Harnessing the post-vaccination era: Do emerging HPV types represent a new threat?

While the HPV vaccine is highly effective, it is unknown whether other, untargeted viral types could occupy empty niches to become an emerging threat. In this issue of Cell Host & Microbe, Pimenoff and colleagues present a community-level epidemiological analysis of HPV types up to 8 years after different vaccination policies.

Acute Cholangitis After Bilioenteric Anastomosis for Bile Duct Injuries.

BACKGROUND/PURPOSE: The study aims to describe the clinical features, microbiology, and associated factors of acute cholangitis (AC) after bilioenteric anastomosis (BEA) for biliary duct injury (BDI). Additionally, we assessed the performance of the Tokyo Guidelines 2013 (TG13) recommendations in these patients. METHODS: We conducted a case-control study of 524 adults with a history of BEA for BDI from January 2000 to January 2014. A propensity score adjustment was performed for the analysis of the independent role of the main factors identified during the univariate logistic regression procedure. RESULTS: We identified 117 episodes of AC in 70 patients; 51.3% were definitive AC according to the TG13 diagnostic criteria, and 39.3% did not fulfill the imaging criteria of AC. A history of post-operative biliary complications (OR 2.55, 95% CI 1.38-4.70) and the bile duct confluence preservation (OR 0.46, 95% CI 0.24-0.87) were associated with AC. Eighty-nine percent of the microorganisms were Enterobacteriaceae; of them, 28% were extended spectrum ß-lactamase (ESBL) producers. CONCLUSIONS: AC is a common complication after BEA and must be suspected even in the absence of imaging findings, particulary in patients with a history of post-operative biliary complications, and/or without bile duct confluence preserved. An empirical treatment for ESBL-producing Enterobacteriaceae may be appropriate in patients living in countries with a high rate of bacterial drug resistance.

Health-Related Quality of Life in leukemia Survivors of Allogeneic Hematopoietic Stem Cell Transplantation Employing the Mexican Reduced-Intensity Conditioning.

BACKGROUND: Quality of life (QOL) is an important consideration in the counseling, implementation, and post-treatment management of arduous treatments for life-threatening conditions such as allogeneic hematopoietic cell transplantation (allo-HCT). OBJECTIVE: To analyze the QOL of leukemia patients allografted with the Mexican reduced-intensity conditioning regimen in two Mexican academic medical centers. MATERIAL AND METHODS: By means of the quality metric short form 36 version 2 to measure generic health concepts, relevant QOL was analyzed in leukemia patients who underwent allo-HCT using reduced-intensity conditioning on an outpatient basis at either the Centro de Hematología y Medicina Interna de Puebla of the Clínica Ruiz or the Hematology Service of the Internal Medicine Department of the Hospital "Dr. José Eleuterio González" of the Universidad Autónoma de Nuevo León, and who had survived more than 12 months after the allograft, who could be approached, who were in a continued complete remission (with or without graft-versus-host disease), and who were willing to respond to the questionnaire. Thirty-five patients fulfilling these requirements were included, and a sex- and age-matched group of 35 reference subjects was also studied. RESULTS: Allografted patients were found to have a slightly better mental component summary than the reference subjects (53.23 vs. 48.66 points; p = 0.01), whereas the physical component summary did not show a difference (54.53 vs. 52.05 points; p = 0.59). Most of the differences between allografted individuals and reference subject controls were not significant. CONCLUSIONS: Despite several sources of bias, these data suggest that allografted individuals employing the Mexican reduced-intensity conditioning regimen enjoy a health-related QOL life similar to that of reference subjects, adding another advantage of this method of conducting stem cell allografts. However, more work needs to be done to elucidate the impact of reduced-intensity conditioning on post allo-HCT QOL.

Genetic variations in toll-like receptor 4 in Mexican-Mestizo patients with intra-abdominal infection and/or pneumonia.

