Renal concentrating ability and glomerular filtration rate in lithium-treated patients.

BACKGROUND: Lithium is the most effective drug for mood stabilization in bipolar disorder. However, lithium exposure has been associated with an impaired renal concentrating ability (RCA) and glomerular filtration rate (GFR). We examined RCA and estimated GFR in a cohort of patients treated with lithium. METHODS: 134 patients (≥ 18 years of age) with a mood disorder treated with lithium were screened; 100 patients were included. Demographic and clinical characteristics and blood and urine samples were collected. Additionally, a dDAVP-test was performed to determine maximal RCA. RESULTS: A dDAVP-test was performed in 98 patients (37 males, 61 females). Mean age was 51 years (SD: 12), median duration of lithium therapy 7 years (IQR: 4-15), mean maximal urine osmolality (Uosmol) 725 mOsmol/kg (SD: 153), and median eGFR 84 ml/min/1.73 m2 (IQR: 68-95). Fifty patients (51%) had an impaired RCA and 17 patients (17%) had nephrogenic diabetes insipidus (Uosmol 600-800 and < 600 mOsmol/kg, respectively). Notably, clinical symptoms did not predict an impaired RCA. Nineteen patients (19%) had an eGFR ≤ 60 ml/min/ 1.73 m2. Multivariable regression analysis showed a significant association between the duration of lithium treatment and maximal Uosmol (B = -6.1, 95%-CI: -9.4, -2.9, p < 0.001) and eGFR (B = -0.6, 95%-CI: 0.2, -3.3; p < 0.01). CONCLUSIONS: RCA is impaired in the majority of lithium-treated patients. Both RCA and eGFR are inversely associated with the duration of lithium therapy. Prospective follow-up will enable us to evaluate if abnormalities in RCA can be used to predict the development of lithium-induced chronic kidney disease.

Targeting multiple pathways reduces renal and cardiac fibrosis in rats with subtotal nephrectomy followed by coronary ligation.

AIM: Multiple interacting pathways contribute to progression of renal and cardiac damage in chronic kidney disease followed by chronic heart failure (renocardiac syndrome). We hypothesized that simultaneous pharmacological modulation of critical pathways implicated in renocardiac syndrome would effectively reduce fibrosis in and preserve function of heart and kidney. METHODS: Rats were subjected to subtotal nephrectomy followed 9 weeks later by coronary artery ligation. From week 11 until week 16, rats received vehicle or losartan, or a combination of the NF-kB inhibitor PDTC, the NO donor molsidomine and superoxide dismutase mimetic tempol, or a combination of all four of these plus metoprolol together. At week 16, renal and cardiac structure, function and gene expression were assessed. RESULTS: Individual and combined treatments were similarly effective in limiting cardiac fibrosis and further decline in systolic function. Combined treatment with all five drugs reduced renal fibrosis and CTGF gene expression more effectively than other strategies. Combining all five drugs reduced heart rate, inotropy and mean arterial pressure (MAP). CONCLUSION: Thus, in our model of chronic renocardiac syndrome, combined treatments similarly decreased cardiac fibrosis and stabilized systolic function as losartan alone, perhaps suggesting a dominant role for a single factor such as angiotensin II type 1 (AT1) receptor activation or inflammation in the network of aberrant systems in the heart. However, tubulointerstitial fibrosis was most effectively reduced by a five-drug regimen, pointing to additive effects of multiple pathophysiological pathways in the kidney.

Feasibility of measuring renal blood flow by phase-contrast magnetic resonance imaging in patients with autosomal dominant polycystic kidney disease.

