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The SARS-CoV-2 pandemic might have increased the risks of healthcare-associated infections (HAIs); however, several studies of HAI such as urinary tract infections (UTIs) and catheter-associated urinary tract infections (CAUTIs) have shown contradictory results. The aim of this study is to assess the clinical features of UTIs and bacterial isolates from urine samples of hospitalized COVID-19 patients. We conducted a retrospective observational study including 87 COVID-19 patients with UTIs admitted to our centre. Bacterial UTIs presented were 87: 9 (10.3%) community-acquired UTIs (coinfection group) and 78 (89.6%) hospital-acquired UTIs (superinfection group). In the coinfection group, the most frequent type was non-CAUTI with 5 (55.5%) patients; however, the most frequent UTI in the superinfection group was CAUTI, with 53 (67.9%) patients. The median number of days of hospitalization in coinfected patients was lower than superinfection patients: 13 (IQR 11, 23) vs. 34 days (IQR 23, 47) p < 0.006. All UTI patients admitted to ICU, 38 (43.7%), belonged to the superinfection group. The mortality rate was 26.4% (23/87), 22/23 in the superinfection group. The most common microorganisms were E. coli 27 (28.4%), E. faecalis 25 (26.3%) and E. faecium 20 (21.1%). There was an increased incidence of E. faecalis and E. faecium in UTIs as well as hospital-acquired UTIs. This can be related to urethral catheterization during hospitalization, UCI admissions and the number of days of hospitalization.

Objectives. Challenges remain and there are still a sufficient number of cases with epidemiological, clinical features and radiological data suggestive of COVID-19 pneumonia that persist negative in their RT-PCR results. The aim of the study was to define the distinguishing characteristics between patients developing a serological response to SARS-CoV-2 and those who did not. Methods. RT-PCR tests used were TaqPath 2019-nCoV Assay Kit v1 (ORF-1ab, N and S genes) from Thermo Fisher Diagnostics and SARS-COV-2 Kit (N and E genes) from Vircell. Serological response was tested using the rapid SARS-CoV2 IgG/IgM Test Cassette from T and D Diagnostics Canada and CMC Medical Devices and Drugs, S.L, CE. Results. In this cross-sectional study, we included a cohort of 52 patients recruited from 31 March 2020 to 23 April 2020. Patients with positive serology had an older average age (73.29) compared to those who were negative (54.82) (P2 in 27 of 34 patients with positive serology were below 94% (P Conclusions. Clinical features and other biomarkers in a context of a positive serology can be considered crucial for diagnosis.

