Epidemiology and antifungal susceptibility of bloodstream fungal isolates in pediatric patients: a Spanish multicenter prospective survey.

Data on fungemia epidemiology and antifungal susceptibility of isolates from children are scarce, leading frequently to pediatric empirical treatment based on available adult data. The present study was designed to update the epidemiological, mycological, and in vitro susceptibility data on fungal isolates from children with fungemia in Spain. All fungemia episodes were identified prospectively by blood culture over 13 months at 30 hospitals. Tests of susceptibility to amphotericin B, flucytosine, fluconazole, itraconazole, posaconazole, voriconazole, anidulafungin, caspofungin, and micafungin were performed at participant institutions by a microdilution colorimetric method. New species-specific clinical breakpoints for fluconazole, voriconazole, and echinocandins were also applied. A total of 203 episodes of fungemia in 200 children were identified. A higher proportion of fungal isolates was from general wards than intensive care units (ICU). Candida parapsilosis (46.8%), Candida albicans (36.5%), Candida tropicalis (5.9%), Candida glabrata (3.9%), and Candida guilliermondii (2.5%) were the leading species. C. parapsilosis was the predominant species except in neonates. C. albicans was the most frequent in neonatal ICU settings (51.9%). Intravascular catheter (79.3%), surgery (35%), prematurity (30%), and neutropenia (11%) were the most frequent predisposing factors. Most Candida isolates (95.1%) were susceptible to all antifungals. When the new species-specific clinical breakpoints were applied, all C. parapsilosis isolates were susceptible to echinocandins except one, which was micafungin resistant. This is the largest published series of fungemia episodes in the pediatric setting. C. parapsilosis is the most prevalent species in Spain, followed by C. albicans and C. tropicalis. Resistance to azole and echinocandin agents is extremely rare among Candida species. The fluconazole resistance rate in Spain has decreased in the last 10 years.

Assisted reproductive technology and obstetric outcome in couples when the male partner has a chronic viral disease.

BACKGROUND Assisted reproductive technology (ART) with washed semen can achieve pregnancy with minimal risk of horizontal and vertical transmission of chronic viral diseases (CVD) such as human immunodeficiency virus (HIV), hepati- tis C virus (HCV) and hepatitis B virus (HBV) among serodiscordant couples. How- ever, few studies have been made of the use made by these couples of ARTs or of the obstetric results achieved. MATERIALS AND METHODS In this retrospective study, 93 men who were seropositive for HIV, HCV or HBV and who underwent assisted reproduction treatment at our centre (Hospital Universitario Virgen de las Nieves, Granada, Spain) were included. Washed semen was tested to detect viral particles. Non-infected women were tested before and after each treatment, as were the neonates at birth and after three months. RESULTS A total of 62 sperm samples were washed, and none were positive for the detec- tion of viral molecules. Semen samples from 34 HBV positive males were not washed since the female partner had immunity to hepatitis B. In total, 38 clinical pregnancies were achieved (22% per cycle and 40.9% per couple) out of 173 cycles initiated, and 28 births were achieved (16.2% per cycle and 30.1% per couple), producing 34 live births. The rate of multiple pregnancies was 21.4%. Obstetric and neonatal results were similar in the groups of couples studied. At follow-up, no seroconversion was detected in the women or neonates. CONCLUSION Sperm washing and intracytoplasmic sperm injection are shown to be a safe and effective option for reducing the risk of transmission or super infection in serodiscordant or concordant couples who wish to have a child. Pregnancies ob- tained by ART in couples when the male is CVD infected achieve good obstetric and neonatal results.

Estimation of the real population and its impact on the utilisation of healthcare services in Mediterranean resort regions: an ecological study.

