Vulvovaginal-swab or first-catch urine specimen to detect Chlamydia trachomatis in women in a community setting?

Screening for chlamydia in women is widely recommended. We evaluated the performance of two nucleic acid amplification tests for detecting Chlamydia trachomatis in self-collected vulvovaginal-swab and first-catch urine specimens from women in a community setting and a strategy for optimizing the sensitivity of an amplified enzyme immunoassay on vulvovaginal-swab specimens. We tested 2,745 paired vulvovaginal-swab and urine specimens by PCR (Roche Cobas) or strand displacement amplification (SDA; Becton Dickinson). There were 146 women infected with chlamydia. The assays detected 97.3% (95% confidence interval [CI], 93.1 to 99.2%) of infected patients with vulvovaginal-swab specimens and 91.8% (86.1 to 95.7%) with urine specimens. We tested 2,749 vulvovaginal-swab specimens with both a nucleic acid amplification test and a polymer conjugate-enhanced enzyme immunoassay with negative-gray-zone testing. The relative sensitivities obtained after retesting specimens in the negative gray zone were 74.3% (95% CI, 62.8 to 83.8%) with PCR and 58.3% (95% CI, 46.1 to 69.8%) with SDA. In community settings, both vulvovaginal-swab and first-catch urine specimens from women are suitable substrates for nucleic acid amplification tests, but enzyme immunoassays, even after negative-gray-zone testing, should not be used in screening programs.

Quantifying leukocytes in first catch urine provides new insights into our understanding of symptomatic and asymptomatic urethritis

We quantitatively investigated inflammatory cells in the male urethra. Leukocytes in the first catch urine (FCU) from 87 men with and without urethritis were quantitated using haemocytometer counts and stained with an anti-CD45 pan-leukocyte antibody. An increased number of leukocytes in FCU specimens was associated with urethritis (P > 0.002), the presence of discharge and/or dysuria (P < 0.001), and detection of Chlamydia trachomatis (P < 0.001) and Neisseria gonorrhoeae (P < 0.001). In men with urethritis, higher leukocyte counts were also observed in the above groups (P = 0.07, 0.03 and P < 0.0001, respectively). As leukocyte number increased, the likelihood of detecting either pathogen increased. This study suggests that symptoms and signs are a surrogate marker for the degree of inflammation present, and that as urethral inflammation increases, the likelihood of detecting a sexually transmitted pathogen also increases. This would explain why men with asymptomatic urethritis are less likely to have a sexually transmitted infection detected than those with discharge and/or dysuria.

[Urinary specific gravity--comparative measurements using reagent strips and refractometer in 340 morning urine samples]

The specific gravity of urine (SG) indicates the number and weight of solute particles in urine; its measurement is helpful in interpreting proteinuria detected by dipstick tests and in monitoring adequate hydration in patients with nephrolithiasis. Four methods for measuring SG or osmolality of urine are currently available (depression of the freezing-point, urometry, refractometry, cation exchange on a reagent strip). Using a recently developed reagent strip, we have measured SG in morning urines of 340 non-selected outpatients and compared the results with SG measurements by refractometry of the same urines. In 86.2% of all urines, a good positive correlation between SG measured by reagent strip and refractometry was noted (r = 0.913, p = 0.0001). In 13.8% of the urines, however, the SG measured by reagent strip deviated by more than +/- 5 from the value obtained by refractometry; in 90% of these urines, glucosuria (reagent strip values too low or too high), proteinuria (values too high), or bacteriuria/leukocyturia (values too low or too high) could be found. In alkaline urine (pH > 7.0), SG values obtained by reagent strip have to be corrected by +5.

Prevalence and densitometric characteristics of incomplete distal renal tubular acidosis in men with recurrent calcium nephrolithiasis