OBJECTIVES: Renal blood flow (RBF) has been shown to predict disease progression in autosomal dominant polycystic kidney disease (ADPKD). We investigated the feasibility and accuracy of phase-contrast RBF by MRI (RBFMRI) in ADPKD patients with a wide range of estimated glomerular filtration rate (eGFR) values. METHODS: First, we validated RBFMRI measurement using phantoms simulating renal artery hemodynamics. Thereafter, we investigated in a test-set of 21 patients intra- and inter-observer coefficient of variation of RBFMRI. After validation, we measured RBFMRI in a cohort of 91 patients and compared the variability explained by characteristics indicative for disease severity for RBFMRI and RBF measured by continuous hippuran infusion. RESULTS: The correlation in flow measurement using phantoms by phase-contrast MRI was high and fluid collection was high (CCC=0.969). Technical problems that precluded RBFMRI measurement occurred predominantly in patients with a lower eGFR (34% vs. 16%). In subjects with higher eGFRs, variability in RBF explained by disease characteristics was similar for RBFMRI compared to RBFHip, whereas in subjects with lower eGFRs, this was significantly less for RBFMRI. CONCLUSIONS: Our study shows that RBF can be measured accurately in ADPKD patients by phase-contrast, but this technique may be less feasible in subjects with a lower eGFR. KEY POINTS: Renal blood flow (RBF) can be accurately measured by phase-contrast MRI in ADPKD patients. RBF measured by phase-contrast is associated with ADPKD disease severity. RBF measurement by phase-contrast MRI may be less feasible in patients with an impaired eGFR.

Hepcidin in chronic kidney disease: not an anaemia management tool, but promising as a cardiovascular biomarker.

Hepcidin is a key regulator of iron homeostasis and plays a role in the pathogenesis of anaemia of chronic disease. Its levels are increased in patients with chronic kidney disease (CKD) due to diminished renal clearance and an inflammatory state. Increased hepcidin levels in CKD patients are supposed to be responsible for functional iron deficiency in these patients and contribute to renal anaemia and resistance to erythropoiesis-stimulating agents. Therefore, hepcidin was purported to be useful as a management tool guiding treatment of renal anaemia. Furthermore, since hepcidin is associated with iron accumulation in macrophages in the vessel wall inducing oxidative stress and atherosclerosis, it has been speculated that hepcidin might function as a biomarker of cardiovascular disease. In this descriptive review, the merits of hepcidin with respect to its role in the pathophysiology of renal anaemia in CKD patients, its presumptive role as a practical diagnostic tool guiding management of renal anaemia, and its possible usefulness as a prognostic biomarker will be discussed.

A tailored implementation strategy to reduce the duration of intravenous antibiotic treatment in community-acquired pneumonia: a controlled before-and-after study.

We previously showed that 40 % of clinically stable patients hospitalised for community-acquired pneumonia (CAP) are not switched to oral therapy in a timely fashion because of physicians' barriers. We aimed to decrease this proportion by implementing a novel protocol. In a multi-centre controlled before-and-after study, we evaluated the effect of an implementation strategy tailored to previously identified barriers to an early switch. In three Dutch hospitals, a protocol dictating a timely switch strategy was implemented using educational sessions, pocket reminders and active involvement of nursing staff. Primary outcomes were the proportion of patients switched timely and the duration of intravenous antibiotic therapy. Length of hospital stay (LOS), patient outcome, education effects 6 months after implementation and implementation costs were secondary outcomes. Statistical analysis was performed using mixed-effects models. Prior to implementation, 146 patients were included and, after implementation, 213 patients were included. The case mix was comparable. The implementation did not change the proportion of patients switched on time (66 %). The median duration of intravenous antibiotic administration decreased from 4 days [interquartile range (IQR) 2-5] to 3 days (IQR 2-4), a decrease of 21 % [95 % confidence interval (CI) 11 %; 30 %) in the multi-variable analysis. LOS and patient outcome were comparable before and after implementation. Forty-three percent (56/129) of physicians attended the educational sessions. After 6 months, 24 % (10/42) of the interviewed attendees remembered the protocol's main message. Cumulative implementation costs were 5,798 (20/reduced intravenous treatment day). An implementation strategy tailored to previously identified barriers reduced the duration of intravenous antibiotic administration in hospitalised CAP patients by 1 day, at minimal cost.

High fall incidence and fracture rate in elderly dialysis patients.