Sepsis is a leading cause of death around the world, and 73-83% of all sepsis cases requiring attention in intensive care units are linked to intra-abdominal infection (IAI) or pneumonia. The activation of innate immunity is central to the manifestation of sepsis, and toll-like receptor (TLR) 4 plays an important role in this activation process. The 299G and 399I alleles of TLR4 have been linked with an increased risk of Gram-negative bacteria (GNB) infections and septic shock in some populations. This case-control study evaluated the prevalence of D299G/T399I polymorphisms in Mexican patients with IAI and/or pneumonia and in healthy controls. Genotyping revealed that 1 in 44 patients (2.3%; CI 95%: 0.05-12.0%) and 4 in 126 controls (3.2%; CI 95%: 0.9-7.9%) were heterozygous for both the D299G and T399l polymorphisms (OR: 0.71, CI 95%: 0.01-7.44, p = NS), confirming the co-segregation of these alleles in this population. Furthermore, the patients with a GNB infection and severe sepsis were not carriers of the risk alleles. In summary, this report shows that the frequency of the D299G and T399I polymorphisms in Mexican-Mestizos is lower than anticipated in comparison with other ethnic groups, emphasizing the variable distribution of TLR4 polymorphisms among different populations. Consequently, this study was not able to detect associations between TLR4 polymorphisms and sepsis in this population.

Comparison of Enterococcus faecium and Enterococcus faecalis Strains isolated from water and clinical samples: antimicrobial susceptibility and genetic relationships.

Enterococci are part of the normal intestinal flora in a large number of mammals, and these microbes are currently used as indicators of fecal contamination in water and food for human consumption. These organisms are considered one of the primary causes of nosocomial and environmental infections due to their ability to survive in the environment and to their intrinsic resistance to antimicrobials. The aims of this study were to determine the biochemical patterns and antimicrobial susceptibilities of Enterococcus faecalis and E. faecium isolates from clinical samples and from water (groundwater, water from the Xochimilco wetland, and treated water from the Mexico City Metropolitan Area) and to determine the genetic relationships among these isolates. A total of 121 enterococcus strains were studied; 31 and 90 strains were isolated from clinical samples and water (groundwater, water from the Xochimilco wetland, and water for agricultural irrigation), respectively. Identification to the species level was performed using a multiplex PCR assay, and antimicrobial profiles were obtained using a commercial kit. Twenty-eight strains were analyzed by pulsed-field gel electrophoresis (PFGE). E. faecium strains isolated from water showed an atypical biochemical pattern. The clinical isolates showed higher resistance to antibiotics than those from water. Both the enterococci isolated from humans, and those isolated from water showed high genetic diversity according to the PFGE analysis, although some strains seemed to be closely related. In conclusion, enterococci isolated from humans and water are genetically different. However, water represents a potential route of transmission to the community and a source of antimicrobial resistance genes that may be readily transmitted to other, different bacterial species.

Pathogenicity island cag, vacA and IS605 genotypes in Mexican strains of Helicobacter pylori associated with peptic ulcers.

BACKGROUND: Helicobacter pylori is associated with chronic gastritis, peptic ulcers, and gastric cancer. Two major virulence factors of H. pylori have been described: the pathogenicity island cag (cag PAI) and the vacuolating cytotoxin gene (vacA). Virtually all strains have a copy of vacA, but its genotype varies. The cag PAI is a region of 32 genes in which the insertion of IS605 elements in its middle region has been associated with partial or total deletions of it that have generated strains with varying virulence. Accordingly, the aim of this work was to determine the cag PAI integrity, vacA genotype and IS605 status in groups of isolates from Mexican patients with non-peptic ulcers (NPU), non-bleeding peptic ulcers (NBPU), and bleeding peptic ulcers (BPU). METHODS: The cag PAI integrity was performed by detection of eleven targeted genes along this locus using dot blot hybridization and PCR assays. The vacA allelic, cag PAI genotype 1 and IS605 status were determined by PCR analysis. RESULTS: Groups of 16-17 isolates (n = 50) from two patients with NPU, NBPU, and BPU, respectively, were studied. 90% (45/50) of the isolates harbored a complete cag PAI. Three BPU isolates lacked the cag PAI, and two of the NBPU had an incomplete cag PAI: the first isolate was negative for three of its genes, including deletion of the cagA gene, whereas the second did not have the cagM gene. Most of the strains (76%) had the vacA s1b/m1 genotype; meanwhile the IS605 was not present within the cag PAI of any strain but was detected elsewhere in the genome of 8% (4/50). CONCLUSION: The patients had highly virulent strains since the most of them possessed a complete cag PAI and had a vacA s1b/m1 genotype. All the isolates presented the cag PAI without any IS605 insertion (genotype 1). Combined vacA genotypes showed that 1 NPU, 2 NBPU, and 1 BPU patients (66.6%) had a mixed infection; coexistence of H. pylori strains with different cag PAI status was observed in 1 NBPU and 2 BPU (50%) of the patients, but only two of these patients (NBPU and BPU) had different vacA genotypes.