GEHEP-004 Study Group: María Dolores Ocete, Miguel Ángel Simón, Pilar Rincón, Sergi Reus, Alberto De la Iglesia, Isabel García-Arata, Miguel Jiménez, Fernando Jiménez, José Hernández-Quero, Carlos Galera, Mohamed Omar Balghata, Joaquín Primo, Mar Masiá, Nuria Espinosa, Marcial Delgado, Miguel Ángel von-Wichmann, Antonio Collado, Jesús Santos, Carlos Mínguez, Felícitas Díaz-Flores, Elisa Fernández, Enrique Bernal, José De Juan, José Joaquín Antón, Mónica Vélez, Antonio Aguilera, Daniel Navarro, Juan Ignacio Arenas, Clotilde Fernández, María Dolores Espinosa, María José Ríos, Roberto Alonso, Carmen Hidalgo, Rosario Hernández, María Jesús Téllez, Francisco Javier Rodríguez, Pedro Antequera, Cristina Delgado, Patricia Martín, Javier Crespo, Berta Becerril, Óscar Pérez, Antonio García-Herola, José Montero, Carolina Freyre, Concepción Grau., [Background & Aims] Most hepatitis C virus (HCV)-infected patients failing NS5A inhibitors develop resistance-associated substitutions (RASs). Here we report the use of resistance-guided retreatment of patients who failed prior NS5A inhibitor-containing regimens in the GEHEP-004 cohort. This is the largest direct-acting antiviral (DAA)-resistance cohort study conducted in Spain. We aim to provide indications on how to use resistance information in settings where sofosbuvir/velpatasvir/voxilaprevir may not be available., [Methods] GEHEP-004 is a prospective multicenter cohort enrolling HCV-infected patients treated with interferon (IFN)-free DAA regimens. Prior to retreatment, population-based sequencing of HCV NS3, NS5A and NS5B genes was performed. After receiving a comprehensive resistance interpretation report, the retreatment regimen was chosen and the sustained virological response (SVR) at 12 weeks after treatment completion (SVR12) was recorded., [Results] A total of 342 patients experiencing virological failure after treatment with sofosbuvir/ledipasvir±ribavirin (54%), sofosbuvir/daclatasvir±ribavirin (23%), or paritaprevir-ritonavir/ombitasvir±dasabuvir±ribavirin (20%) were studied. After a resistance report, 186 patients were retreated. An SVR12 was achieved for 88.1% of the patients who failed after sofosbuvir/ledipasvir±ribavirin, 83.3% of the patients who failed after sofosbuvir/daclatasvir±ribavirin, 93.7% of the patients who failed after paritaprevir-ritonavir+ombitasvir±dasabuvir±ribavirin., [Conclusions] In our study, we show how resistance-guided retreatment in conjunction with an interpreted report allows patients to achieve SVR rates close to 90%. We hypothesize that SVR rates may even be improved if resistance data are discussed between experienced virologists and treating clinicians. We believe that our data may be relevant for countries where the access to new DAA combination regimens is limited., [Lay summary] Hepatitis C infection can be cured with currently available antiviral agents. Only a small proportion of patients experience treatment failure, however, in absolute numbers, a high number of patients may require retreatment. Highly effective combinations of antivirals are also available for retreatment. However, these antivirals might not be available in resource-limited settings. Herein, we show how, by analyzing the cause of resistance, retreatment efficacy with old drugs can get very close to the efficacy of new drug combinations., This work was supported in part by grants from Fondo de Investigación Sanitaria (www.isciii.es) (PI15/00713), Plan Nacional de I+D+I and Fondo Europeo de Desarrollo Regional-FEDER (www.redes/redes/inicio) (RD16/0025/0040), Fundación Progreso y Salud, Junta de Andalucia (http://www.juntadeandalucia.es/fundacionprogresoysalud/es) (PI-0411-2014), and GEHEP-SEIMC (GEHEP-004).

REIPI-SEIMC COVID-19 group and COVID@HULP group., [Background] The clinical presentation of COVID-19 in patients admitted to hospital is heterogeneous. We aimed to determine whether clinical phenotypes of patients with COVID-19 can be derived from clinical data, to assess the reproducibility of these phenotypes and correlation with prognosis, and to derive and validate a simplified probabilistic model for phenotype assignment. Phenotype identification was not primarily intended as a predictive tool for mortality., [Methods] In this study, we used data from two cohorts: the COVID-19@Spain cohort, a retrospective cohort including 4035 consecutive adult patients admitted to 127 hospitals in Spain with COVID-19 between Feb 2 and March 17, 2020, and the COVID-19@HULP cohort, including 2226 consecutive adult patients admitted to a teaching hospital in Madrid between Feb 25 and April 19, 2020. The COVID-19@Spain cohort was divided into a derivation cohort, comprising 2667 randomly selected patients, and an internal validation cohort, comprising the remaining 1368 patients. The COVID-19@HULP cohort was used as an external validation cohort. A probabilistic model for phenotype assignment was derived in the derivation cohort using multinomial logistic regression and validated in the internal validation cohort. The model was also applied to the external validation cohort. 30-day mortality and other prognostic variables were assessed in the derived phenotypes and in the phenotypes assigned by the probabilistic model., [Findings] Three distinct phenotypes were derived in the derivation cohort (n=2667)—phenotype A (516 [19%] patients), phenotype B (1955 [73%]) and phenotype C (196 [7%])—and reproduced in the internal validation cohort (n=1368)—phenotype A (233 [17%] patients), phenotype B (1019 [74%]), and phenotype C (116 [8%]). Patients with phenotype A were younger, were less frequently male, had mild viral symptoms, and had normal inflammatory parameters. Patients with phenotype B included more patients with obesity, lymphocytopenia, and moderately elevated inflammatory parameters. Patients with phenotype C included older patients with more comorbidities and even higher inflammatory parameters than phenotype B. We developed a simplified probabilistic model (validated in the internal validation cohort) for phenotype assignment, including 16 variables. In the derivation cohort, 30-day mortality rates were 2·5% (95% CI 1·4–4·3) for patients with phenotype A, 30·5% (28·5–32·6) for patients with phenotype B, and 60·7% (53·7–67·2) for patients with phenotype C (log-rank test p, [Interpretation] Patients admitted to hospital with COVID-19 can be classified into three phenotypes that correlate with mortality. We developed and validated a simplified tool for the probabilistic assignment of patients into phenotypes. These results might help to better classify patients for clinical management, but the pathophysiological mechanisms of the phenotypes must be investigated., [Funding] Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation, and Fundación SEIMC/GeSIDA., Funding: Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation, and Fundación SEIMC/GeSIDA.