BACKGROUND: Although it is recognised that the dialysis population is ageing rapidly, geriatric complications such as falls are poorly appreciated, despite the many risk factors for falls in this population. The objective of this study was to determine the incidence, complications and risk factors for falls in an elderly dialysis population. METHODS: A one-year observational study of chronic dialysis patients aged ≥ 70 years. At baseline, patient characteristics were noted and during follow-up the vital parameters and laboratory values were recorded. Patients were questioned weekly about falls, fall circumstances and consequences by trained nurses. RESULTS: 49 patients were included with a median age of 79.3 years (70-89 years). During follow-up 40 fall accidents occurred in 27 (55%) patients. Falls resulted in fractures in 15% of cases and in hospital admissions in 15%. In haemodialysis (HD) patients, the mean systolic blood pressure (SBP) before HD was lower in fallers compared with non-fallers (130 vs. 143 mmHg). Several patients in the lower blood pressure category received antihypertensive medication. For every 5 mmHg lower SBP (before HD) the fall risk increased by 30% (hazard ratio (HR) 1.30, 95% CI 1.03-1.65, p = 0.03). Furthermore, fall risk increased by 22% for every 10 pmol/l rise of parathyroid hormone (HR 1.22, 95% CI 1.06-1.39, p = 0.004). CONCLUSIONS: Elderly dialysis patients have a high incidence of falls accompanied by a high fracture rate. Given the high complication rate, elderly patients at risk of falling should be identified and managed. Reduction of blood pressure-lowering medication might be a treatment strategy to reduce falls.

Dutch guideline for the management of hypertensive crisis -- 2010 revision.

Hypertensive crises are divided into hypertensive urgencies and emergencies. Together they form a heterogeneous group of acute hypertensive disorders depending on the presence or type of target organs involved. Despite better treatment options for hypertension, hypertensive crisis and its associated complications remain relatively common. In the Netherlands the number of patients starting renal replacement therapy because of 'malignant hypertension' has increased in the past two decades. In 2003, the first Dutch guideline on hypertensive crisis was released to allow a standardised evidence-based approach for patients presenting with a hypertensive crisis. In this paper we give an overview of the current management of hypertensive crisis and discuss several important changes incorporated in the 2010 revision. These changes include a modification in terminology replacing 'malignant hypertension' with 'hypertensive crisis with retinopathy and reclassification of hypertensive crisis with retinopathy under hypertensive emergencies instead of urgencies. With regard to the treatment of hypertensive emergencies, nicardipine instead of nitroprusside or labetalol is favoured for the management of perioperative hypertension, whereas labetalol has become the drug of choice for the treatment of hypertension associated with pre-eclampsia. For the treatment of hypertensive urgencies, oral administration of nifedipine retard instead of captopril is recommended as first-line therapy. In addition, a section on the management of hypertensive emergencies according to the type of target organ involved has been added. Efforts to increase the awareness and treatment of hypertension in the population at large may lower the incidence of hypertensive crisis and its complications.

Vascular disease and chronic renal failure: new insights.

Premature cardiovascular disease (CVD) is a frequent complication in patients with chronic kidney disease (CKD). The traditional (Framingham) risk factors only partly explain the high prevalence of CVD in these patients and nontraditional risk factors/markers such as oxidative stress, persistent inflammation, cardiovascular ossification, endothelial dysfunction and anaemia are prevalent and seem to play an important role in the pathogenesis of CVD in CKD patients. In addition, the so-called reverse epidemiology phenomenon, which occurs in advanced kidney disease, complicates the search for causative mechanisms. Here we review a few recently developed concepts regarding the high incidence of CVD in CKD patients.

Transient nitric oxide reduction induces permanent cardiac systolic dysfunction and worsens kidney damage in rats with chronic kidney disease.

Left ventricular systolic dysfunction (LVSD) in patients with chronic kidney disease (CKD) is associated with poorer prognosis. Because patients with CKD often exhibit progressively decreased nitric oxide (NO) availability and inhibition of NO production can reduce cardiac output, we hypothesized that loss of NO availability in CKD contributes to pathogenesis of LVSD. Subtotally nephrectomized (SNX) rats were treated with a low dose of the NO synthase inhibitor N(omega)-nitro-L-arginine (L-NNA; 20 mg/l water; SNX+L-NNA) and compared with relevant control groups. To study permanent changes separate from hemodynamic effects, L-NNA was stopped after week 8 and rats were followed up to week 15, until blood pressure was similar in SNX+L-NNA and SNX groups. To study effects of NO depletion alone, a control group with high-dose L-NNA (L-NNA-High: 100 mg/l) was included. Mild systolic dysfunction developed at week 13 after SNX. In SNX+L-NNA, systolic function decreased by almost 50% already from week 4 onward, together with markedly reduced whole body NO production and high mortality. In L-NNA-High, LVSD was not as severe as in SNX+L-NNA, and renal function was not affected. Both LVSD and NO depletion were reversible in L-NNA-High after L-NNA was stopped, but both were persistently low in SNX+L-NNA. Proteinuria increased compared with rats with SNX, and glomerulosclerosis and cardiac fibrosis were worsened. We conclude that SNX+L-NNA induced accelerated and permanent LVSD that was functionally and structurally different from CKD or NO depletion alone. Availability of NO appears to play a pivotal role in maintaining cardiac function in CKD.