Validation of the psychometric hepatic encephalopathy score (PHES) for identifying patients with minimal hepatic encephalopathy.

BACKGROUND: The psychometric hepatic encephalopathy score (PHES) is a battery of neuropsychological tests used in the diagnosis of minimal hepatic encephalopathy (MHE). AIM: The aim of this study was to construct and validate a dataset of normal values for the PHES. METHODS: Volunteers and patients with cirrhosis with and without low-grade overt hepatic encephalopathy (OHE) were enrolled. All subjects completed the PHES battery, and possible modifying factors were assessed. Formulas to predict expected scores in cirrhotics were constructed, and MHE was diagnosed whenever a deviation of <-4 SDs occurred across the five tests. RESULTS: Among the 743 volunteers, age and years of education influenced the scores of all tests. Eighty-four patients with cirrhosis lacked evidence of OHE, whereas 20 had OHE: median PHES were -1 [0 to -3] and -9 [-6.5 to -11.8] (P < 0.001), respectively. Thirteen of the 84 patients (15%) with cirrhosis but without OHE had MHE. Patients with MHE were older and less educated than those without MHE (61 ± 8 and 52 ± 10 years old, P = 0.003; 7 ± 4 and 12 ± 5 years education, P = 0.002), whereas liver function was not different (MELD, 8 ± 5 and 8 ± 5). A very strong correlation between these norms and those derived from Spain was observed (r = 0.964, P < 0.001). CONCLUSIONS: PHES performance was mostly influenced by age and education, and expected results in cirrhotics need to be adjusted for these factors. Our validation of Mexican norms for PHES (PHES-Mex) establishes a practical method for assessing MHE and contributes to international attempts to standardize diagnostic protocols for MHE.

Geographical variation in human papillomavirus prevalence in Mexican women with normal cytology.

OBJECTIVE: To determine the prevalence of human papillomavirus (HPV) infection and genotype distribution in Mexican women with similar lifestyles from two geographical regions who receive medical care from the Mexican Navy Health System, and to identify the associated sociodemographic and reproductive characteristics. METHODS: Cervical swabs from 671 women, beneficiaries of the Mexican Navy Health System, from two distinct southern coast regions of Mexico, were analyzed. Data were obtained regarding sociodemographic variables and sexual and reproductive history. For HPV detection and typing, PCR with general primers and direct sequencing were performed on extracted DNA. Association with clinical variables was evaluated. RESULTS: Most patients had a normal cytology or low-grade intraepithelial neoplasia. A high prevalence of HPV was found (43.6%), with a significant difference between the two regions studied from the southwest Pacific coast of Mexico (37.6% in Acapulco, Guerrero vs. 49.7% in Lázaro Cárdenas, Michoacán). Some differences were also found associated to HPV type distribution, particularly related to genotypes 18, 58, and 53. Factors influencing these differences could not be identified with the analysis of typical risk factors linked to the acquisition of an HPV infection. CONCLUSIONS: Regional differences in HPV prevalence and distribution show an apparent geographic boundary between the studied populations that deserves further analysis, taking into account other factors such as those related to the sexual partners.

Prospective evaluation of oral mucositis in acute leukemia patients receiving chemotherapy.