The aim of this study was to assess the prevalence of incomplete distal renal tubular acidosis (idRTA) in men with recurrent calcium nephrolithiasis and its potential impact on bone mineral density. We conducted a retrospective analysis of 150 consecutive, male idiopathic recurrent calcium stone formers (RCSFs), which had originally been referred to the tertiary care stone center of the University Hospital of Berne for further metabolic evaluation. All RCSFs had been maintained on a free-choice diet while collecting two 24-h urine samples and delivered second morning urine samples after 12 h fasting. Among 12 RCSFs with a fasting urine pH >5.8, a modified 3-day ammonium chloride loading test identified idRTA in 10 patients (urine pH >5.32, idRTA group). We matched to each idRTA subject 5 control subjects from the 150 RCSFs, primary by BMI and then by age, i.e., 50 patients, without any acidification defect (non-RTA group) for comparative biochemistry and dual energy X-ray absorptiometry (DEXA) analyses. The prevalence of primary idRTA among RCSFs was 6.7% (10/150). Patients with idRTA had significantly higher 2-h fasting and 24-h urine pH (2-h urine pH: 6.6 ± 0.4 vs. 5.2 ± 0.1, p = 0.001; 24-h urine pH: 6.1 ± 0.2 vs. 5.3 ± 0.3, p = 0.001), 24-h urinary calcium excretion (7.70 ± 1.75 vs. 5.69 ± 1.73 mmol/d, p = 0.02), but significantly lower 24-h urinary urea excretion (323 ± 53 vs. 399 ± 114 mmol/d, p = 0.01), urinary citrate levels (2.32 ± 0.82 vs. 3.01 ± 0.72 mmol/d, p = 0.04) and renal phosphate threshold normalized for the glomerular filtration rate (TmPO(4)/GFR: 0.66 ± 0.17 vs. 0.82 ± 0.21, p = 0.03) compared to non-RTA patients. No significant difference in bone mineral density (BMD) was found between idRTA and non-RTA patients for the lumbar spine (LS BMD (g/cm(2)): 1.046 ± 0.245 SD vs. 1.005 ± 0.119 SD, p = 0.42) or femoral neck (FN BMD (g/cm(2)): 0.830 ± 0.135 SD vs. 0.852 ± 0.127 SD). Thus, idRTA occurs in 1 in 15 male RCSFs and should be sought in all recurrent calcium nephrolithiasis patients. Bone mineral density, however, does not appear to be significantly affected by idRTA.

Epidemiological, social, diagnostic and economic evaluation of population screening for genital chlamydial infection

OBJECTIVES: To investigate epidemiological, social, diagnostic and economic aspects of chlamydia screening in non-genitourinary medicine settings. METHODS: Linked studies around a cross-sectional population-based survey of adult men and women invited to collect urine and (for women) vulvovaginal swab specimens at home and mail these to a laboratory for testing for Chlamydia trachomatis. Specimens were used in laboratory evaluations of an amplified enzyme immunoassay (PCE EIA) and two nucleic acid amplification tests [Cobas polymerase chain reaction (PCR), Becton Dickinson strand displacement amplification (SDA)]. Chlamydia-positive cases and two negative controls completed a risk factor questionnaire. Chlamydia-positive cases were invited into a randomised controlled trial of partner notification strategies. Samples of individuals testing negative completed psychological questionnaires before and after screening. In-depth interviews were conducted at all stages of screening. Chlamydia transmission and cost-effectiveness of screening were investigated in a transmission dynamic model. SETTING AND PARTICIPANTS: General population in the Bristol and Birmingham areas of England. In total, 19,773 women and men aged 16-39 years were randomly selected from 27 general practice lists. RESULTS: Screening invitations reached 73% (14,382/19,773). Uptake (4731 participants), weighted for sampling, was 39.5% (95% CI 37.7, 40.8%) in women and 29.5% (95% CI 28.0, 31.0%) in men aged 16-39 years. Chlamydia prevalence (219 positive results) in 16-24 year olds was 6.2% (95% CI 4.9, 7.8%) in women and 5.3% (95% CI 4.4, 6.3%) in men. The case-control study did not identify any additional factors that would help target screening. Screening did not adversely affect anxiety, depression or self-esteem. Participants welcomed the convenience and privacy of home-sampling. The relative sensitivity of PCR on male urine specimens was 100% (95% CI 89.1, 100%). The combined relative sensitivities of PCR and SDA using female urine and vulvovaginal swabs were 91.8% (86.1, 95.7, 134/146) and 97.3% (93.1, 99.2%, 142/146). A total of 140 people (74% of eligible) participated in the randomised trial. Compared with referral to a genitourinary medicine clinic, partner notification by practice nurses resulted in 12.4% (95% CI -3.7, 28.6%) more patients with at least one partner treated and 22.0% (95% CI 6.1, 37.8%) more patients with all partners treated. The health service and patients costs (2005 prices) of home-based postal chlamydia screening were 21.47 pounds (95% CI 19.91 pounds, 25.99) per screening invitation and 28.56 pounds (95% CI 22.10 pounds, 30.43) per accepted offer. Preliminary modelling found an incremental cost-effectiveness ratio (2003 prices) comparing screening men and women annually to no screening in the base case of 27,000 pounds/major outcome averted at 8 years. If estimated screening uptake and pelvic inflammatory disease incidence were increased, the cost-effectiveness ratio fell to 3700 pounds/major outcome averted. CONCLUSIONS: Proactive screening for chlamydia in women and men using home-collected specimens was feasible and acceptable. Chlamydia prevalence rates in men and women in the general population are similar. Nucleic acid amplification tests can be used on first-catch urine specimens and vulvovaginal swabs. The administrative costs of proactive screening were similar to those for opportunistic screening. Using empirical estimates of screening uptake and incidence of complications, screening was not cost-effective.

