamydia trachomatis, Neisseria gonorrhea, and herpes simplex virus are etiological important. These agents are found most frequently in young, sexually active women with new sexual partners.
The causative role of non-bacterial pathogens in Urinary Tract Infection's remains poorly defined. Urea plasma urealyticum has frequently been isolated from the urethra and urine of patients with acute dysuria and frequency but is also found in specimens from many patients without urinary symptoms.
Mycoplasma hominis have been isolated from prostatic and renal tissues of patients with acute prostatitis and pyelonephritis, and are probably irresponsible for some of the infections as well. Candida and other fungal infection is common and sometimes progressive to symptomatic invasive infection.
Mycobacterial infection of Urinary Tract Infection is also a common cause of Asymptomatic Bacteriuria.
PATHOGENESIS AND SOURCES OF INFECTION
The urinary tract should be viewed as a single anatomic unit that is united by a single column of urine extending from the urethra to the kidney.
Routes of Entry to the urinary tract
Mostly the infections of kidney units from organisms desired from gastrointestinal tract to the urethra and periurethra tissues into the bladder and then by the catheter to renal pelvis with subsequent invasion of renal medulla.
Accounts for less than 3% cases of Urinary Tract Infection and pyelonephritis. The major cases of hematogenous infection are staphylococcus aureus, salmonella species, pseudomonas aeruginosa, Enterococcus faecalis
Spread of infection along the lymphatic channels connecting bowel and urinary tract is possible.
GENDER AND SEXUAL ACTIVITY
In females the urethra is prone for gram negative bacilli infection because it is close to the perineum and its short length and its termination beneath the labia. In addition use of spermicidal compounds with a diaphragm or a cervical cap or of spermicide coated condoms dramatically alters the normal introital bacterial flora and has been associated with marked increase of colonization of E. coli and the risk of Urinary Tract Infection. In males who are a 50 years old and who have no H/o Heterosexual or Homosexual rectal intercourse, Urinary Tract Infection is exceedingly uncommon. Men & women who are infected with HIV are at increased risk of both bacteriuria and Urinary Tract Infection. Lack of circumcision has been identified as a risk factor for Urinary Tract Infection in both neonates & young men.
Pregnancy: 2-9 of Pregnant women 20-30% of pregnant women with Asymptomatic Bacteriuria subsequently develop pyelonephritis. Catheterization during or after delivery causes additional infections.
Obstruction: Any obstruction in free flow of urine tumour, stricture, stone, or prostatic hypertrophy results in increased frequency of Urinary Tract Infection.
NEUROGENIC BLADDER DYSFUNTION
Interference with nerve supply to the bladder, as in spinal cord onjury, Tabes dorsalis, multiple sclerosis. Diabetes and other diseases may be associated with Urinary Tract Infection. In additional factor operative in there causes is bone demineralization due to immobilization, which causes hypercalciuria, calculus formation & obstructive uropathy.
Vesicoureteral Reflux: Anatomically impaired vesicoureteral function facilities reflux of bacteria and thus Urinary Tract Infection.
BACTERIAL VIRULENCE FACTORS
Virulence factors of E.coli - surface antigen & toxins
Somatic Polysaccharide surface 0 antigen
Exerts endotoxic activity
Protects bacillus from phagocytosis
Protects bacillus from bactericidal effects of complement.
K antigens or envelop
P fibriae binds specifically to the P blood group substance on human erythrocytes and uroepithelial cells.
The E.coli serotypes commonly responsible for Urinary tract infections are those normally found in the faces, o group 1,2,4,6,7 strains carrying K antigens are more commonly responsible for pyelonephritis.
Increasing evidence suggests that host genetic factors influence susceptibility to Urinary Tract Infection. A maternal History of Urinary Tract Infections is found more among women who have experienced recurrent Urinary Tract Infection's than among controls. It has also been demonstrated that non-secretions of blood group antigens are at increased risk of Urinary Tract Infection.
DEFENSE MECHANISMS OF URINARY TRACT
Normal flora of the vagina
Flushing effect of urine flow and voiding
IgA, IgM, IgG Antibodies
METHOD OF URINE SAMPLE COLLECTION
Collection of midstream specimens of urine.
Suprapubic aspiration of urine
Collection of Midstream Urine collection specimen
Patient must have a full bladder
Retract the fore skin if present
Clean glans penis with swab
Void into toilet with foreskin retracted until half done
Without interrupting stream catch sample in sterile bottle
Patient must have a full bladder
Patient removes underclothing and stands legs either side of toilet
Separate labia with left hand
Cleans vulva front to back with sterile swab
Void downward into toilet until half done
Without interrupting stream catch urine in sterile bottle
The method of urine sample collection is followed because the distal urethra contains bacteria normally so voided urine is contaminated so Midstream Urine collection is done.