PURPOSE: Knowledge of oral mucositis (OM) in patients with acute leukemia (AL) and chemotherapy (CT) has remained limited. Thus, a prospective, longitudinal study was undertaken to characterize clinical features, associated risk factors, and behavior of OM in a cohort of AL patients starting CT. METHODS: Prospective and longitudinal study. A cohort of patients, older than 15 years of age with AL, scheduled to receive CT, was followed from March 2006 to October 2007. At baseline and three times per week, for 21 days, patients had an oral examination performed using the Oral Mucositis Assessment Scale (OMAS); also, oral pain and difficulty to swallow were recorded using a visual analog scale. Weekly, salivary flow measurements (Schirmer's test modified version) were done. RESULTS: A cohort of 29 AL patients was followed for a median time of 21 (range, 14-53) days; 12 (41.4%) developed OM, with a mean OMAS score of 0.181 (SD +/- 0.56) and a mean peak OMAS score of 1.8 (SD +/- 0.56). The OM onset mean time was 9.8 (range, 2-20, SD +/- 6.09) days, with a mean duration of 7 (range, 3-14, SD +/- 4.15) days. OM was significantly correlated with salivary flow [rs = 0.420 (P = 0.0051)], oral pain [rs = 0.47 (P < 0.0001)], ability to swallow [rs = 0.36 (P = 0.0001)], and type of food intake [rs = 0.38 (P < 0.0001)]. CONCLUSIONS: OM is a frequent and early side effect of CT closely correlated with oral pain, difficulty to swallow, and impairment in food intake.

Diagnostic accuracy of fine needle aspiration biopsy in preoperative diagnosis of patients with parotid gland masses.

BACKGROUND: The use of fine needle aspiration biopsy (FNAB) for diagnosis of parotid gland masses (PGM) is questioned, because of low sensitivity and the generalized belief requiring surgery for most parotid masses. Information available is retrospective. Our objective was to evaluate the diagnostic accuracy of FNAB for diagnosis of patients with PGM. METHODS: A prospective diagnostic test study was conducted in a cancer center from 2003 to 2007. FNAB was obtained from patients older than 18 years with PGM. Cytopathologist and histopathologist were blinded for all clinical information. The reference standard for diagnosis was the surgical pathology report. RESULTS: FNAB sensitivity and specificity values in diagnosis of malignancy were 0.923 (95% confidence interval [CI], 0.85-0.99) and 0.986 (95% CI, 0.96-1.00), respectively. Positive and negative likelihood ratios (LRs) were 64.6 (95% CI, 9.22-453) and 0.078 (95% CI 0.03-0.18), respectively. Negative LR of FNAB was strengthened (0.078-0.029) when negative diagnosis of FNAB was associated with tumor size <4 cm, definite borders, and homogeneous tumor mass observed by computed tomography (CT). CONCLUSION: Diagnostic accuracy for FNAB was very high. No clinical or radiological factors improved the positive LR of FNAB alone. Liberal use of FNAB of PGM is recommended.

Functional implication of sequence variation in the long control region and E2 gene among human papillomavirus type 18 variants.

Cervical cancer incidence remains highly frequent in developing countries. It is possible that populations of these countries are exposed to more oncogenic human papillomavirus (HPV) variants. Functional differences among high-risk HPV variants have been described, suggesting repercussions on their oncogenic potential. In this report, we demonstrate that the long control region (LCR) of HPV18 variants has distinct transcriptional activities in different cervical cancer cell lines. African (Af)-LCR possessed the lowest transcriptional activity; its sequence harbors the highest number of nucleotide changes among the HPV18 variants analyzed. Some of these embedded in identified transcription-factor-binding sites, suggesting a less aggressive biological activity possibly involved in a slower progression of cervical lesions. Asian-Amerindian LCR showed distinct activities among cell types, while European LCR activity was similar in cell lines tested. Despite multiple nucleotide substitutions found in HPV18 E2 variant genes, their repressive activities over homologous LCRs were not distinct among variants.

High diversity of vacA and cagA Helicobacter pylori genotypes in patients with and without gastric cancer.

BACKGROUND: Helicobacter pylori is associated with chronic gastritis, peptic ulcers, and gastric cancer. The aim of this study was to assess the topographical distribution of H. pylori in the stomach as well as the vacA and cagA genotypes in patients with and without gastric cancer. METHODOLOGY/PRINCIPAL FINDINGS: Three gastric biopsies, from predetermined regions, were evaluated in 16 patients with gastric cancer and 14 patients with dyspeptic symptoms. From cancer patients, additional biopsy specimens were obtained from tumor centers and margins; among these samples, the presence of H. pylori vacA and cagA genotypes was evaluated. Positive H. pylori was 38% and 26% in biopsies obtained from the gastric cancer and non-cancer groups, respectively (p = 0.008), and 36% in tumor sites. In cancer patients, we found a preferential distribution of H. pylori in the fundus and corpus, whereas, in the non-cancer group, the distribution was uniform (p = 0.003). A majority of the biopsies were simultaneously cagA gene-positive and -negative. The fundus and corpus demonstrated a higher positivity rate for the cagA gene in the non-cancer group (p = 0.036). A mixture of cagA gene sizes was also significantly more frequent in this group (p = 0.003). Ninety-two percent of all the subjects showed more than one vacA gene genotype; s1b and m1 vacA genotypes were predominantly found in the gastric cancer group. The highest vacA-genotype signal-sequence diversity was found in the corpus and 5 cm from tumor margins. CONCLUSION/SIGNIFICANCE: High H. pylori colonization diversity, along with the cagA gene, was found predominantly in the fundus and corpus of patients with gastric cancer. The genotype diversity observed across systematic whole-organ and tumor sampling was remarkable. We find that there is insufficient evidence to support the association of one isolate with a specific disease, due to the multistrain nature of H. pylori infection shown in this work.