Associations of Urinary Uromodulin with Clinical Characteristics and Markers of Tubular Function in the General Population

BACKGROUND AND OBJECTIVES Allelic variants in UMOD, the gene coding for uromodulin, are associated with rare tubulointerstitial kidney disorders and risk of CKD and hypertension in the general population. The factors associated with uromodulin excretion in the normal population remain largely unknown, and were therefore explored in this study. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Urinary uromodulin excretion was measured using a validated ELISA in two population-based cohorts that included more than 6500 individuals. The Swiss Kidney Project on Genes in Hypertension study (SKIPOGH) included 817 adults (mean age±SD, 45±17 years) who underwent renal ultrasonography and performed a 24-hour urine collection. The Cohorte Lausannoise study included 5706 adults (mean age, 53±11 years) with fresh spot morning urine samples. We calculated eGFRs using the CKD-Epidemiology Collaboration formula and by 24-hour creatinine clearance. RESULTS In both studies, positive associations were found between uromodulin and urinary sodium, chloride, and potassium excretion and osmolality. In SKIPOGH, 24-hour uromodulin excretion (median, 41 [interquartile range, 29-57] mg/24 h) was positively associated with kidney length and volume and with creatinine excretion and urine volume. It was negatively associated with age and diabetes. Both spot uromodulin concentration and 24-hour uromodulin excretion were linearly and positively associated (multivariate analyses) with eGFR<90 ml/min per 1.73 m(2). CONCLUSION Age, creatinine excretion, diabetes, and urinary volume are independent clinical correlates of urinary uromodulin excretion. The associations of uromodulin excretion with markers of tubular functions and kidney dimensions suggest that it may reflect tubule activity in the general population.

Urine tested positive for ethyl glucuronide and ethyl sulphate after the consumption of "non-alcoholic" beer

In abstinence maintenance programs, for reissuing the driving licence and in workplace monitoring programs abstinence from ethanol and its proof are demanded. Various monitoring programs that mainly use ethyl glucuronide (EtG) as alcohol consumption marker have been established. To abstain from ethanol, but not from the taste of alcoholic beverages, in particular non-alcoholic beer has become more and more popular. In Germany, these "alcohol-free" beverages may still have an ethanol content of up to 0.5vol.% without the duty of declaration. Due to severe negative consequences resulting from positive EtG tests, a drinking experiment with 2.5L of non-alcoholic beer per person was performed to address the question of measurable concentrations of the direct metabolites EtG and EtS (ethyl sulphate) in urine and blood. Both alcohol consumption markers - determined by LC-MS/MS - were found in high concentrations: maximum concentrations in urine found in three volunteers were EtG 0.30-0.87mg/L and EtS 0.04-0.07mg/L, i.e., above the often applied cut-off value for the proof of abstinence of 0.1mg EtG/L. In the urine samples of one further volunteer, EtG and EtS concentrations cumulated over-night and reached up to 14.1mg/L EtG and 16.1mg/L EtS in the next morning's urine. Ethanol concentrations in blood and urine samples were negative (determined by HS-GC-FID and by an ADH-based method).