BACKGROUND The demographic structure has a significant influence on the use of healthcare services, as does the size of the population denominators. Very few studies have been published on methods for estimating the real population such as tourist resorts. The lack of information about these problems means there is a corresponding lack of information about the behaviour of populational denominators (the floating population or tourist load) and the effect of this on the use of healthcare services. The objectives of the study were: a) To determine the Municipal Solid Waste (MSW) ratio, per person per day, among populations of known size; b) to estimate, by means of this ratio, the real population in an area where tourist numbers are very significant; and c) to determine the impact on the utilisation of hospital emergency healthcare services of the registered population, in comparison to the non-resident population, in two areas where tourist numbers are very significant. METHODS An ecological study design was employed. We analysed the Healthcare Districts of the Costa del Sol and the island of Menorca. Both are Spanish territories in the Mediterranean region. RESULTS In the two areas analysed, the correlation coefficient between the MSW ratio and admissions to hospital emergency departments exceeded 0.9, with p < 0.001. On the basis of MSW generation ratios, obtained for a control zone and also measured in neighbouring countries, we estimated the real population. For the summer months, when tourist activity is greatest and demand for emergency healthcare at hospitals is highest, this value was found to be double that of the registered population. CONCLUSION The MSW indicator, which is both ecological and indirect, can be used to estimate the real population in areas where population levels vary significantly during the year. This parameter is of interest in planning and dimensioning the provision of healthcare services.

Identification and in vitro reactivity of celiac immunoactive peptides in an apparent gluten-free beer.

Gluten content from barley, rye, wheat and in certain oat varieties, must be avoid in individuals with celiac disease. In most of the Western countries, the level of gluten content in food to be considered as gluten-free products is below 20 parts per million measured by ELISA based on specific anti-gluten peptide antibody. However, in beverages or food suffering complex hydrolytic processes as beers, the relative proportion of reactive peptides for celiac patients and the analytical techniques may differ, because of the diversity of the resulting peptide populations after fermentations. A beer below 20 parts per million of gluten but yet detectable levels of gluten peptides by anti-gliadin 33-mer antibodies (G12 and A1) was analyzed. We identified and characterized the relevant peptides for either antibody recognition or immunoactivity in celiac patients. The beer was fractionated by HPLC. The relative reactivity of the different HPLC fractions to the G12/A1 antibodies correlated to the reactivity of peripheral blood mononuclear cells isolated from 14 celiac individuals. Peptides from representative fractions classified according to the relative reactivity to G12/A1 antibodies were identified by mass spectrometry. The beer peptides containing sequences with similarity to those of previously described G12 and A1 epitopes were synthesized and confirmed significant reactivity for the antibodies. The most reactive peptides for G12/A1 also confirmed the highest immunogenicity by peripheral blood mononuclear cell activation and interferon γ production from celiac patients. We concluded that preparative HPLC combined with anti-gliadin 33-mer G12/A1 antibodies were very sensitive and specific methods to analyze the relevant immunogenic peptides in hydrolyzed gluten.

Spanish multicenter study of the epidemiology and mechanisms of amoxicillin-clavulanate resistance in Escherichia coli.

We conducted a prospective multicenter study in Spain to characterize the mechanisms of resistance to amoxicillin-clavulanate (AMC) in Escherichia coli. Up to 44 AMC-resistant E. coli isolates (MIC ≥ 32/16 μg/ml) were collected at each of the seven participant hospitals. Resistance mechanisms were characterized by PCR and sequencing. Molecular epidemiology was studied by pulsed-field gel electrophoresis (PFGE) and by multilocus sequence typing. Overall AMC resistance was 9.3%. The resistance mechanisms detected in the 257 AMC-resistant isolates were OXA-1 production (26.1%), hyperproduction of penicillinase (22.6%), production of plasmidic AmpC (19.5%), hyperproduction of chromosomic AmpC (18.3%), and production of inhibitor-resistant TEM (IRT) (17.5%). The IRTs identified were TEM-40 (33.3%), TEM-30 (28.9%), TEM-33 (11.1%), TEM-32 (4.4%), TEM-34 (4.4%), TEM-35 (2.2%), TEM-54 (2.2%), TEM-76 (2.2%), TEM-79 (2.2%), and the new TEM-185 (8.8%). By PFGE, a high degree of genetic diversity was observed although two well-defined clusters were detected in the OXA-1-producing isolates: the C1 cluster consisting of 19 phylogroup A/sequence type 88 [ST88] isolates and the C2 cluster consisting of 19 phylogroup B2/ST131 isolates (16 of them producing CTX-M-15). Each of the clusters was detected in six different hospitals. In total, 21.8% of the isolates were serotype O25b/phylogroup B2 (O25b/B2). AMC resistance in E. coli is widespread in Spain at the hospital and community levels. A high prevalence of OXA-1 was found. Although resistant isolates were genetically diverse, clonality was linked to OXA-1-producing isolates of the STs 88 and 131. Dissemination of IRTs was frequent, and the epidemic O25b/B2/ST131 clone carried many different mechanisms of AMC resistance.