Resumen Introduccion Nuestro objetivo principal fue revisar los aspectos clinicos, microbiologicos y epidemiologicos de la infeccion asociada a Clostridium difficile en pediatria (2010-2015), comparando los diagnosticos realizados por deteccion de toxinas en heces y por PCR a tiempo real del gen de la toxina B. Metodos Este estudio retrospectivo incluyo a 82 pacientes pediatricos. La deteccion de C. difficile toxigenico se realizo de manera secuencial, en heces diarreicas y bajo solicitud expresa. Resultados El 39% de los pacientes procedian de Hemato-Oncologia y >50% recibio previamente cefalosporinas. La presencia de fiebre asociada a diarrea fue mas frecuente en el grupo de deteccion positiva de toxinas y no recibir antibioterapia especifica fue mas frecuente en el grupo con PCR positiva, sin diferencias estadisticamente significativas. Conclusion Destacamos la presencia de infecciones en ninos menores de 2 anos. Seria recomendable realizar un diagnostico de infeccion asociada a C. difficile en pacientes pediatricos, siempre que la sospecha clinica lo requiera.

Introduction Our main objective was a revision of clinical, microbiological and epidemiological results of Clostridium difficile-associated infection in paediatric patients (2010–2015). We compared the diagnoses performed by detection of toxins in faeces and those performed by real-time PCR. Methods This retrospective study included 82 paediatric patients. Detection of toxigenic C. difficile was performed sequentially, in diarrheal faeces and under clinical request. Results A total of 39% of the patients were attended at Haematology–Oncology Unit and >50% of them had previously received cephalosporins. Fever associated with diarrhoea was more frequent in the group of toxin detection, whereas not receiving specific antibiotic treatment was more frequent in the group of positive PCR, without statistically significant differences. Conclusions We highlight the presence of C. difficile infection in children under 2 years old. A diagnostic testing in selected paediatric patients would be advisable when there is clinical suspicion of infection.

Introduction A considerable increase of imported Zika virus (ZIKV) infection has been reported in Europe in the last year. This is the result of the large outbreak of the disease in the Americas, along with the increase in the numbers of travellers and immigrants arriving from ZIKV endemic areas. Methods A descriptive study was conducted in the Tropical Medicine Unit of Hospital La Paz-Carlos III in Madrid on travellers returning from an endemic area for ZIKV from January to April 2016. Demographic, clinical and microbiological data were analyzed. Results A total of 185 patients were screened for ZIKV (59.9% women, median age of 37.7 ± 10.3 years). Main purpose of the travel was tourism to Colombia, Brazil, and Mexico. Just under three-quarters (73%) were symptomatic, mostly with fever and headache. A total of 13 patients (7% of those screened) were diagnosed with ZIKV infections, of which four of them were pregnant. All of them were symptomatic patients, the majority immigrants, and mainly from Colombia. Diagnostic tests were based on positive neutralization antibodies (8 cases, 61.6%) and a positive RT-PCR in different organic fluids (7 cases, 53.8%) The four infected pregnant women underwent a neurosonography every 3 weeks, and no alterations were detected. RT-PCR in amniotic fluid was performed in three of them, with negative results. One of the children has already been born healthy. Conclusions Our cases series represents the largest cohort of imported ZIKV to Spain described until now. Clinicians must increase awareness about the progression of the ZIKV outbreak and the affected areas so that they can include Zika virus infection in their differential diagnosis for travellers from those areas.