The selective determination of sulfates, sulfonates and phosphates in urine by CE-MS

Metabolite identification and metabolite profiling are of major importance in the pharmaceutical and clinical context. However, highly polar and ionic substances are rarely included as analytical tools are missing. In this study, we present a new method for the determination of urinary sulfates, sulfonates, phosphates and other anions of strong acids. The method comprises a CE separation using an acidic BGE (pHfirst part of the electropherogram, followed by phosphates and potentially highly acidic carboxylates. The selectivity for sulfur-containing species is proved using extracted ion electropherograms based on certain isotopic ratios. Ethyl sulfate can be determined by this method and, thus, CE-MS can be used for determination of this alcohol consumption marker. An SPE method was developed for the extraction of ethyl sulfate and other organic anions. Several additional compounds can be identified based on the accurate mass determined by the TOF MS in conjunction with databases. However, numerous detected compounds have not been reported in urinary metabolite databases so far. Thus, it is demonstrated that the presented method is complementary to the existing methods for metabolite characterization in urine.

Analysis of lorazepam and its 30-glucuronide in human urine by capillary electrophoresis: evidence for the formation of two distinct diastereoisomeric glucuronides

Lorazepam (LOR) is a 3-hydroxy-1,4-benzodiazepine that is chiral and undergoes enantiomerization at room temperature. In humans, about 75% of the administered dose of LOR is excreted in the urine as its 30-glucuronide. CE-MS with negative ESI was used to confirm the presence of LOR-30-glucuronide in urines that stemmed from a healthy individual who ingested 1 or 2 mg LOR, whereas free LOR could be detected in extracts prepared from enzymatically hydrolyzed urines. As the 30-glucuronidation reaction occurs at the chiral center of the molecule, two diastereoisomers can theoretically be formed, molecules that can no longer interconvert. The stereoselective formation of LOR glucuronides in humans and in vitro was investigated. MEKC analysis of extracts of the nonhydrolyzed urines suggested the presence of the two different LOR glucuronides in the urine. The formation of the same two diastereoisomers was also observed in vitro employing incubations of LOR with human liver microsomes in the presence of uridine 5'-diphospho-glucuronic acid as coenzyme. The absence of other coenzymes excluded the formation of phase I or other phase II metabolites of LOR. Both results revealed a stereoselectivity, one diastereoisomer being formed in a higher amount than the other. After enzymatic hydrolysis using beta-glucuronidase, these peaks could not be detected any more. Instead, LOR was monitored. Analysis of the extracts prepared from enzymatically hydrolyzed urines by MEKC in the presence of 2-hydroxypropyl-beta-CD revealed the enantiomerization process of LOR (observation of two peaks of equal magnitude connected with a plateau zone). The data presented provide for the first time the evidence of the stereoselectivity of the LOR glucuronidation in humans.

Microalbuminuria in diabetes mellitus: efficacy of a new screening method in comparison with timed overnight urine collection

Diabetic nephropathy and end-stage renal failure are still a major cause of mortality amongst patients with diabetes mellitus (DM). In this study, we evaluated the Clinitek-Microalbumin (CM) screening test strip for the detection of microalbuminuria (MA) in a random morning spot urine in comparison with the quantitative assessment of albuminuria in the timed overnight urine collection ("gold standard"). One hundred thirty-four children, adolescents, and young adults with insulin-dependent DM Type 1 were studied at 222 outpatient visits. Because of urinary tract infection and/or haematuria, the data of 13 visits were excluded. Finally, 165 timed overnight urine were collected in the remaining 209 visits (79% sample per visit rate). Ten (6.1%) patients presented MA of > or =15 microg/min. In comparison however, 200 spot urine could be screened (96% sample/visit rate) yielding a significant increase in compliance and screening rate (P<.001, McNemar test). Furthermore, at 156 occasions, the gold standard and CM could be directly compared. The sensitivity and the specificity for CM in the spot urine (cut-off > or =30 mg albumin/l) were 0.89 [95% confidence interval (CI) 0.56-0.99] and 0.73 (CI 0.66-0.80), respectively. The positive and negative predictive value were 0.17 (CI 0.08-0.30) and 0.99 (CI 0.95-1.00), respectively. Considering CM albumin-to-creatinine ratio, the results were poorer than with the albumin concentration alone. Using CM instead of quantitative assessment of albuminuria is not cost-effective (35 US dollars versus 60 US dollars/patient/year). In conclusion, to exclude MA, the CM used in the random spot urine is reliable and easy to handle, but positive screening results of > or =30 mg albumin/l must be confirmed by analyses in the timed overnight collected urine. Although the screening compliance is improved, in terms of analysing random morning spot urine for MA, we cannot recommend CM in a paediatric diabetic outpatient setting because the specificity is far too low.