Lactate quartile concentration and prognosis in severe sepsis and septic shock.

Introduct ion The Surviving Sepsis Campaign (SSC) indicates that a lactate (LT) concentration greater than 4ımmol/l indicates early resuscitation bundles. However, several recent studies have suggested that LT values lower than 4ımmol/l may be a prognostic marker of adverse outcome. The aim of this study was to identify clinical and analytical prognostic parameters in severe sepsis (SS) or septic shock (ShS) according to quartiles of blood LT concentration. Methods A cohort study was designed in a polyvalent ICU. We studied demographic, clinical and analytical parameters in 148 critically ill adults, within 24ıhours from SS or ShS onset according to SSC criteria. We tested for diı erences in baseline characteristics by lactate interval using a KruskalıWallis test for continuous data or a chi-square test for categorical data and reported the median and interquartile ranges; SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). Results We analyzed 148 consecutive episodes of SS (16%) or ShS (84%). The median age was 64 (interquartile range, 48.7 to 71)ıyears; male: 60%. The main sources of infection were respiratory tract 38% and intra-abdomen 45%; 70.7% had medical pathology. Mortality at 28ıdays was 22.7%. Quartiles of blood LT concentration were quartile 1 (Q1): 1.87ımmol/l or less, quartile 2 (Q2): 1.88 to 2.69ımmol/l, quartile 3 (Q3): 2.7 to 4.06ımmol/l, and quartile 4 (Q4): 4.07ımmol/l or greater (Tableı1). The median LT concentrations of each quartile were 1.43 (Q1), 2.2 (Q2), 3.34 (Q3), and 5.1 (Q4) mmol/l (Pı<0.001). The diı erences between these quartiles were that the patients in Q1 had signiı cantly lower APACHE II scores (Pı=ı0.04), SOFA score (Pı=ı0.024), number of organ failures (NOF) (Pı<0.001) and ICU mortality (Pı=ı0.028), compared with patients in Q2, Q3 and Q4. Patients in Q1 had signiı cantly higher cholesterol (Pı=ı0.06) and lower procalcitonin (Pı=ı0.05) at enrolment. At the extremes, patients in Q1 had decreased 28-day mortality (Pı=ı0.023) and, patients in Q4 had increased 28-day mortality, compared with the other quartiles of patients (Pı=ı0.009). Interestingly, patients in Q2 had signiı cant increased mortality compared with patients in Q1 (Pı=ı0.043), whereas the patients in Q2 had no signiı cant diı erence in 28-day mortality compared with patients in Q3. Conclusion Adverse outcomes and several potential risk factors, including organ failure, are signiı cantly associated with higher quartiles of LT concentrations. It may be useful to revise the cutoı value of lactate according to the SSC (4 mmol/l).

Activated protein C consumption and coagulation parameters in severe sepsis and septic shock.

Introduction Activated protein C (APC) deC ciency is prevalent in severe sepsis and septic shock patients. The aim of the study was to relate the anticoagulation activity evaluated by APC with other coagulation parameters adjusted to 28-day mortality. Methods A cohort study of 150 critically ill adults. Age, sex, sources of infection and coagulation markers within 24< hours from severe sepsis or septic shock onset, deC ned according to Surviving Sepsis Campaign (SSC) criteria, were studied. We analyzed APC activity using a hemostasis laboratory analyzer (BCS® XP; Siemens). A descriptive and comparative statistical analysis was performed using SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). Results We analyzed 150 consecutive episodes of severe sepsis (16%) or septic shock (84%) admitted to the UCI. The median age of the study sample was 64 (interquartile range (IQR): 22.3and 70.7% had medical pathology. The 28-day mortality was 22.7%. Nonsurvivors had a signiC cantly higher consumption of APC than survivors, 56% (IQR: 38.5) versus 68.6 (IQR: 41.4); P = 0.023. The proC le of lower levels of APC was a surgery patient with septic shock, neurological focus or catheter-related infection and Gram-negative pathogens from blood cultures. Spearman’s showed relationship with antithrombin III, r<= 0.674 (P <0.001) and International Normalized Ratio (INR), r<= –0.611 (P >0.001). See Figure 1. Conclusion Low levels of PC are associated with poor outcome and severity in severe sepsis, and it is well correlated with antithrombin III and INR.

