Dynamics of gliotoxin and bis(methylthio)gliotoxin production during the course of Aspergillus fumigatus infection.

As recently described, fungal secondary metabolism activates during infection in response to a hostile host environment. Gliotoxin and bis(methylthio)gliotoxin are two recognized secondary metabolites produced by Aspergillus fumigatus with differential cytotoxicity and involved in virulence. We sought to describe the temporal dynamics of gliotoxin and bis(methylthio)gliotoxin during A. fumigatus progression to further explore their role in the infection. First, we optimized the production of the mycotoxins under different in vitro growth conditions and then specifically measured them using an UHPLC/PDA method. The analytical conditions were selected after testing different parameters such as extraction procedures, column type, and mobile phase composition. We found that gliotoxin and bis(methylthio)gliotoxin are differentially excreted to the extracellular media during the course of A. fumigatus infection regardless of the growth format tested. Dynamic profiles show an early production of gliotoxin, which, after reaching a maximum, decreases coinciding with the increase in the production of the inactive derivative bis(methylthio)gliotoxin. Presence of gliotoxin may indicate an early phase of fungal development, whereas detection of bis(methylthio)gliotoxin may correspond to a more advanced stage of infection. Our chromatographic method successfully characterizes these secondary metabolites. Thus, it may potentially be used to further understand Aspergillus infection. LAY SUMMARY: Aspergillus fumigatus secondary metabolites may contribute to fungal survival. A new chromatographic method was applied to simultaneously characterize two relevant metabolites. Presence of toxic gliotoxin may indicate an early phase of development, whereas the detection of the inactive derivate may represent an advanced infection stage.

Misdiagnosis of Babesiosis as Malaria, Equatorial Guinea, 2014.

We report a case of babesiosis, caused by Babesia microti, in a missionary who worked in Equatorial Guinea but also visited rural Spain. The initial diagnosis, based on clinical features and microscopy, was malaria. The patient's recovery was delayed until she received appropriate treatment for babesiosis.

Yu's Technique: An Optimal Local Flap for Lower Lip Reconstruction.

PURPOSE: The purpose of this study was to describe the authors' experience using the Yu flap, a rarely reported reconstructive technique that is an excellent method for lower lip reconstruction after sustaining large defects. MATERIALS AND METHODS: An observational retrospective study was designed to record all patients from 2010 through 2015 who had any lower lip disease that required wide resection and subsequent lip reconstruction using local flaps. The sample was supplied from the operating room database of the Department of Oral and Maxillofacial Surgery at Rio Hortega University Hospital (Valladolid, Spain). Patients with lower lip injuries treated using reconstructive techniques other than the Yu technique were excluded from the study. RESULTS: Seventeen patients (15 men, 2 women; age range, 41 to 91 yr) were treated by the Yu technique and the follow-up period was at least 9 months. Six left unilateral, 5 right unilateral, and 6 bilateral Yu flaps were performed during this period. No major complications occurred during follow-up. Good functional and esthetic results were observed after surgery in most patients. CONCLUSION: Although the Yu technique is a seldom used reconstructive method, it is a straightforward technique based on local flaps that offers excellent results for medium and large lower lip defects.

Oxidative stress in follicular fluid of young women with low response compared with fertile oocyte donors.

The aim of this study was to determine the concentrations of oxidative stress markers, antioxidant enzymes and cytokines in the follicular fluid of young women with low response in ovarian stimulation cycles compared with high responders and fertile oocyte donors of the same age, to assess the impact of oxidative stress on ovarian reserve. The activity of follicular fluid antioxidant enzymes glutathione transferase, glutathione reductase and glutathione peroxidase was significantly lower in young women with reduced ovarian reserve compared with that in high responders and oocyte donors. Follicular fluid concentrations of oxidative stress marker malondialdehyde combined with 4-hydroxyalkenals and nitric oxide were higher in low responders than in high responders and oocyte donors. Significant differences between low responders and donors in concentrations of IL-2, IL-6, IL-8 and vascular endothelial growth factor were observed, with higher concentrations in low responders. However, IL-10 concentration was lower in low responders than in high responders and donors. No significant differences were found in follicular fluid concentrations of tumour necrosis factor alpha between the three groups. These results demonstrate that different concentrations of oxidative stress markers, oxidant enzymes and cytokines in low responders compared with high responders and oocyte donors may negatively impact ovarian response.