Time for first antibiotic dose is not predictive for the early clinical failure of moderate-severe community-acquired pneumonia.

The time to first antibiotic dose (TFAD) has been mentioned as an important performance indicator in community-acquired pneumonia (CAP). However, the advice to minimise TFAD to 4 hours (4 h) is only based on database studies. We prospectively studied the effect of minimising the TFAD on the early clinical outcome of moderate-severe CAP. On admission, patients' medical data and TFAD were recorded. Early clinical failure was expressed as the proportion of patients with clinical instability, admission to the intensive care unit (ICU) or mortality on day three. Of 166 patients included in the study, 27 patients (29.7%) with TFAD <4 h had early clinical failure compared to 23 patients (37.7%) with TFAD >4 h (odds ratio [OR] 0.69; 95% confidence interval [CI] 0.35-1.35). In multivariate analysis, the pneumonia severity index (OR 1.03; 95%CI 1.01-1.04), confusion (OR 2.63; 95%CI 1.14-6.06), Staphylococcus aureus infection (OR 7.26; 95%CI 1.33-39.69) and multilobar pneumonia (OR 2.40; 95%CI 1.11-5.22) but not TFAD were independently associated with early clinical failure. Clinical parameters on admission other than the TFAD predict early clinical outcome in moderate-severe CAP. In contrast to severe CAP necessitating treatment in the ICU directly, in the case of suspected moderate-severe CAP, there is time to establish a reliable diagnosis of CAP before antibiotics are administered. Therefore, the implementation of the TFAD as a performance indicator is not desirable.

Disparate systemic and renal blocking properties of angiotensin II antagonists during exogenous angiotensin II administration: implications for treatment.

Generation of angiotensin II (Ang II) contributes to the pathogenesis of cardiovascular diseases. Owing to the existence of high levels of Ang II within the kidney, blockade of the intrarenal Ang II levels may be important since long term outcome seems not only to be related to blood pressure per se. This was a prospective, randomized, double-blind, placebo-controlled study, with crossover design. We examined in 13 patients with mild to moderate hypertension the specific systemic and renal blocking properties of two different Ang II receptor blockers during a wide range of Ang II concentrations, 24 h post dose. The effects were evaluated after 4 weeks treatment with candesartan cilexetil (16 mg OD), losartan (50 mg OD) and placebo using clearance techniques. Candesartan reduced the 24 h blood pressure better than losartan (138(*)/87+/-12/8 vs 145/89+/-12/7 mmHg, (*)P<0.05 vs losartan) and placebo. Despite the lower blood pressure, candesartan attenuated the Ang II-induced response on ERPF and RVR markedly better than losartan or placebo. The GFR decreased, as expected, with placebo, but remained stable with candesartan. The present study demonstrates that in hypertensive patients candesartan and to a lesser degree losartan are effective in blocking the systemic and renal effects of Ang II during a wide range of Ang II infusion rates. Interestingly, 24 h post dose, candesartan effectively diminished the change in ERPF as well as GFR. This sustained renal effect of candesartan may be of importance, especially in pathophysiological circumstances in which (high renal levels of) Ang II contributes to cardiovascular damage.

Hypertension management in primary care: standard care and attitude towards a disease management model.