[Objective] To study the prevalence and distribution of HBV genotypes in Spain for the period 2000–2016., [Methods] Retrospective study recruiting 2559 patients from 17 hospitals. Distribution of HBV genotypes, as well as sex, age, geographical origin, mode of transmission, HDV-, HIV- and/or HCV-coinfection, and treatment were recorded., [Results] 1924 chronically HBV native Spanish patients have been recruited. Median age was 54 years (IQR: 41–62), 69.6% male, 6.3% HIV-coinfected, 3.1% were HCV-coinfected, 1.7% HDV-co/superinfected. Genotype distribution was: 55.9% D, 33.5% A, 5.6% F, 0.8% G, and 1.9% other genotypes (E, B, H and C). HBV genotype A was closely associated with male sex, sexual transmission, and HIV-coinfection. In contrast, HBV genotype D was associated with female sex and vertical transmission. Different patterns of genotype distribution and diversity were found between different geographical regions. In addition, HBV epidemiological patterns are evolving in Spain, mainly because of immigration. Finally, similar overall rates of treatment success across all HBV genotypes were found., [Conclusions] We present here the most recent data on molecular epidemiology of HBV in Spain (GEHEP010 Study). This study confirms that the HBV genotype distribution in Spain varies based on age, sex, origin, HIV-coinfection, geographical regions and epidemiological groups., This study has been funded in part by the funds of the research project GEHEP-2018-010, granted by the Hepatitis Group of the Spanish Society of Infectious Diseases and Clinical Microbiology (Grupo de Hepatitis de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica, GEHEP/SEIMC).

Background: Integrase strand-transfer inhibitors (INSTIs) constitute at present one of the pillars of first-line ART. Objectives: To study the prevalence of and the trend in transmitted drug resistance (TDR) to INSTIs in ART-naive patients in Spain. Methods: During the period 2012-17, 1109 patients from CoRIS were analysed. The Stanford algorithm v8.7 was used to evaluate TDR and transmission of clinically relevant resistance. To describe individual mutations/polymorphisms, the most recent IAS list (for INSTIs) and the 2009 WHO list update (for the backbone NRTIs used in combination with INSTIs in first-line treatment) were used. Results: Clinically relevant resistance to the INSTI class was 0.2%: T66I, 0.1%, resistance to elvitegravir and intermediate resistance to raltegravir; and G163K, 0.1%, intermediate resistance to raltegravir and elvitegravir. No clinical resistance to dolutegravir or bictegravir was observed. The prevalence of INSTI TDR following the IAS-USA INSTI mutation list was 2.6%, with no trend towards changes in the prevalence throughout the study period. The overall prevalence of NRTI WHO mutations was 4.3%, whereas clinically relevant resistance to tenofovir, abacavir and emtricitabine/ lamivudine was 1.7%, 1.9% and 0.7%, respectively. Conclusions: Given the low prevalence of clinically relevant resistance to INSTIs and first-line NRTIs in Spain, it is very unlikely that a newly diagnosed patient will present with clinical resistance to a first-line INSTI-based regimen. These patients may not benefit from INSTI and NRTI baseline resistance testing.

Background From the first Zika virus (ZIKV) description, it has progressively widespread worldwide. We analyzed demographic, clinical, microbiologic and travel-related characteristic from returned patients from a ZIKV endemic country in a referral Tropical Medicine Unit. Method A prospective cohort study performed in a Spanish referral center with the aim of determining the significant factors associated with confirmed Zika virus (ZIKV) infection. Results 817 patients, (56% women, median age 36 [IQR, Interquartile Range: 32–42]) were enrolled. Most had returned from Latin America (n = 486; 59.4%), travelled for tourism (n = 404; 49.4%) and stayed a median of 18 days (IQR: 10–30). 602 (73.6%) presented symptoms, but only 25 (4%) were finally diagnosed with confirmed ZIKV infection (including two pregnant women, without adverse fetal outcomes), 88% (n:22) presented with fever and 92% (n:23) with rash. 56% (n:14) arthralgia and/or myalgia and 28% (n:7) conjunctivitis. The presence of conjunctivitis, fever and rash were associated with an 8.9 (95% CI: 2.2–34.9), 6.4 (95% CI: 1.2–33.3) and 72.3 (95% CI: 9.2–563.5) times greater probability of confirmed ZIKV infection, respectively. Conclusion Travel characteristics and clinical presentation may help clinicians to optimize requests for microbiological testing. Diagnosis of arboviriasis in travellers arriving form endemic areas remains a challenge for clinicians, but must be detected for the possible transmission outside endemic areas, where the vector is present.