Radiation metabolomics. 1. Identification of minimally invasive urine biomarkers for gamma-radiation exposure in mice

Gamma-radiation exposure has both short- and long-term adverse health effects. The threat of modern terrorism places human populations at risk for radiological exposures, yet current medical countermeasures to radiation exposure are limited. Here we describe metabolomics for gamma-radiation biodosimetry in a mouse model. Mice were gamma-irradiated at doses of 0, 3 and 8 Gy (2.57 Gy/min), and urine samples collected over the first 24 h after exposure were analyzed by ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOFMS). Multivariate data were analyzed by orthogonal partial least squares (OPLS). Both 3- and 8-Gy exposures yielded distinct urine metabolomic phenotypes. The top 22 ions for 3 and 8 Gy were analyzed further, including tandem mass spectrometric comparison with authentic standards, revealing that N-hexanoylglycine and beta-thymidine are urinary biomarkers of exposure to 3 and 8 Gy, 3-hydroxy-2-methylbenzoic acid 3-O-sulfate is elevated in urine of mice exposed to 3 but not 8 Gy, and taurine is elevated after 8 but not 3 Gy. Gene Expression Dynamics Inspector (GEDI) self-organizing maps showed clear dose-response relationships for subsets of the urine metabolome. This approach is useful for identifying mice exposed to gamma radiation and for developing metabolomic strategies for noninvasive radiation biodosimetry in humans.

Comparison of direct and indirect alcohol markers with PEth in blood and Urine in alcohol dependent inpatients during detoxication

The importance of direct and indirect alcohol markers to evaluate alcohol consumption in clinical and forensic settings is increasingly recognized. While some markers are used to prove abstinence from ethanol, other markers are suitable for detection of alcohol misuse. Phosphatidyl ethanol (PEth) is ranked among the latter. There is only little information about the correlation between PEth and other currently used markers (ethyl glucuronide, ethyl sulfate, carbohydrate deficient transferrin, gamma-glutamyl transpeptidase, and methanol) and about their decline during detoxification. To get more information, 18 alcohol-dependent patients in withdrawal therapy were monitored for These parameters in blood and urine for up to 19 days. There was no correlation between the different markers. PEth showed a rapid decrease at the beginning of the intervention, a slow decline after the first few days, and could still be detected after 19 days of abstinence from ethanol.

Plasma and urine neutrophil gelatinase-associated lipocalin (NGAL) in dogs with acute kidney injury or chronic kidney disease.

BACKGROUND Neutrophil gelatinase-associated lipocalin (NGAL) is a protein that is used in human medicine as a real-time indicator of acute kidney injury (AKI). HYPOTHESIS Dogs with AKI have significantly higher plasma NGAL concentration and urine NGAL-to-creatinine ratio (UNCR) compared with healthy dogs and dogs with chronic kidney disease (CKD). ANIMALS 18 healthy control dogs, 17 dogs with CKD, and 48 dogs with AKI. METHODS Over a period of 1 year, all dogs with renal azotemia were prospectively included. Urine and plasma samples were collected during the first 24 hours after presentation or after development of renal azotemia. Plasma and urine NGAL concentrations were measured with a commercially available canine NGAL Elisa Kit (Bioporto® Diagnostic) and UNCR was calculated. A single-injection plasma inulin clearance was performed in the healthy dogs. RESULTS Median (range) NGAL plasma concentration in healthy dogs, dogs with CKD, and AKI were 10.7 ng/mL (2.5-21.2), 22.0 ng/mL (7.7-62.3), and 48.3 ng/mL (5.7-469.0), respectively. UNCR was 2 × 10(-8) (0-46), 1,424 × 10(-8) (385-18,347), and 2,366 × 10(-8) (36-994,669), respectively. Dogs with renal azotemia had significantly higher NGAL concentrations and UNCR than did healthy dogs (P < .0001 for both). Plasma NGAL concentration was significantly higher in dogs with AKI compared with dogs with CKD (P = .027). CONCLUSIONS AND CLINICAL IMPORTANCE Plasma NGAL could be helpful to differentiate AKI from CKD in dogs with renal azotemia.