INTRODUCTION: Cardiovascular risk control has become one of the hallmarks in the treatment of diabetes and coronary heart disease, yet assessment of individual risk factors is suboptimal. We have designed a new Hypertension Screening Facility (HSF) for the evaluation of cardiovascular risk in hypertensive patients, based on 1) systematic, protocol-driven (WHO/ISH-based) analysis by nurse practitioners, 2) computer-assisted reporting of results and advice, 3) risk assessment using a Decision Support System (DSS), 4) maintenance of the autonomy of the GP. In a pilot study we wanted to investigate this HSF. METHODS: Survey 1 addressed a. how general practitioners deal with hypertension, b. whether they intend to and do use existing clinical guidelines, c. what their opinions are towards changes in the current process of care. In survey 2, we evaluated the attitude of GPs using the HSF. Responses were 43% (51 out of 120) to the first survey and 100% (20 out of 20) to the second. RESULTS: The majority of physicians included lifestyle in their assessment of risk factors and management of hypertension. Consideration of age and a positive family history was extremely high. In contrast, vision disturbances, ECG and microalbuminuria were not often considered. In the absence of additional risk factors, drug treatment was initiated in patients with a mean systolic blood pressure of 162+/-6 over 99+/-4 mmHg. In the presence of risk factors (obesity, smoking and a positive family history of cardiovascular disease) treatment is started at an average blood pressure of 154+/-8 over 96+/-4 mmHg. Opinions towards a change in management of hypertensive patients were generally positive. The opinions about the new HSF and the cardiovascular risk were reported to the general practitioner and considered useful or very useful by 79%. CONCLUSION: The present study thus confirms that cardiovascular risk evaluation by GPs is suboptimal, but there is a positive attitude towards an improvement in their assessment by HSF. The novelty of the HSF is that it respects the autonomy of the GP and brings the expertise to the GP.

Impaired NO-dependent vasodilation in patients with Type II (non-insulin-dependent) diabetes mellitus is restored by acute administration of folate.

AIMS/HYPOTHESIS: Patients with diabetes are characterised by endothelial dysfunction and cardiovascular mortality. In particular endothelium-derived nitric oxide has emerged as a first line mechanism against atherosclerosis. Hyperglycaemia causes oxygen radical stress but has also been associated with endothelial nitric oxide synthase uncoupling, both lead to decreased nitric oxide-availability. We recently showed that folate reverses eNOS uncoupling in vitro. Therefore we hypothesise that folate improves endothelial function in Type II (non-insulin-dependent) diabetes mellitus in vivo. METHODS: Using forearm plethysmography, we evaluated the effect of local, intra-arterial administration of 5-methyltetrahydrofolate (5-MTHF, the active form of folic acid, 1 microg/100 ml FAV/min) on forearm blood flow in 23 patients with Type II diabetes and 21 control subjects, matched for age, sex, blood pressure, body mass index, weight and smoking habits. Serotonin as a stimulator of nitric oxide-dependent vasodilation and sodium nitroprusside as a stimulator of endothelium-independent vasodilation were infused. RESULTS: Serotonin-induced vasodilation was blunted (53+/-30 vs 102+/-66 M/C%, p<0.005) and nitroprusside-induced vasodilation was mildly reduced (275+/-146 vs 391+/-203 M/C%, p<0.05) in patients with Type II diabetes compared to control subjects. 5-MTHF improved nitric oxide-mediated vasodilation (from 53+/-30 to 88+/-59 M/C%, p<0.05) in patients with Type II diabetes mellitus. As expected, 5-MTHF had no effect on forearm blood flow in control subjects. CONCLUSION/INTERPRETATION: These data imply that folate can be used to improve nitric oxide status and to restore endothelial dysfunction in patients with Type II diabetes. Our results provide a strong rationale for the initiation of studies that investigate whether supplementation with folic acid prevents future cardiovascular events in this patient group.

Prevention of ventilator-associated pneumonia by oral decontamination: a prospective, randomized, double-blind, placebo-controlled study.

UNLABELLED: Colonization of the intestinal tract has been assumed to be important in the pathogenesis of ventilator-associated pneumonia (VAP), but relative impacts of oropharyngeal, gastric, or intestinal colonization have not been elucidated. Our aim was to prevent VAP by modulation of oropharyngeal colonization, without influencing gastric and intestinal colonization and without systemic prophylaxis. In a prospective, randomized, placebo-controlled, double-blind study, 87 patients received topical antimicrobial prophylaxis (gentamicin/ colistin/vancomycin 2% in Orabase, every 6 h) in the oropharynx and 139 patients, divided over two control groups, received placebo (78 patients were studied in the presence of patients receiving topical prophylaxis [control group A] and 61 patients were studied in an intensive care unit where no topical prophylaxis was used [control group B]). Baseline characteristics were comparable in all three groups. Topical prophylaxis eradicated colonization present on admission in oropharynx (75% in study group versus 0% in control group A [p < 0.00001] and 9% in control group B patients [p < 0.00001]) and in trachea (52% versus 22% in A [p = 0.03] and 7% in B [p = 0.004]). Moreover, topical prophylaxis prevented acquired oropharyngeal colonization (10% versus 59% in A [p < 0.00001] and 63% in B [p < 0.00001]). Colonization rates in stomach and intestine were not affected. Incidences of VAP were 10% in study patients, 31% in Group A, and 23% in Group B patients (p = 0.001 and p = 0.04, respectively). This was not associated with shorter durations of ventilation or ICU stay or better survival. Oropharyngeal colonization is of paramount importance in the pathogenesis of VAP, and a targeted approach to prevent colonization at this site is a very effective method of infection prevention. KEYWORDS: cross infection, prevention and control; respiration, artificial, adverse effects; antibiotics, administration and dosage infection control methods; pneumonia, etiology, prevention and control; intubation, intratracheal, adverse effects