Acute hepatitis C virus (HCV) is now a major health problem, mainly in men who have sexual relations with other men (MSM). Hepatitis E virus (HEV) causes sporadic cases of acute hepatitis. In this article, we describe two cases of acute hepatitis due to HCV with an interesting clinical progress.

Background There are limited data about the persistence and infectivity of Zika virus in semen of symptomatic travelers returning from endemic areas and even less data in asymptomatic cases. Objective We investigated the persistence and infectivity of ZIKA virus in semen in five patients with Zika virus infection returning to Spain from endemic areas. Study design We evaluated the epidemiological, clinical and virological characteristic of the five patients. In semen we detected ZIKA virus by PCR, partial sequencing and cell culture. We also performed phylogenetic analysis. Results We detected Zika virus RNA (Asian lineage) by PCR in semen samples from day 14th to day 96th since the day of illness onset. Semen viral culture was positive for Zika virus in two patients at days of illness 30 and 69 by virus propagation. Phylogenetic analysis strongly suggested male to female sexual transmission in a couple returning from Maldives. Conclusion This case series confirms that Zika virus RNA can be detected in semen up to three months after infection. Viral culture of semen samples shows prolonged infectivity that can lead to sexual transmission of Zika virus.

We report the largest study on the prevalence and distribution of HCV genotypes in Spain (2000-2015), and we relate them with clinical, epidemiological and virological factors. Patients from 29 hospitals in 10 autonomous communities (Andalusia, Aragon, Castilla-Leon, Catalonia, Galicia, Canary Islands, Madrid Community, Valencian Community, Murcia Region and Basque Country) have been studied. Annual distribution of HCV genotypes and subtypes, as well as gender, age, transmission route, HIV and/or HBV coinfection, and treatment details were recorded. We included 48595 chronically HCV-infected patients with the following characteristics: median age 51 years (IQR, 44-58), 67.9% male, 19.1% HIV-coinfected, 23.5% HBV-coinfected. Parenteral transmission route was the most frequent (58.7%). Genotype distribution was 66.9% GT1 (24.9% subtype 1a and 37.9% subtype 1b), 2.8% GT2, 17.3% GT3, 11.4% GT4 and 0.1% GT5 and 0.02% GT6. LiPA was the most widely HCV genotyping test used (52.4%). HCV subtype 1a and genotypes 3 and 4 were closely associated with male gender, parenteral route of infection and HIV and HBV coinfection; in contrast, subtype 1b and genotype 2 were associated with female gender, nonparenteral route and mono-infection. Age was related to genotype distribution, and different patterns of distribution and biodiversity index were observed between different geographical areas. Finally, we describe how treatment and changes in transmission routes may have affected HCV genotype prevalence and distribution patterns. We present the most recent data on molecular epidemiology of hepatitis C virus in Spain. This study confirms that genotype distributions vary with age, sex, HIV and HBV coinfection and within geographical areas and epidemiological groups.

Prolonged treatment of human immunodeficiency virus (HIV)–infected patients with nonnucleoside reverse transcriptase inhibitors (NNRTIs) might result in the selection of resistant mutants, the most frequent being the K103N mutation in reverse transcriptase. Resistance mutations are routinely detected by Sanger sequencing of the whole viral population, which does not detect sequence variants with frequencies below 20%. We have developed a pyrosequencing approach for the analysis of codon 103 of the HIV reverse transcriptase gene in the circulating viral population that detects variants below the limit of conventional sequencing. The method was tested with samples from 5 controls (not exposed to NNRTIs), 6 from patients exposed to NNRTIs and having a K103N mutant virus population detected by conventional sequencing, and 9 from patients previously exposed to NNRTIs that had a wild-type virus population by conventional sequencing. In 7 of 9, samples the mutation could not be detected by either the standard assay or pyrosequencing, while in 2 samples persistence of the mutation could be detected by pyrosequencing. The method might be of practical use in detecting minority variants of HIV in the clinical setting, in epidemiological studies with large numbers of samples, or as a complement to more complex approaches.