SUPRAPUBIC ASPIRATION OF URINE
This method is used when it is impossible to obtain uncontaminated samples or in symptomatic patients with low bacterial counts. This method is not usually followed
Patient must have a full bladder which can be percussed if not percussible or in doubt, give 300ml of water and 20mg of furosemide orally and wait for 1 hr. If still in doubt and especially in obese subjects, localize bladder using ultrasound. When patient supine chose site in midline 2.5cm above symphysis pubis, clean skin with spirit impregnated sterile gauze. Insert a 21 gauge 1.5" needle, attached to a 10ml syringe, directly downwards and aspirate urine. Withdraw needle and collect urine local anesthetics may be used.
It is nearly unnecessary to catheterize patients for collection of urine sample because catheter may introduce infection in the bladder and results in false positive cultures.
Urine being an excellent superfine medium for growth of most bacteria, it must be plated immediately or refrigerated at 4oc. Bacterial counts in refrigerated urine remains constant for as long as 24 hrs.
BD urine culture kit, a urine transport tube containing boric acid, glycerol, sodium formate, preserve bacteria without refrigeration for as long as 24 hrs when greater than 105 CFU/ml were present in initial urine specimen.
SAGE products care of III preservative system also available.
Both above products preserve bacterial inability in urine for 24 hours in the absence of antibodies. A new lyophilized system appears to stabilize microbial population for 24 hrs in the presence of antibiotics. For population of patients from whom colony counts of organisms of less than 105 /ml might be clinically significant, planting within 2 hour collection is recommended. None of the kits have any advantage over refrigeration.
TESTS FOR URINARY TRACT INFECTION
Grams stain method
Earliest least expensive and probably the most sensitive and reliable screening method for identifying urine samples that contain greater than 10 CFU/ ml
A drop of well mixed urine is allowed to dry. The smear is gram stained and examined under oil immersion (1000 x ) . Presence of atleast one organism per oil immersion field, examining 20 fields correlated with significant bacteriuria in 90% cases. The gram stain should not be relied on for detecting polymorphonuclear leucocytes in urine.
GRIESS NITRITE test
The test is based on ;the absence of nitrite in normal urine. The presence of nitrite, Detected by a simple test indicates the presence of nitrate inducing bacteria in urine. A positive test suggests the presence of atleast 105 organisms per ml of urine. This test detects Escherchia coli, klebsiella, proteus, staplylococcus, and pseudomonas species. False negative tests occurs in the presence of yeast, some gram positive cocci, urinary ascorbic acid, frequent voiding, urobilinogen.
This test depends on the generation of oxygen bubbles by catalase produced by the bacteria when hydrogen peroxide is added to the infected urine. False positive results occur in the hematuria.
4. Triphenyl Tetrazolium chloride test
The respiratory activity of growing bacteria, reduce 2,3,5 triphenlytetrazolium chloride to red insoluble triphenyl formogen false positive, false negative results are in the range of 5-10%. This test not used widely.
Glucose oxidase test
Depends on the bacterial metabolism of glucose normally present in urine. In the presence of infection glucose is not detected. False negative tests occur with high urine low rather and frequent voiding, an infected urine must be present in the bladder for some 1 hour before the glucose is metabolized completely. False positive results occur in glycosuric patients.
6.Leucocyte esterase test
This test detects the presence of pyuria by measurement of esterase activity within leucocytes, even in ;the absence of intact neutrophils. On its own it is relatively sensitive. However this test has been combined with griess test sensitivity and ESS-SS-Specificity. On study showed the negative predictive value of the combined tests to be 97.5% therefore if both tests are negative the possibility of a positive urine culture is remote.
7 . Dipslide culture methods
Agar counted slides are immersed in urine or even exposed to the stream of urine during voiding, incubated and growth is estimated by colony counting, by colour change of indications.
Based on detection of adenosine triphosphate (ATP) by measuring light emitted by the reaction of lacifenin luciterase. These tests are expensive and takes time.
9. BAC- T screen bacteriuria detection device
In this method the urine is forced through a filter paper, which retains microorganisms, somatic cells and other particles. A dye is then added to the filter paper to visualize the particulate matter that has adhered. The intensity of colour relates to number of particles. This procedure takes approximately one minute, has been shown to detect greater than 90% of all positive urine specimens even in 102 organisms per ml are consider to be significant.
A manual filtration method using the reagents on ;the Bac-T screen in the filtrate checks out Urinary Tract Infection.
Another promising recently introduced manual system combines filtration with differential media to quantitate and identify presumptively uropathogens with results available within 4 hrs.
None of the screening methods are as sensitive or as reliable on a culture. These tests may have a role in ;the immediate diagnostic. Screening of symptomatic patients and may be if some value in mass screening programs. They are not a substitute for urine culture.