Response of erythropoiesis and iron metabolism to recombinant human erythropoietin in intensive care unit patients.

OBJECTIVES: Critically ill patients often are anemic, which may impair oxygen delivery. Transfusion of red cells and supplementation with vitamins or iron are the usual therapeutic strategies, whereas only sporadic data are available on the use of epoetin alfa in these patients. We investigated endogenous erythropoietin (EPO) production and the response to epoetin alfa in anemic intensive care unit (ICU) patients. DESIGN: Randomized, open trial. SETTING: Multidisciplinary ICU in a single secondary care center. PATIENTS: Thirty-six critically ill patients admitted to the ICU who became anemic (hemoglobin concentration, <11.2 g/dL or <12.1 g/dL in case of cardiac disease) were randomized to one of three study groups. INTERVENTIONS: All patients received folic acid (1 mg) daily. The control group received no additional therapy, the iron group received 20 mg of iron saccharate intravenously (iv) daily for 14 days. The EPO group received iv iron and epoetin alfa (300 IU/kg) subcutaneously on days 1, 3, 5, 7, and 9. MEASUREMENTS AND MAIN RESULTS: Blood and reticulocyte counts were measured daily for 22 days. Serum EPO, C-reactive protein, serum transferrin receptor, and iron variables were measured on days 0, 2, 6, 10, and 21. Blood loss and red cell transfusions were recorded. Serum EPO concentrations were inappropriately low for the degree of anemia at baseline, with no difference between patients with and without renal failure. Exogenous administration of EPO increased EPO concentrations from 23+/-13 to a maximum of 166+/-98 units/L on day 10 (p < .05). Reticulocyte count increased exclusively in the EPO group from 56+/-33 x 10(9)/L to a maximum of 189+/-97 on day 13 (p < .05). Serum transferrin receptor rose only in the EPO group from 3.7+/-1.4 to 8.6+/-3.1 mg/L on day 10 (p < .05) and remained elevated on day 21, indicating an increase in erythropoiesis. Hemoglobin concentration and platelet count remained identical in the three study groups. CONCLUSION: Endogenous EPO concentrations are low in critically ill patients. The bone marrow of these patients is able to respond to exogenous epoetin alfa, as shown by elevated concentrations of reticulocytes and serum transferrin receptors.

[What is to be done with vancomycin-resistant enterococcal infections?]. / Wat te doen als men vancomycineresistente enterokokken aantreft?

Recently, three epidemics in Dutch hospitals were caused by vancomycin-resistant enterococci (VRE). Although the number of infections was small, spread of colonization was extensive and many infection control measures were necessary to prevent further spread. VRE are relatively avirulent bacteria. However, few, if any, antibiotics are available for treatment of infections caused by VRE and the genetic code for resistance may be transferable to other, more virulent, bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). Although colonization and infection with MRSA have become endemic in many surrounding countries, such a situation has been prevented in the Netherlands by employing an aggressive 'search and destroy' policy. Although many questions regarding the optimal approach of VRE remain unanswered, a similar policy as employed for MRSA will not be possible. In contrast to MRSA, colonization with VRE occurs in the open population, no populations with increased risk for colonization appear to be definable and colonization cannot be eradicated. Based on common sense, a differentiated approach seems indicated in which extensive infection control measures should only be implemented when spread of a single genotype has been demonstrated. A reference laboratory should be created for uniform genotyping.