High-resolution phylogeography of zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1 with an emphasis on its distribution in Turkey, Italy and Spain.

Echinococcus granulosus is the causative agent of cystic echinococcosis. The disease is a significant global public health concern and human infections are most commonly associated with E. granulosus sensu stricto (s. s.) genotype G1. The objectives of this study were to: (i) analyse the genetic variation and phylogeography of E. granulosus s. s. G1 in part of its main distribution range in Europe using 8274 bp of mtDNA; (ii) compare the results with those derived from previously used shorter mtDNA sequences and highlight the major differences. We sequenced a total of 91 E. granulosus s. s. G1 isolates from six different intermediate host species, including humans. The isolates originated from seven countries representing primarily Turkey, Italy and Spain. Few samples were also from Albania, Greece, Romania and from a patient originating from Algeria, but diagnosed in Finland. The analysed 91 sequences were divided into 83 haplotypes, revealing complex phylogeography and high genetic variation of E. granulosus s. s. G1 in Europe, particularly in the high-diversity domestication centre of western Asia. Comparisons with shorter mtDNA datasets revealed that 8274 bp sequences provided significantly higher phylogenetic resolution and thus more power to reveal the genetic relations between different haplotypes.

The efficacy of the ultraviolet C pathogen inactivation system in the reduction of Babesia divergens in pooled buffy coat platelets.

BACKGROUND: Babesia spp. is an intraerythrocytic parasite that causes human babesiosis and its transmission by transfusion has been extensively demonstrated. The aim of this study was to ascertain the efficacy of an ultraviolet C (UVC)-based pathogen inactivation system in the reduction of Babesia divergens-infected platelet (PLT) concentrates and to determine the parasite's ability to survive in PLT concentrates stored under blood bank conditions. STUDY DESIGN AND METHODS: This study was conducted using in vitro cultures of B. divergens. The detection limit of the culture assay was established and, subsequently, 15 buffy coat-derived PLT concentrates (BC-PCs) were inoculated with 10(7) B. divergens-infected red blood cells. Infected BC-PCs were irradiated with 0.2 J/cm(2) UVC light using the THERAFLEX UV-Platelets method (Macopharma). Viability and parasite growth were evaluated before and after inactivation. Culture growth kinetics were monitored by DNA incorporation of [(3) H]thymidine. The ability of B. divergens to survive in PLT concentrates was also analyzed. RESULTS: The limit of detection in cultures was established at 0.1 × 10(-6) % parasites. The THERAFLEX UV-Platelets system inactivated B. divergens to below the limit of detection in 12 of 15 BC-PCs (log reduction, >6.0) and to the limit of detection (log reduction, 5.0) in three of 15. It was also demonstrated that B. divergens remains viable in BC-PCs stored up to 7 days. CONCLUSION: Since B. divergens can survive in PLT concentrates and given the performance of UVC, this system could be considered as an alternative to prevent B. divergens and other Babesia species from being transmitted through PLT transfusions.

Multiomics analysis reveals B. MO1 as a distinct Babesia species and provides insights into its evolution and virulence.

Babesiosis, caused by protozoan parasites of the genus Babesia , is an emerging tick-borne disease of significance for both human and animal health. Babesia parasites infect erythrocytes of vertebrate hosts where they develop and multiply rapidly to cause the pathological symptoms associated with the disease. The identification of various Babesia species underscores the ongoing risk of new zoonotic pathogens capable of infecting humans, a concern amplified by anthropogenic activities and environmental shifts impacting the distribution and transmission dynamics of parasites, their vectors, and reservoir hosts. One such species, Babesia MO1, previously implicated in severe cases of human babesiosis in the midwestern United States, was initially considered closely related to B. divergens , the predominant agent of human babesiosis in Europe. Yet, uncertainties persist regarding whether these pathogens represent distinct variants of the same species or are entirely separate species. We show that although both B. MO1 and B. divergens share similar genome sizes, comprising three nuclear chromosomes, one linear mitochondrial chromosome, and one circular apicoplast chromosome, major differences exist in terms of genomic sequence divergence, gene functions, transcription profiles, replication rates and susceptibility to antiparasitic drugs. Furthermore, both pathogens have evolved distinct classes of multigene families, crucial for their pathogenicity and adaptation to specific mammalian hosts. Leveraging genomic information for B. MO1, B. divergens , and other members of the Babesiidae family within Apicomplexa provides valuable insights into the evolution, diversity, and virulence of these parasites. This knowledge serves as a critical tool in preemptively addressing the emergence and rapid transmission of more virulent strains.