10.NITRITE DIPSTICK TEST
Nitrite dipstick is subject to false-negatives, because 4 to 6 hours is required for bacteria to convert nitrate to nitrite in bladder urine, and some infecting organisms are nitrite negative. In a study of bacteriuria screening in infants ,85% of nitrite tests were false negative compared with culture. A 53% false-negative rate was also reported in an obstetric population with dipstick screening of nitrite.
QUANTITATIVE URINE CULTURE
The quantitative urine culture remains the optimal screening test.
Pour plate dilution technique:
This is an extremely accurate method but time consuming. It is used as a standard of comparison for other methods. Here double dilution series of urine or two fold dilution of urine are spread over the culture plate. The number of colonies in each plate in head in 24 hours and 48 hours and colonies calculated.
Surface culture methods:
Serial 10 fold dilution of urine are plated by surface culture method Number of colonies are calculated at end of 24 hr & 48 hrs.
Both the above methods are too complicated for routine diagnostic ---- for which semi quantitative techniques are more conveniently calibrated bacteriologic loop technique.
Most commonly employed method. In this standard platinum loops or disposable sterile loops are designed to deliver either 0.01 ml or 0.001ml of urine used.
The urine should be mixed thoroughly before plating flame a wire calibrated inoculating loop, allow it to cool without touching any surface if disposable plastic tips are not used. Insert the loop vertically into the urine to allow urine to adhere to the loop spread the loopful of urine to the surface of blood agar loop is touched to the center of the plate, from which the inoculums is spread in a line across the diameter in the plate, without flaming or reentering urine loop in drawn across the entire plate crossing the first inoculums without inflaming insert the loop vertically into the urine again for transfer of a loopful to an indicator medium. Incubate plates for atleast 24 hours at 35o to 37o c in air. The colonies are counted on each plate. The number of colonies CFUs are multiplied by 1000 ( if a 0.001ml loop is used) or by 100 if a 0.01ml of loop was used to determine the number of microorganisms per ml in the original specimen. The former medium gives quantitative measurement of bacteriuria while the later a presumptive diagnosis of the bacterium. The isolated are identified by their properties.
Reincubate plates with no growth or tiny colonies for an additional 24 hours before discarding plates. Since antimicrobial treatment or other factors may inhabit initial growth.
Antibiotic sensitivity test
Antibiotic sensitivity tests may be done directly using the urine samples as inocula and the results confirmed by repeating the tests with individual isolates.
Localization of urinary tract infection
Localisation of urinary tract infection to the bladder or kidney in women and to the bladder, kidney or prostate in men, importantly influences the clinical manifestation, response to treatment likelihood and pattern of recurrent infection and long term prognosis associated with these patients. In diabetic patients with urinary tract infection half of patients have upper urinary tract infection. This stratification of patients by site of infection becomes critical. While an ideal procedure for localization of urinary tract infection does not exist, the following techniques are available.
This method was cystoscopy followed by collection of bladder urine samples for quantitative culture. The bladder is then irrigated, repeatedly to wash out bladder organisms. This is confirmed by collecting further samples at the end of the washout procedure. Catheters are then placed along the ureters and left in the place to collect uretheral urine for quantitative culture. A diagnosis of upper tract infection is based on evidence of a 10 fold increase in bacterial counts in ureteral urine compared with post wash out bladder urine. This technique is invasive, not without morbidity and with considerable urological exploration. This method can identify unilateral Upper Urinary Tract Instrumentation.
Bladder washout technique
This method is now usually considered to be the most acceptable gold standard against which all newer techniques should be compared.
To do the washout test, a triple human catheter is inserted, a specimen is collected for culture and the bladder is emptied of urine. Next 100ml of sterile saline, containing 5mg gentamicin or 2 mg of neomycin and 1,25,000 units of topical streptokinase - stretodornace ( two ampoules of the drug) is injected into the catheter and allowed to remain for 30 min. The bladder is then emptied of urine, washed out with two litres of sterile saline and a post washout culture specimen is obtained. Subsequently, five additional urine specimens are collected 10 min are collected 10 minutes apart and the catheter is withdrawn. After quantitative urine cultures have been done in all specimen patients are classified to have lower tract infection if all post-washout culture specimen or upper tract infection if bacterial count > 102/ml occur in at least 4 of specimen 3 to 7 and there is a long increase in count between specimen 2 and the later specimen.
False positive results occur in those patients who have intermittent shedding of microorganisms from kidney and in patients with vesicoureteric reflux.
NON INVASIVE METHODS
URINARY CONCENTRATING ABILITY
The ability to concentrate urine is used to localize urinary tract infection. Renal infection results in a decreased concentrating ability; but not the bladder infection. Bilateral infection produces greater concentrating defect. Treatment usually produces a return of normal concentrating ability.