The prevalence and predictive value of dipstick urine protein in HIV-positive persons in Europe.

INTRODUCTION Proteinuria (PTU) is an important marker for the development and progression of renal disease, cardiovascular disease and death, but there is limited information about the prevalence and factors associated with confirmed PTU in predominantly white European HIV+ persons, especially in those with an estimated glomerular filtration rate (eGFR) of 60 mL/min/1.73 m(2). PATIENTS AND METHODS Baseline was defined as the first of two consecutive dipstick urine protein (DPU) measurements during prospective follow-up >1/6/2011 (when systematic data collection began). PTU was defined as two consecutive DUP >1+ (>30 mg/dL) >3 months apart; persons with eGFR <60 at either DPU measurement were excluded. Logistic regression investigated factors associated with PTU. RESULTS A total of 1,640 persons were included, participants were mainly white (n=1,517, 92.5%), male (n=1296, 79.0%) and men having sex with men (n=809; 49.3%). Median age at baseline was 45 (IQR 37-52 years), and CD4 was 570 (IQR 406-760/mm(3)). The median baseline date was 2/12 (IQR 11/11-6/12), and median eGFR was 99 (IQR 88-109 mL/min/1.73 m(2)). Sixty-nine persons had PTU (4.2%, 95% CI 3.2-4.7%). Persons with diabetes had increased odds of PTU, as were those with a prior non-AIDS (1) or AIDS event and those with prior exposure to indinavir. Among females, those with a normal eGFR (>90) and those with prior abacavir use had lower odds of PTU (Figure 1). CONCLUSIONS One in 25 persons with eGFR>60 had confirmed proteinuria at baseline. Factors associated with PTU were similar to those associated with CKD. The lack of association with antiretrovirals, particularly tenofovir, may be due to the cross-sectional design of this study, and additional follow-up is required to address progression to PTU in those without PTU at baseline. It may also suggest other markers are needed to capture the deteriorating renal function associated with antiretrovirals may be needed at higher eGFRs. Our findings suggest PTU is an early marker for impaired renal function.

Estimation of reference curves for the urinary steroid metabolome in the first year of life in healthy children: tracing the complexity of human postnatal steroidogenesis

CONTEXT Complex steroid disorders such as P450 oxidoreductase deficiency or apparent cortisone reductase deficiency may be recognized by steroid profiling using chromatographic mass spectrometric methods. These methods are highly specific and sensitive, and provide a complete spectrum of steroid metabolites in a single measurement of one sample which makes them superior to immunoassays. The steroid metabolome during the fetal-neonatal transition is characterized by a) the metabolites of the fetal-placental unit at birth, b) the fetal adrenal androgens until its involution 3-6 months postnatally, and c) the steroid metabolites produced by the developing endocrine organs. All these developmental events change the steroid metabolome in an age- and sex-dependent manner during the first year of life. OBJECTIVE The aim of this study was to provide normative values for the urinary steroid metabolome of healthy newborns at short time intervals in the first year of life. METHODS We conducted a prospective, longitudinal study to measure 67 urinary steroid metabolites in 21 male and 22 female term healthy newborn infants at 13 time-points from week 1 to week 49 of life. Urine samples were collected from newborn infants before discharge from hospital and from healthy infants at home. Steroid metabolites were measured by gas chromatography-mass spectrometry (GC-MS) and steroid concentrations corrected for urinary creatinine excretion were calculated. RESULTS 61 steroids showed age and 15 steroids sex specificity. Highest urinary steroid concentrations were found in both sexes for progesterone derivatives, in particular 20α-DH-5α-DH-progesterone, and for highly polar 6α-hydroxylated glucocorticoids. The steroids peaked at week 3 and decreased by ∼80% at week 25 in both sexes. The decline of progestins, androgens and estrogens was more pronounced than of glucocorticoids whereas the excretion of corticosterone and its metabolites and of mineralocorticoids remained constant during the first year of life. CONCLUSION The urinary steroid profile changes dramatically during the first year of life and correlates with the physiologic developmental changes during the fetal-neonatal transition. Thus detailed normative data during this time period permit the use of steroid profiling as a powerful diagnostic tool.