Retrospective study of the epidemiological risk and serological diagnosis of human babesiosis in Asturias, Northwestern Spain.

BACKGROUND: Babesiosis is a globally growing tick-borne disease in humans. Severe babesiosis caused by Babesia divergens has been reported in two patients from Asturias (Northwestern Spain), suggesting an undetected risk for the disease. To analyze this risk, we retrospectively evaluated the seroprevalence of babesiosis in the Asturian population from 2015 through 2017, a period covering the intermediate years in which these two severe cases occurred. METHODS: Indirect fluorescent assay (IFA) and Western blot (WB) were performed to detect B. divergens IgG antibodies in 120 serum samples from Asturian patients infected with the tick-transmitted spirochete Borrelia burgdorferi sensu lato, a condition that indicates exposure to tick bites. RESULTS: This retrospective study confirmed a B. divergens seroprevalence rate of 39.2% according to IFA results. B. divergens incidence was 7.14 cases/100,000 population, exceeding previously reported seroprevalence rates. No differences in epidemiology and risk factors were found between patients infected solely with B. burgdorferi s.l. and those infected with B. burgdorferi s.l. and with IgG antibodies against B. divergens. This last group of patients lived in Central Asturias, had a milder clinical course and, according to WB results, developed different humoral responses against B. divergens. CONCLUSIONS: Babesia divergens parasites have circulated for several years in Asturias. Epidemiological evidence of babesiosis makes Asturias an emerging risk area for this zoonosis. Human babesiosis could also be relevant in other Spanish and European regions affected by borreliosis. Hence, the potential risk of babesiosis on human health in Asturias and other European forest regions needs to be addressed by the health authorities.

Babesia duncani multi-omics identifies virulence factors and drug targets.

Babesiosis is a malaria-like disease in humans and animals that is caused by Babesia species, which are tick-transmitted apicomplexan pathogens. Babesia duncani causes severe to lethal infection in humans, but despite the risk that this parasite poses as an emerging pathogen, little is known about its biology, metabolic requirements or pathogenesis. Unlike other apicomplexan parasites that infect red blood cells, B. duncani can be continuously cultured in vitro in human erythrocytes and can infect mice resulting in fulminant babesiosis and death. We report comprehensive, detailed molecular, genomic, transcriptomic and epigenetic analyses to gain insights into the biology of B. duncani. We completed the assembly, 3D structure and annotation of its nuclear genome, and analysed its transcriptomic and epigenetics profiles during its asexual life cycle stages in human erythrocytes. We used RNA-seq data to produce an atlas of parasite metabolism during its intraerythrocytic life cycle. Characterization of the B. duncani genome, epigenome and transcriptome identified classes of candidate virulence factors, antigens for diagnosis of active infection and several attractive drug targets. Furthermore, metabolic reconstitutions from genome annotation and in vitro efficacy studies identified antifolates, pyrimethamine and WR-99210 as potent inhibitors of B. duncani to establish a pipeline of small molecules that could be developed as effective therapies for the treatment of human babesiosis.

Diagnostic epitope variability within Taenia solium 8 kDa antigen family: implications for cysticercosis immunodetection.

To study diagnostic epitopes within the Taenia solium 8 kDa antigen family, six overlapping synthetic peptides from an 8 kDa family member (Ts8B2) were synthesized and evaluated by ELISA and MABA with sera from patients with neurocysticercosis (NCC), from infected pigs and from rabbits immunized with recombinant Ts8B2 protein. The pre-immune rabbit sera and the Ts8B2 recombinant protein served as negative and positive controls, respectively. A similar analysis was done with the already described antigenic peptides from another member of the 8 kDa family, highly similar to Ts8B2, the CyDA antigen. Surprisingly, neither the Ts8B2 peptides nor the CyDA peptides were recognized by infected human and porcine sera. However, the entire Ts8B2 recombinant, as well as amino and carboxy-terminal halves were recognized by the positive serum samples. The observed lack of recognition of linear Ts8B2 peptides suggests that the principal serological response to the Ts8B2 family is focused on conformational epitopes in contrast to the previously observed antigenicity of the CyDA peptides. This differential antigenicity of 8 kDa family peptides could be related with parasite antigenic variability. The fact that rabbits experimentally immunized with Ts8B2 did make anti-peptide antibodies to peptides Ts8B2-6 and CyDA-6, located in the carboxy-terminal region demonstrated that the Ts8B2 peptides are not intrinsically non-immunogenic.