MEASUREMENT OF URINARY ENZYMES
Wacker and Dorfman found that urinary lactate dyhydrogenase activity was elevated in upper urinary tractr infection. Recently LDH - iso - enzyme 5 has been investigated as a localization tool.
False positive results occur in the presence of pyuria, haematuria, proteinuria. So that test is insensitive and non specific.
Measurement of urinary Î² glucuronidase activity as a localization tool is suggested by Ronald. In patients with upper urinary tract infection, this enzyme level is high
Vigano and associates suggests measuring the renal tubule cell enzyme N Actyl-Î²-D- Glucosaminidase to localize upper urinary tract infection.
MEASUREMENT OF SERUM OR URINARY ANTIBODIES
Serum levels of antibody directed against the lipopolysaccharide antigen present on bacteria, particularly that of E.Coli are commonly raised in patients with upper tract infection and absent in those with bladder infection. This is most diagnostically useful when an acute increase in antibody titres is demonstrated on serial samples taken over the period of the infection. Sensitivity and specificity of this test is still doubtful.
MEASUREMENT OF C-REACTIVE PROTEIN
Jodal and colleagues reported that consistently elevated level of C - reactive protein in serum, detected by immune diffusion technique were seen in children with pyelonephritis, not with acute cystitis. This test is less sensitive in evaluating adult Urinary Tract Infection.
ANTIBODY COATED BACTERIA ASSAY
Described by Thomas in 1974. This method is widely spread and used now because of its simplicity and apparent reliability.
The test depends upon the demonstration of immunoglobin to somatic or O Antigen on the bacterial cell surface. The presence of immunoglobulins is taken as evidence of invasion of tissues, especially the kidney by bacteria, resulting in an antibody response. IgG, IgM, IgA have all been shown to participate in the antibody coating phenomenon, IgG being predominant. Fluorescein-labelled anti-human immunoglobulin is incubated with infected urine and the number of fluorescent - bacteria present s recorded. Different criterias are used for a positive result. The original criterion of Thomas required 25% of all bacteria seen to be fluorescent to qualify as a positive assay. Subsequent criteria have ranged from 1 to 20 fluorescing bacteria in a search of 200 fields to 2 to 5 fluorescing bacteria in a five minute search.
False positive results occur when vaginal or rectal flora contaminate a urine specimen, proteinuria, prostatitis, haemorrhagic cystitis or bladder infection in the presence of bladder tumors or catheters.
False negative results occur in the range of 16 to 38%, if there is delay in performing the test, particularly if bacterial multiplication continues.
In a first inflection the test may not become positive for 2 weeks.
RESPONSE TO SINGLE DOSE TEATMENT
Described by Robin, using the antibody coated bacteria test in conjunction with single dose therapy, the response to single dose of antibiotics may be used as a localization tool.
Renal scanning with 67Ga citrate has been used to localize infection. A false positive rate of 15% and a false negative rate of 13% have been reported.
MEASUREMENT OF SERUM ANTI TAMM-HORSFALL CLYCOPROTEIN AUTOANTIBODIES
Significant concentration of IgG and IgA anti Tamm-Horsfall glycoprotein antibodies have been observed in patients with acute pyelonephritis, especially in the presence of vesicoureteric reflux. But this is not the case in lower urinary tact infection.
Asymptomatic bacteriuria is common in neonates, preschool children, pregnant women, elderly patients, in diabetics, in catheterized patients and in patients with abnormal urinary tracts or renal disease. Asymptomatic bacteriuria is uncommon in non elderly, non pregnant women and in men.
The patients with diabetes mellitus have many potential reasons to have bacteriuria which in many instances may be asymptomatic, including poor control of blood glucose levels, diabetic neuropathy with neurogenic bladder and chronic urinary retention, impairment of leucocyte function, frequent instrumentation of urinary tract, recurrent vaginitis and diabetic microangiopathy, and large vessel renal vascular disease.
The prevalence of asymptomatic bacteriuria is not significantly influenced by the duration of diabetes or the quality of diabetic control. A recent study that evaluated haemoglobin A1c levels in diabetic patients with and without bacteriuria was unble to relate the risk of bacteriuria to the level of haemoglobin A1c at the time of urine culture, thus concluding that factors other than reversible metabolic derangement place the diabetic at risk of bacteriuria.
The prevalence of asymptomatic bacteriuria increase as diabetic retinopathy becomes more severe, as heart disease and peripheral vascular disease become apparent .
Locaization techniques indicate that approximately half of all diabetic patients with bacteriuria have upper urinary tract involvements. Most of these patients are asymptomatic .
The long term consequences of asymptomatic bacteriuria in patients with diabetes mellitus are poorly documented. These patients are at high risk of developing.
2. Renal corticomedullary abscess
3. Renal carbuncle
4. Emphysematous pyelonephritis
5. Emphysematous cystitis
8. Perinephric abscess