Microbiological implications of periurban agriculture and water reuse in Mexico City.

BACKGROUND: Recycled treated or untreated wastewater represents an important health challenge in developing countries due to potential water related microbiological exposure. Our aim was to assess water quality and health implications in a Mexico City periurban agricultural area. METHODOLOGY/PRINCIPAL FINDINGS: A longitudinal study in the Xochimilco wetland area was conducted, and 42 sites were randomly selected from 211, including irrigation water canals and effluents of treatment plants. Sample collection took place during rainy and dry seasons (2000-2001). Microbiological parameters (total coliforms, fecal coliforms, streptococci/enterococci, and bacteria other than Vibrio grown on TCBS), Helicobacter pylori, and physicochemical parameters including trihalomethanes (THM) were determined. Fecal coliforms and fecal streptococci are appropriate indicators of human or animal fecal contamination. Fecal coliform counts surpass Mexican and World Health Organization irrigation water guidelines. Identified microorganisms associated with various pathologies in humans and domestic animals comprise Escherichia coli, Klebsiella spp., Salmonella spp., Enterobacter spp., Enterococcus spp., and Pseudomonas spp; H. pylori was also present in the water. An environmental characteristic of the canal system showed high Total Organic Carbon content and relatively low dissolved oxygen concentration; residual chlorine as a disinfection control is not efficient, but THMs do not represent a problem. During the rainy season, temperature and conductivity were higher; in contrast, pH, dissolved oxygen, ammonia, and residual chlorine were lower. This is related with the continuous load of feces from human and animal sources, and to the aquatic systems, which vary seasonally and exhibit evidence of lower water quality in effluents from treatment plants. CONCLUSIONS/SIGNIFICANCE: There is a need for improvement of wastewater treatment systems, as well as more efficient monitoring, regulation, and enforcement procedures for wastewater disposal into bodies of water.

Oral lesions as clinical markers of highly active antiretroviral therapy failure: a nested case-control study in Mexico City.

BACKGROUND: Clinical markers that may predict virological failure during highly active antiretroviral therapy (HAART) have not been evaluated adequately. The aim of the present study was to evaluate the usefulness of human immunodeficiency virus (HIV)-related oral lesions as clinical predictors of virological failure in HIV-infected patients receiving HAART. METHODS: A nested case-control study was conducted within a cohort of 1134 HIV-infected patients receiving HAART who attended the AIDS Clinic of the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán in Mexico City during the period 1997-2005. Case patients were patients who, after achieving an undetectable viral load, had at least 1 viral load determination > or = 2000 copies/mL while receiving treatment. Control subjects were patients who, after achieving an undetectable viral load, continued to have undetectable viral loads during the follow-up period. There were 2-3 control subjects for each case patient, matched according to duration of follow-up. Oral examinations were blinded to viral loads and CD4+ lymphocyte counts. Analyses were performed with multivariate conditional logistic regression models, and associations were shown as odds ratios (ORs) with 95% confidence intervals (CI). Positive predictive values were calculated. RESULTS: The target cohort consisted of 431 HIV-infected individuals; 47 case patients and 132 control subjects underwent complete oral examinations and formed the basis of the analysis. At the visit at which an undetectable viral load was determined, case patients and control subjects showed a similar frequency of HIV-related oral lesions (21.3% vs. 17.4%) (OR, 1.39; 95% CI, 0.57-3.38; P=.47). At the visit at which virological failure was determined, case patients showed a higher risk for HIV-related oral lesions (OR, 14.5; 95% CI, 4.21-49.94; P<.001) and oral candidosis (OR, 26.2; 95% CI, 3.34-205.9; P<.001) than did control subjects. The positive predictive value of HIV-related oral lesions and oral candidosis to identify patients who experienced virological failure while receiving HAART was 80% and 83%, respectively. CONCLUSIONS: HIV-related oral lesions and, specifically, oral candidosis may be considered to be clinical markers of virological failure in HIV-infected patients receiving HAART.