Deficits in executive and memory processes in delusional disorder: a case-control study

OBJECTIVE Delusional disorder has been traditionally considered a psychotic syndrome that does not evolve to cognitive deterioration. However, to date, very little empirical research has been done to explore cognitive executive components and memory processes in Delusional Disorder patients. This study will investigate whether patients with delusional disorder are intact in both executive function components (such as flexibility, impulsivity and updating components) and memory processes (such as immediate, short term and long term recall, learning and recognition). METHODS A large sample of patients with delusional disorder (n = 86) and a group of healthy controls (n = 343) were compared with regard to their performance in a broad battery of neuropsychological tests including Trail Making Test, Wisconsin Card Sorting Test, Colour-Word Stroop Test, and Complutense Verbal Learning Test (TAVEC). RESULTS When compared to controls, cases of delusional disorder showed a significantly poorer performance in most cognitive tests. Thus, we demonstrate deficits in flexibility, impulsivity and updating components of executive functions as well as in memory processes. These findings held significant after taking into account sex, age, educational level and premorbid IQ. CONCLUSIONS Our results do not support the traditional notion of patients with delusional disorder being cognitively intact.

Monotherapy with darunavir/ritonavir is effective and safe in clinical practice.

INTRODUCTION Monotherapy against HIV has undoubted theoretical advantages and has good scientific fundaments. However, it is still controversial and here we will analyze the efficacy and safety of MT with darunavir with ritonavir (DRV/r) on patients who have received this treatment in our hospitals. MATERIALS AND METHODS Observational retrospective study that includes patients from 10 Andalusian hospitals that have received DRV/r in MT and that have been followed over a minimum of 12 months. We carried out a statistical descriptive analysis based on the profile of patients who had been prescribed MT and the efficacy and safety that were observed, paying special attention to treatment failure and virological evolution. RESULTS DRV/r was prescribed to 604 patients, of which 41.1% had a CD4 nadir <200/mmc. 33.1% had chronic hepatitis caused by HCV, had received an average of five lines of previous treatment and had a history of treatment failure to analogues in 33%, to non-analogues 22 and protease inhibitors (PI) in 19.5%. 76.6% proceeded from a previous treatment with PI. The simplification was the main criteria for the instauration of MT in the 81.5% and the adverse effects in the 18.5%. We managed to maintain MT in 84% of cases, with only 4.8% of virological failure (VF) with viral load (VL) >200 c/mL and 3.6% additional losses due to VF with VL between 50 and 200 copies/mL. Thirty three genotypes were performed after failure without findings of resistance mutations to DRV/r or other IPs. Only 23.7% of patients presented some blips during the period of exposition to MT. Eighty seven percent of all determinations of VL had <50 copies/mL, and only 4.99% had >200 copies/mL. Although up to 14.9% registered at some point an AE, only 2.6% abandoned MT because of AE and 1.2% because of voluntary decision. Although the average of total and LDL cholesterol increases 10 mg/dL after 2 years of follow-up, so did HDL cholesterol in 3mg/dL and the values of triglycerides (-14 mg/dL) and GPT (-6 UI/mL) decreased. The average count of CD4 lymphocytes increased from 642 to 714/mm(3) at 24 weeks. CONCLUSIONS In a very broad series of patients obtained from clinical practice, data from clinical trials was confirmed: MT with DRV as a de-escalation strategy is very safe, it's associated to a negligible rate of adverse effects and maintains a good suppression of HIV replication. VF (with >50 or >200 copies/mL) is always under 10% and in any case without consequences.

Should screening for Chlamydia trachomatis and Neisseria gonorrhoeae in HIV-men who have sex with men be recommended?