Babesia and Human Babesiosis.

Babesia is a genus of intraerythrocytic protozoan parasites belonging to the exclusively parasitic phylum Apicomplexa [...].

Babesia and Theileria Identification in Adult Ixodid Ticks from Tapada Nature Reserve, Portugal.

This study, conducted in a nature reserve in southern Portugal, investigated the frequency and diversity of tick-borne piroplasms in six species of adult ixodid ticks removed from 71 fallow deer (Dama dama) and 12 red deer (Cervus elaphus), collected over the period 2012-2019. The majority of 520 ticks were Ixodes ricinus (78.5%), followed by Rhipicephalus sanguineus sensu lato, Hyalomma lusitanicum, Haemaphysalis punctata, Dermacentor marginatus, and Ixodes hexagonus. The R. sanguineus ticks collected from the deer were clearly exophilic, in contrast to the endophilic species usually associated with dogs. Four tick-borne piroplasms, including Theileria spp., and the zoonotic species, Babesia divergens and Babesia microti, were detected. B. divergens 18S rDNA, identical to that of the bovine reference strain U16370 and to certain strains from red deer, was detected in I. ricinus ticks removed from fallow deer. The sporadic detection of infections in ticks removed from the same individual hosts suggests that the piroplasms were present in the ticks rather than the hosts. Theileria sp. OT3 was found in I. ricinus and, along with T. capreoli, was also detected in some of the other tick species. The natural vector and pathogenic significance of this piroplasm are unknown.

Detection and discrimination of Loa loa, Mansonella perstans and Wuchereria bancrofti by PCR-RFLP and nested-PCR of ribosomal DNA ITS1 region.

The ribosomal deoxyribonucleic acid (DNA) internal transcribed spacer region (ITS1) of two filarial nematodes, Loa loa and Mansonella perstans, was amplified and further sequenced to develop an species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol for the differentiation of both species from Wuchereria bancrofti, three filarial nematodes with blood circulating microfilariae. The ITS1-PCR product digested with the restriction endonuclease Ase I generated an specific diagnostic pattern for each of the three species. Moreover, three new specific nested-PCRs, targeting the ITS1 region, for differential detection of L. loa, M. perstans and W. bancrofti were developed and used when the ITS1-PCR products were insufficient for the Ase I enzymatic digestion. These filarial species-specific molecular protocols were evaluated in forty blood samples from African adult immigrants attending in the Hospital Insular of Gran Canaria, Canarias, Spain.

[Differential diagnosis of imported filariasis by molecular techniques (2006-2009)]. / Diagnóstico diferencial de filariasis importada mediante técnicas moleculares (2006-2009).

INTRODUCTION: The last few years has seen an increase in the number of immigrants and travellers from endemic areas where filariasis are mainly caused by Loa loa (L. loa), Mansonella perstans (M. perstans) and Wuchereria bancrofti (W. bancrofti) species. These demographic changes has led to the need for better filariae species-specific molecular diagnostic tests to solve problems, as alternatives to the more time consuming classic parasitology methods. Thus, the objective of the present work was the implementation of optimised molecular protocols (nested-PCR and ITS1-RFLP) developed in our laboratory, for the differential diagnosis of filarial parasites. The results obtained were compared with those obtained using the conventional parasitological methods. MATERIAL AND METHODS: A total of 523 samples (517 peripheral blood, 5 adult worms and one vitreous body) were sent to Parasitology Department of the National Microbiology Centre, Carlos II Research Institute (ISCIII), from 47 Health Centres in the Autonomous Regions of Spain, from 2006 to 2009. The samples were studied by the Knott technique, nested-PCR and ITS1-RFLP. RESULTS: The molecular techniques applied on blood samples showed to be more sensitive that Knott's concentration technique in the diagnosis of both L. loa (n=12 versus n=4) and M. perstans (n=57 versus n=25) infections. CONCLUSIONS: The nested-PCR and ITS1-RFLP are potential diagnostic tools for daily routine laboratory species-specific and sensitive detection of L. loa and M. perstans filarial species in immigrant population and travellers from endemic areas where these filarial species are co-endemic. Knott's concentration technique was less sensitive than molecular methods and should be carried out as a complementary diagnostic assay.