Colchicine delays the development of hepatocellular carcinoma in patients with hepatitis virus-related liver cirrhosis.

BACKGROUND: Hepatocellular carcinoma (HCC) is a malignant neoplasm associated with liver cirrhosis, with an annual incidence of 3% to 9%, which is one of the main causes of death in patients with cirrhosis. Viral hepatitis is associated with an increased risk of HCC, probably due to an inflammatory reaction. Colchicine is an antiinflammatory agent that inhibits the formation of intracellular microtubules, affecting mitosis and fibrogenesis. Diverse clinical studies have failed to demonstrate the benefit of colchicine over the progression of fibrosis in patients with liver cirrhosis; nevertheless, to the authors' knowledge there are no studies that evaluate its effect in the development of HCC. METHODS: The effect of the administration of colchicine on the development of HCC was evaluated in 186 patients with hepatitis virus-related liver cirrhosis in a retrospective cohort study. The minimum follow-up time was 3 years (median, 84 months +/- 2.8 months). One hundred sixteen patients received treatment with colchicine. The characteristics of both groups were similar. RESULTS: The percentage of patients who developed HCC was significantly smaller in the colchicine group when compared with the noncolchicine group (9% vs. 29%; P = .001). On multivariate analysis, an alpha-fetoprotein level > or = 5 ng/dL (P = .03), a platelet count < 100,000 at diagnosis (P = .05), alanine aminotransferase > or = 52 IU (P = .006), and a lack of treatment with colchicine (P = .0001) were found to be associated with an earlier development of HCC. The average time for the development of HCC was 222 months +/- 15 months and 150 months +/- 12 months in the patients who received and who did not receive colchicine, respectively. CONCLUSIONS: The results suggest that treatment with colchicine prevents and delays the development of HCC in patients with hepatitis virus-related cirrhosis. The protective mechanisms of colchicine over the development of HCC could be related to antiinflammatory properties and inhibition of mitosis. Prospective studies to confirm this observation with a greater number of patients and long-term follow-up may be indicated.

Synchronous kinetics of CD4+ lymphocytes and viral load before the onset of oral candidosis and hairy leukoplakia in a cohort of Mexican HIV-infected patients.

An observational, prospective, longitudinal cohort study was performed at the AIDS Clinic of a tertiary care institution in Mexico City to determine the association of viral load (VL) and CD4+ lymphocyte kinetics with the development of oral candidosis (OC) and hairy leukoplakia (HL). Participants were HIV-infected adult subjects, without a history of or current OC or HL, not receiving HAART. Oral examinations were performed at baseline and every month for evidence of OC or HL; CD4+ and VL determinations were done at baseline, at 6-month intervals, when oral lesions were detected, and 2 months later. Affected subjects (OL group) by OC or HL had clinical intervals defined before (antecedent), during (concurrent), and after their development. In the nonaffected individuals (NA group), 6-month intervals were determined. Differences (changes) along the clinical and study intervals were calculated for CD4+ and VL. The median study time was 178 (range: 31-924) days; 99 patients were included. The 2-year cumulative incidence of either oral lesion was 54% (49.5% for OC and 33.2% for HL). In the OL group (31 patients) a progressive and continuous decrease of CD4+ was found in the antecedent interval followed by a significant increase in VL in the concurrent period. The NA group showed a significant fall in CD4+ by semester 3, without a significant rise of VL in the following semester. The effect of CD4+ remained significant in a multivariate analysis. This study has shown that the onset of OC and/or HL is heralded by the sequence of a sustained reduction of CD4+, followed by a sharp increase of VL. In the multivariate analysis, the decrease in CD4+ lymphocytes appeared to be the predominant factor predicting the appearance of these oral lesions. Their potential use as markers of a recent change in the immunologic and virologic status of HIV-infected individuals is emphasized.