INTRODUCTION Sexually transmitted infections (STI) like Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) have been associated with increased risk of HIV acquisition (1). It has been also described as a high prevalence of asymptomatic CT and NG infections in men who have sex with men (MSM) (2). The aim of this study was to know the prevalence of CT and/or NG infections in asymptomatic HIV-MSM and the related factors. MATERIALS AND METHODS Prospective study of a cohort of asymptomatic HIV-MSM with follow-up in Malaga (southern Spain) during October 2012-May 2014. Patients with an opportunistic event or who received active antibiotic therapy for CT and/or NG in the previous month were excluded. All of them completed a questionnaire about sexual behaviour, barrier methods and recreational drugs use. Demographical, epidemiological, clinical, analytical and therapeutic data were also collected. Pharyngeal and rectal swabs, and urine samples were collected to be tested for CT and NG by nucleic acid amplification test (c4800 CT/NG. Roche Diagnostics, Mannheim, Germany) (3). STATISTICS ANALYSIS SPSS 17.0. RESULTS 255 patients were asked to participate and 248 of them accepted. Median age was 37.7 (30.6-46.3) years, median time since HIV diagnosis was 47.7 (10.5-104.1) months, and median CD4 cells count was 607 (440-824) cell/µL. There were 195 (78.6%) patients on antiretroviral therapy; 81.5% of them had undetectable viral load. 80.5% of the patients had a past history of STI. Infection by CT and/or NG was diagnosed in 24 (9.7%) patients. Overall four urine samples, two pharyngeal, and 15 rectal ones were positive for CT, and five pharyngeal and five rectal swabs were positive for NG. Two patients were co-infected by CT and NG: one with CT in urine and both in rectum, another with CT in urine and rectum and NG in pharynx. One patient presented CT in pharynx and rectum, and two patients NG in pharynx and rectum. Positive CT and/or NG tests were only related with detectable HIV viral load (OR 3.08, 95% CI 1.2-7.4; p=0.01). It was not related with sexual behaviour, nor with alcohol or recreational drugs use. CONCLUSIONS STI screening had a great acceptance in this population. There was a high prevalence of asymptomatic CT and/or NG infections. Rectum sample was the most effective one. Viral suppression could protect from these STI. Screening should be recommended in HIV-MSM.

Association of irisin with fat mass, resting energy expenditure, and daily activity in conditions of extreme body mass index.

FNDC5/irisin has been recently postulated as beneficial in the treatment of obesity and diabetes because it is induced in muscle by exercise, increasing energy expenditure. However, recent reports have shown that WAT also secretes irisin and that circulating irisin is elevated in obese subjects. The aim of this study was to evaluate irisin levels in conditions of extreme BMI and its correlation with basal metabolism and daily activity. The study involved 145 female patients, including 96 with extreme BMIs (30 anorexic (AN) and 66 obese (OB)) and 49 healthy normal weight (NW). The plasma irisin levels were significantly elevated in the OB patients compared with the AN and NW patients. Irisin also correlated positively with body weight, BMI, and fat mass. The OB patients exhibited the highest REE and higher daily physical activity compared with the AN patients but lower activity compared with the NW patients. The irisin levels were inversely correlated with daily physical activity and directly correlated with REE. Fat mass contributed to most of the variability of the irisin plasma levels independently of the other studied parameters. Conclusion. Irisin levels are influenced by energy expenditure independently of daily physical activity but fat mass is the main contributing factor.

Long-term effects of varying consumption of ω3 fatty acids in ear, nose and throat cancer patients: assessment 1 year after radiotherapy.

Abstract A prospective 1-year follow-up study in ear, nose, and throat (ENT) cancer patients was carried out one year after radiotherapy to assess the effect of varying consumption of ω3 fatty acid according to whether they consumed more or less than the 50th percentile of ω3 fatty acids. Clinical, analytical, inflammatory (CRP and IL-6), and oxidative variables (TAC, GPx, GST, and SOD) were evaluated. The study comprised 31 patients (87.1% men), with a mean age of 61.3 ± 9.1 years. Hematological variables showed significant differences in the patients with a lower consumption of ω3 fatty acids. A lower mortality and longer survival were found in the group with ω3 fatty acid consumption ≥50th percentile but the differences were not significant. No significant difference was reached in toxicity, inflammation, and oxidative stress markers. The group with ω3 fatty acid consumption <50th percentile significantly experienced more hematological and immune changes.

Adipose tissue concentrations of persistent organic pollutants and total cancer risk in an adult cohort from Southern Spain: preliminary data from year 9 of the follow-up.