Sequencing data from Plasmodium ovale genotypes co-circulating in multiple countries support the hypothesis that P. ovale curtisi and P. ovale wallikeri are 2 separate species. We conducted a multicenter, retrospective, comparative study in Spain of 21 patients who had imported P. ovale curtisi infections and 14 who had imported P. ovale wallikeri infections confirmed by PCR and gene sequencing during June 2005-December 2011. The only significant finding was more severe thrombocytopenia among patients with P. ovale wallikeri infection than among those with P. ovale curtisi infection (p = 0.031). However, we also found nonsignificant trends showing that patients with P. ovale wallikeri infection had shorter time from arrival in Spain to onset of symptoms, lower level of albumin, higher median maximum core temperature, and more markers of hemolysis than did those with P. ovale curtisi infection. Larger, prospective studies are needed to confirm these findings. We thank Francisco Javier Vilar Izquierdo for his assistance in the translation and critical review of this manuscript.Dr Rojo-Marcos is a consultant in tropical and travel medicine with the Department of Internal Medicine, Príncipe de Asturias Uni-versity Hospital, Alcalá de Henares, Madrid, Spain. His research interests include malaria, leishmaniasis, schistosomiasis, imported tropical diseases, and social and health problems of immigration Sí

Chagas disease is a chronic and systemic infection caused by Trypanosoma cruzi. According to estimates from WHO, 10 million people are affected by this parasite. In the last years, birthrate among the immigrant women from Latin America settled in the Comunidad Autónoma de Madrid has been increasing, and as T. cruzi can be transmitted from mother to child, in fact 11 cases of congenital Chagas disease have been confirmed. Therefore, the aim of this paper is encouraging improvements in the coverage of the anti-T. cruzi antibodies detection in pregnant women from endemic areas. By this strategy, an active search for infected pregnant women and early detection of her infected newborns could be conducted, and then an early specific treatment could be administrated. Thus, there could be an important contribution to the control of Chagas disease in non-endemic area.

We report long-term virologic response to etravirine and tenofovir/emtricitabine in four HIV-1-infected patients who had prior standard genotypic resistance testing showing an isolated K103N mutation (three acquired, one transmitted). In three patients tested, the K103N mutation was detected in cellular HIV-1 DNA whereas remaining suppressed on etravirine plus tenofovir/emtricitabine.

As HIV becomes a chronic infection, an increasing number of HIV-infected patients are travelling to malaria-endemic areas. Association of malaria with HIV/AIDS can be clinically severe. Severe falciparum malaria is a medical emergency that is associated with a high mortality, even when treated in an Intensive Care Unit. This article describes two cases of HIV-positive patients, who returned from malaria-endemic areas and presented a parasitaemia > 5% of erythrocytes and clinical signs of severe falciparum malaria, both with > 350 CD4 cell count/μl, absence of chemoprophylaxis and successful response. Factors like drug interactions and the possible implication of anti-malarial therapy bioavailability are all especially interesting in HIV-malaria co-infections.