Seminal Plasma and Serum Afamin Levels Are Associated with Infertility in Men with Oligoasthenoteratozoospermia.

The plasma glycoprotein afamin has been previously identified as an alternative carrier protein for vitamin E in extravascular fluids such as plasma and cerebrospinal, ovarian follicular, and seminal fluids. However, to date, no study has established a relationship between afamin levels and infertility in women or men. The purposes of our study were (i) to assess the level of afamin in serum and seminal fluids in infertile men compared to healthy controls and (ii) to study the association between polymorphisms in afamin genes and male infertility. This observational, prospective study evaluated the afamin levels in serum and seminal fluids from infertile men (n = 39) and compared them to those in healthy controls (n = 30). We studied the association between single-nucleotide polymorphisms (SNPs) in the 5`-untranslated region (5`-UTR) of the afamin gene and infertility and analyzed a total of 1000 base pairs from the untranslated region of the afamin gene. Subjects with low sperm motility and low sperm concentration had higher median seminal afamin (18.9 ± 2.9 ng/mg of proteins) and serum afamin concentrations (24.1 ± 4.0 ng/mg of proteins) than subjects with normal sperm parameters (10.6 ± 1.4 ng/mg of proteins) (p < 0.02) (15.6 ± 1.4 ng/mg of proteins) (p < 0.002). A total of five different polymorphisms were found, including one deletion and four single-nucleotide polymorphisms (SNPs). A new transversion (A/T) (position 4:73481093) was identified in an oligoasthenoteratozoospermic patient and was associated with high levels of afamin in plasma and seminal fluids. The prevalence of this variant in our study in the case homozygous for TT is 0.985 (98.5%), and in the case heterozygous for TA is 0.015 (1.5%). Our results suggest that genetic variations in afamin might be associated with male infertility. These findings could significantly enhance our understanding of the molecular genetic causes of infertility.

Integration of Genomic and Transcriptomic Data to Elucidate Molecular Processes in Babesia divergens.

Emerging pathogens have developed ingenious life cycles to facilitate their growth and survival in the host organism. Detailed knowledge of the life cycle of these pathogens is increasingly necessary if we are to design new strategies to prevent infection and transmission. Multi-omics platforms provide useful data at different biological levels, and integration of these data into current approaches can facilitate holistic assessment of emerging pathogens. In this chapter, we bring together various methods and apply an integrative approach for analysis of genomic and transcriptomic data in Babesia divergens, an Apicomplexa emerging parasite that invades red blood cells and causes redwater fever in cattle and the most severe form of babesiosis in humans in Europe. The integrative methodology described herein can be helpful to identify genes active at specific points during life cycle of Apicomplexa parasites.

Integration of Functional Genomic, Transcriptomic, and Metabolomic Data to Identify Key Features in Genomic Expression, Metabolites, and Metabolic Pathways of Babesia divergens.

Upon invasion of red blood cells (RBCs), the Apicomplexa parasite Babesia divergens remains within the RBC for several hours and reproduces asexually, resulting in infective free merozoites that egress and destroy the host cell. Free merozoites rapidly seek and invade new uninfected RBCs. This repetitive cycle allows B. divergens to build a complex population of intraerythrocytic and extracellular stages in the bloodstream of humans and cattle, thus causing babesiosis. To compare biological aspects between B. divergens stages, including the different nature of their metabolism, could be key to our understanding of pathogenesis. Thus, we are currently assessing differences in the B. divergens metabolism of intra- and extracellular (free merozoites) life stages by the use of an integrative approach combining functional genomic, transcriptomic, differential expression, and metabolomic data acquired from sequencing and various analytical platforms. To our knowledge, this is the first effort to describe, in detail, the experimental procedures and integration of different omics to explore the regulation of the metabolism, invasion and proliferation mechanisms of B. divergens. This integrative approach can be used as a reference to study other Apicomplexa parasites.