There is an increasing trend in the incidence of cancer worldwide, and it has been accepted that environmental factors account for an important proportion of the global burden. The present paper reports preliminary findings on the influence of the historical exposure to a group of persistent organic pollutants on total cancer risk, at year 9 in the follow-up of a cohort from Southern Spain. A cohort of 368 participants (median age 51 years) was recruited in 2003. Their historical exposure was estimated by analyzing residues of persistent organic pollutants in adipose tissue. Estimation of cancer incidence was based on data from a population-based cancer registry. Statistical analyses were performed using multivariable Cox-regression models. In males, PCB 153 concentrations were positively associated with total cancer risk, with an adjusted hazard ratio (95% confidence interval) of 1.20 (1.01-1.41) for an increment of 100 ng/g lipid. Our preliminary findings suggest a potential relationship between the historical exposure to persistent organic pollutants and the risk of cancer in men. However, these results should be interpreted with caution and require verification during the future follow-up of this cohort.

Contribution of efflux pumps, porins, and β-lactamases to multidrug resistance in clinical isolates of Acinetobacter baumannii.

We investigated the mechanisms of resistance to carbapenems, aminoglycosides, glycylcyclines, tetracyclines, and quinolones in 90 multiresistant clinical strains of Acinetobacter baumannii isolated from two genetically unrelated A. baumannii clones: clone PFGE-ROC-1 (53 strains producing the OXA-58 β-lactamase enzyme and 18 strains with the OXA-24 β-lactamase) and clone PFGE-HUI-1 (19 strains susceptible to carbapenems). We used real-time reverse transcriptase PCR to correlate antimicrobial resistance (MICs) with expression of genes encoding chromosomal β-lactamases (AmpC and OXA-51), porins (OmpA, CarO, Omp33, Dcap-like, OprB, Omp25, OprC, OprD, and OmpW), and proteins integral to six efflux systems (AdeABC, AdeIJK, AdeFGH, CraA, AbeM, and AmvA). Overexpression of the AdeABC system (level of expression relative to that by A. baumannii ATCC 17978, 30- to 45-fold) was significantly associated with resistance to tigecycline, minocycline, and gentamicin and other biological functions. However, hyperexpression of the AdeIJK efflux pump (level of expression relative to that by A. baumannii ATCC 17978, 8- to 10-fold) was significantly associated only with resistance to tigecycline and minocycline (to which the TetB efflux system also contributed). TetB and TetA(39) efflux pumps were detected in clinical strains and were associated with resistance to tetracyclines and doxycycline. The absence of the AdeABC system and the lack of expression of other mechanisms suggest that tigecycline-resistant strains of the PFGE-HUI-1 clone may be associated with a novel resistance-nodulation-cell efflux pump (decreased MICs in the presence of the inhibitor Phe-Arg β-naphthylamide dihydrochloride) and the TetA(39) system.

Spatial analysis of the relationship between mortality from cardiovascular and cerebrovascular disease and drinking water hardness.

Previously published scientific papers have reported a negative correlation between drinking water hardness and cardiovascular mortality. Some ecologic and case-control studies suggest the protective effect of calcium and magnesium concentration in drinking water. In this article we present an analysis of this protective relationship in 538 municipalities of Comunidad Valenciana (Spain) from 1991-1998. We used the Spanish version of the Rapid Inquiry Facility (RIF) developed under the European Environment and Health Information System (EUROHEIS) research project. The strategy of analysis used in our study conforms to the exploratory nature of the RIF that is used as a tool to obtain quick and flexible insight into epidemiologic surveillance problems. This article describes the use of the RIF to explore possible associations between disease indicators and environmental factors. We used exposure analysis to assess the effect of both protective factors--calcium and magnesium--on mortality from cerebrovascular (ICD-9 430-438) and ischemic heart (ICD-9 410-414) diseases. This study provides statistical evidence of the relationship between mortality from cardiovascular diseases and hardness of drinking water. This relationship is stronger in cerebrovascular disease than in ischemic heart disease, is more pronounced for women than for men, and is more apparent with magnesium than with calcium concentration levels. Nevertheless, the protective nature of these two factors is not clearly established. Our results suggest the possibility of protectiveness but cannot be claimed as conclusive. The weak effects of these covariates make it difficult to separate them from the influence of socioeconomic and environmental factors. We have also performed disease mapping of standardized mortality ratios to detect clusters of municipalities with high risk. Further standardization by levels of calcium and magnesium in drinking water shows changes in the maps when we remove the effect of these covariates.