The molecular epidemiology of HIV-1 is constantly changing, mainly as a result of human migratory flows and the high adaptive ability of the virus. In recent years, Spain has become one of Europe's main destinations for immigrants and one of the western European countries with the highest rates of HIV-positive patients. Using a phylogeographic approach, we have analyzed the relationship between HIV-1 variants detected in immigrant and native populations of the urban area of Madrid. Our project was based on two coincidental facts. First, resistance tests were extended to naïve and newly diagnosed patients, and second, the Spanish government legislated the provision of legal status to many immigrants. This allowed us to obtain a large data set ( n = 2,792) from 11 Madrid hospitals of viral pol sequences from the two populations, and with this unique material, we explored the impact of immigration in the epidemiological trends of HIV-1 variants circulating in the largest Spanish city. The prevalence of infections by non-B HIV-1 variants in the studied cohort was 9%, rising to 25% among native Spanish patients. Multiple transmission events involving different lineages and subsubtypes were observed in all the subtypes and recombinant forms studied. Our results also revealed strong social clustering among the most recent immigrant groups, such as Russians and Romanians, but not in those groups who have lived in Madrid for many years. Additionally, we document for the first time the presence of CRF47_BF and CRF38_BF in Europe, and a new BG recombinant form found in Spaniards and Africans is tentatively proposed. These results suggest that the HIV-1 epidemic will evolve toward a more complex epidemiological landscape.

Human immunodeficiency virus type 1 (HIV-1) antiviral drug resistance is a major consequence of therapy failure and compromises future therapeutic options. Nelfinavir and lopinavir/ritonavir-based therapies have been widely used in the treatment of HIV-infected patients, in combination with reverse transcriptase inhibitors. The aim of this observational study was the identification and characterization of mutations or combinations of mutations associated with resistance to nelfinavir and lopinavir/ritonavir in treated patients. Nucleotide sequences of 1,515 subtype B HIV-1 isolates from 1,313 persons with different treatment histories (including naive and treated patients) were collected in 31 Spanish hospitals over the years 2002–2005. Chi-square contingency tests were performed to detect mutations associated with failure to protease inhibitor-based therapies, and correlated mutations were identified using statistical methods. Virological failure to nelfinavir was associated with two different mutational pathways. D30N and N88D appeared mostly in patients without previous exposure to protease inhibitors, while K20T was identified as a secondary resistance mutation in those patients. On the other hand, L90M together with L10I, I54V, A71V, G73S, and V82A were selected in protease inhibitor-experienced patients. A series of correlated mutations including L10I, M46I, I54V, A71V, G73S, and L90M appeared as a common cluster of amino acid substitutions, associated with failure to lopinavir/ritonavir-based treatments. Despite the relatively high genetic barrier of some protease inhibitors, a relatively small cluster of mutations, previously selected under drug pressure, can seriously compromise the efficiency of nelfinavir- and lopinavir/ritonavir-based therapies. J. Med. Virol. 79:1617–1628, 2007. © 2007 Wiley-Liss, Inc.

Malaria is the most potentially dangerous imported parasitic disease in Spain [1, 2] and other European countries [3], and laboratories must be prepared round the clock to diagnose the disease quickly to start treatment and avoid complications [4]. In recent years, a second generation of rapid diagnostic tests combining the detection of a soluble Plasmodium antigen, the histidine-rich protein II (HRP-2) and aldolase, an antigen common to all species of Plasmodium, has been marketed and proved to be useful [5]. As a recent meta-analysis showed that HRP-2-based tests are superior to the lactate dehydrogenase (LDH) tests in diagnosis of malaria in nonimmune returned travellers [6], we decided to perform a multicentric study to determine the performance of a HRP-2-based diagnostic kit (NOW ICT) and expert microscopy in comparison with multiplex polymerase chain reaction (PCR) as the gold standard, with a target population mainly composed of febrile semi- and nonimmune and travellers returned from Africa. Eligible for the study were all febrile patients (>=38°C),presenting in emergency rooms or ambulatory consults of five hospitals of the Community of Madrid (Spain) who had visited or migrated from an endemic area for malaria in the previous year, to whom the physician in charge requested a malaria test to the laboratory. Patients were excluded if they had received at least one dose of an antimalarial drug in the preceding 4 weeks. Whole-blood samples were collected on ethylenediamine tetraacetic acid (EDTA) tubes for microscopy (thick and thin films stained with Giemsa stain [7]), PCR and HRP-2 detection. The procedure of the NOW ICT (detection of PfHPR2 for P. falciparum and aldolase, a common antigen to the four species of malaria) was performed by laboratory technicians as recommended by the manufacturer [5]. Slides were examined by one experienced microscopist for at least 500 oil fields (x 1,000) before reporting them as negative. Parasite count was rated as very low ( 50,000). The malaria PCR was carried out in the Parasitology Department of the National Microbiology Center following the seminested multiplex malaria PCR method [8] without knowledge of the results of previous thick film and rapid tests. True positives were defined taking multiplex PCR as the gold standard. Sensitivities and specificities and positive (PPV) and negative (NPV) predictive values with 95% confidence interval (CI) were calculated for expert microscopy and NOW ICT.