Tracking the Antibody Immunome in Sporadic Colorectal Cancer by Using Antigen Self-Assembled Protein Arrays.

Sporadic Colorectal Cancer (sCRC) is the third leading cause of cancer death in the Western world, and the sCRC patients presenting with synchronic metastasis have the poorest prognosis. Genetic alterations accumulated in sCRC tumor cells translate into mutated proteins and/or abnormal protein expression levels, which contribute to the development of sCRC. Then, the tumor-associated proteins (TAAs) might induce the production of auto-antibodies (aAb) via humoral immune response. Here, Nucleic Acid Programmable Protein Arrays (NAPPArray) are employed to identify aAb in plasma samples from a set of 50 sCRC patients compared to seven healthy donors. Our goal was to establish a systematic workflow based on NAPPArray to define differential aAb profiles between healthy individuals and sCRC patients as well as between non-metastatic (n = 38) and metastatic (n = 12) sCRC, in order to gain insight into the role of the humoral immune system in controlling the development and progression of sCRC. Our results showed aAb profile based on 141 TAA including TAAs associated with biological cellular processes altered in genesis and progress of sCRC (e.g., FSCN1, VTI2 and RPS28) that discriminated healthy donors vs. sCRC patients. In addition, the potential capacity of discrimination (between non-metastatic vs. metastatic sCRC) of 7 TAAs (USP5, ML4, MARCKSL1, CKMT1B, HMOX2, VTI2, TP53) have been analyzed individually in an independent cohort of sCRC patients, where two of them (VTI2 and TP53) were validated (AUC ~75%). In turn, these findings provided novel insights into the immunome of sCRC, in combination with transcriptomics profiles and protein antigenicity characterizations, wich might lead to the identification of novel sCRC biomarkers that might be of clinical utility for early diagnosis of the tumor. These results explore the immunomic analysis as potent source for biomarkers with diagnostic and prognostic value in CRC. Additional prospective studies in larger series of patients are required to confirm the clinical utility of these novel sCRC immunomic biomarkers.

A Growth Modulation Index-Based GEISTRA Score as a New Prognostic Tool for Trabectedin Efficacy in Patients with Advanced Soft Tissue Sarcomas: A Spanish Group for Sarcoma Research (GEIS) Retrospective Study.

The aim of this study was to identify an easily reliable prognostic score that selects the subset of advanced soft tissue sarcoma (ASTS) patients with a higher benefit with trabectedin in terms of time to progression and overall survival. A retrospective series of 357 patients with ASTS treated with trabectedin as second- or further-line in 19 centers across Spain was analyzed. First, it was confirmed that patients with high growth modulation index (GMI > 1.33) were associated with the better clinical outcome. Univariate and multivariate analyses were performed to identify factors associated with a GMI > 1.33. Thus, GEISTRA score was based on metastasis free-interval (MFI ≤ 9.7 months), Karnofsky < 80%, Non L-sarcomas and better response in the previous systemic line. The median GMI was 0.82 (0-69), with 198 patients (55%) with a GMI < 1, 41 (11.5%) with a GMI 1-1.33 and 118 (33.1%) with a GMI > 1.33. The lowest GEISTRA score showed a median of time-to-progression (TTP) and overall survival (OS) of 5.7 and 19.5 months, respectively, whereas it was 1.8 and 3.1 months for TTP and OS, respectively, for the GEISTRA 4 score. This prognostic tool can contribute to better selecting candidates for trabectedin treatment in ASTS.

Molecular cloning and characterisation of the RESA gene, a marker of genetic diversity of Plasmodium falciparum.

To identity immunodiagnostic antigen genes, a Plasmodium falciparum (Dd2 clone) expression library was screened using human immune sera. The ring-infected erythrocyte surface antigen (RESA) was isolated: this antigen of the resistant clone presents repeat tandem sequences like the 3D7 clone, albeit in different numbers. RESA has been studied as a marker of genetic diversity, with different sizes being observed in different isolates and clones of Plasmodium falciparum. The native protein was localised in cultures by western-blot and immuno-transmission electron microscopy. The antigenicity of RESA was evaluated by ELISA, using the carboxy-terminal repeat region as antigen. The assay's sensitivity and specificity were 78.2 and 94% respectively.