HIV-1 viral load testing from 51 patients was compared in plasma samples simultaneously processed and stored in primary Vacutainer plasma preparation tubes (PPTs) and PPT sample aliquots transferred in secondary tubes before freezing, using RT-PCR quantification with an ultrasensitive method (the Roche AMPLICOR HIV-1 MONITOR). We concluded that freezing the primary sample in the PPT collection tube can artificially elevate the HIV-1 viral load. We therefore store samples as frozen aliquots in a second tube.

The purpose of this study was to compare a line probe assay (LiPA) with sequence analysis for the detection of mutations conferring resistance to nucleoside and non-nucleoside inhibitors in human immunodeficiency reverse transcriptase and protease inhibitors. The limitations for interpreting LiPA make it unacceptable for routine clinical practice.

The purpose of this study was to analyze the quantitation of the human immunodeficiency virus type 1 RNA (HIV-1 RNA) in the genital tract of HIV-1-infected pregnant women and to evaluate a possible correlation with the viral load in blood plasma (Spearman's rank correlation coefficient). A total of 38 each of cervical, vaginal, and blood samples from 38 women were obtained during the third trimester of pregnancy for quantitation of the HIV-1 RNA load. Viral loads were determined by reverse transcription-polymerase chain reaction. The HIV-1 RNA viral load was detectable in 29 of the 38 (76.3%) blood samples, in 6 of the 38 (15.7%) cervical secretion samples, and in 8 of the 38 (21%) vaginal secretion samples. Overall, the correlation between the HIV-1 RNA viral load in the blood plasma and in cervical secretion samples was 0.51 ( P0.001). However, the correlation disappeared ( r=0.27) when three patients with high blood plasma viral loads were eliminated from the statistical study. The viral load in the vaginal secretions did not correlate with that in the blood samples ( r=0.26). There were two cases in which HIV-1 RNA was undetectable in the blood and cervix but was detectable in vaginal secretions: one woman had 220 copies/ml and the other 68 copies/ml. These results suggest that pregnant women with undetectable viral loads in blood plasma are still at risk of transmitting the virus vertically during vaginal delivery. Because of this, antiretroviral prophylaxis during vaginal delivery must be administered to HIV-1-infected women and their newborns, regardless of the mother's viral load in plasma. In conclusion, quantification of cervicovaginal levels of HIV-1 may represent a useful tool for assessing the individual risk associated with a vaginal delivery and for guiding decisions about whether a scheduled caesarean should be recommended.

ingreso del paciente para iniciar tratamiento intravenoso con anfotericina B liposomal 165 mg/dia, que se mantiene durante 9 dias. La evolucion fue favorable. Los ensayos de PCR para Leishmania son positivos y, el estudio de las secuencias SSUrRNA e ITS-1 obtenidas con las distintas PCR indican que la infeccion esta causada por un parasito perteneciente al complejo Leishmania braziliensis. Descripcion clinica del caso

esporulado. La visualizacion en fresco de heces permite ver estructuras esfericas y de un tamano aproximado de 8 a 10 μm de diametro. A causa de que la eliminacion de los ooquistes puede ser intermitente, esporadica y muchas veces en poco numero, las tecnicas de concentrado de heces forman parte de los procedimientos de diagnostico de rutina. Los ooquistes de C. cayetanensis son aproximadamente 2 veces mayores que los de Cryptosporidium spp. y al menos un tercio mas pequenos que los de Isospora spp. que son los otros dos coccidios de esta familia mas frecuentes en el hombre. La esporulacion de los ooquistes confirma la identificacion del parasito. Para ello, se puede incubar la suspension de heces en 2,5% de dicromato potasico a temperatura ambiente (25-32 °C) en una placa de Petri y Caso clinico