Francisco Jesús Merino

Application of real-time PCR for the detection of Strongyloides spp. in clinical samples in a reference center in Spain

Strongyloidiasis is one of the major intestinal helminthic infections in humans with a worldwide distribution, affecting especially tropical and subtropical regions. This disease can occur without any symptoms or as a potentially fatal hyperinfection or disseminated infection. Definitive diagnosis of Strongyloides stercoralis infection relies mainly on demonstration of larvae in stool, but at present there is no gold standard for this diagnosis. Our main objective was to evaluate a real-time PCR targeting the 18S rRNA gene of Strongyloides spp. and to compare it with routine parasitological methods. DNA from Strongyloides venezuelensis was used to optimize PCR protocols obtaining an analytical sensitivity of 0.1 pg of parasite DNA per sample. Sensitivity and specificity of real-time PCR on fecal samples from 231 patients screened for suspected strongyloidiasis attending two hospitals in Madrid were 93.8% and 86.5%, respectively. No significant differences were found when comparing Ct-values of positive PCR between parasitological positive and negative samples. This study showed that real-time PCR is an effective tool for diagnosing strongyloidiasis and could be applied in association with parasitological methods in epidemiological studies in endemic areas. It would be also important to assess its performance in immunocompromised populations who are at risk of fatal disease.

Epidemiological characteristics of dogs with Lyme borreliosis in the province of Soria (Spain)

From the point of view of the human disease, dogs are the most important animal reservoir of Lyme borreliosis; therefore, they are used as ‘sentinel animals’. In order to know the epidemiological characteristics of dogs with antibodies against Borrelia burgdorferi, 146 canine sera samples collected during 1993–94 have been studied. The antibody detection was made by an indirect immunofluorescence method and confirmed by a haemagglutination test. Seventeen dogs (11.6%) were seropositive, of which seven were hunting dogs, three were shepherd dogs, five were domestic pets, one was a watchdog and one was a stray dog. Seven dogs had longhair phenotype, 2 medium length type, 4 hard-hair and the other 4 were short-haired. Twelve seropositive dogs were males and five were females. All seropositive dogs were more than 1 year old. The dogs with greater seroprevalence were those that harboured ticks more often than the rest. Spring and summer were the seasons when more ticks were observed. The seroprevalence found in dogs was similar to that previously obtained in humans in this area. This finding gave evidence to an intimate association between human and canine seroprevalence.

Tick species and tick-borne infections identified in population from a rural area of Spain.

To determine the tick species that bite humans in the province of Soria (Spain) and ascertain the tick-borne pathogens that threaten peoples health in that province, 185 tick specimens were collected from 179 patients who sought medical advice at health-care centres. The ticks were identified, and their DNA examined by PCR for pathogens. Most ticks were collected in autumn and spring (59 and 57 respectively). Nine species of ticks were identified, the most frequent being Dermacentor marginatus (55.7%), Ixodes ricinus (12.4%) and Rhipicephalus bursa (11.9%). Ninety-seven females, 66 males, 21 nymphs and one larva were identified. Twenty-six ticks carried DNA from Rickettsia spp. (11 Rickettsia slovaca, 6 Rickettsia spp. RpA4/DnS14, 1 Rickettsia massiliae/Bar29, and 8 unidentified); two ticks carried DNA from Borrelia burgdorferi sensu lato and seven ticks harboured DNA from Anaplasma phagocytophilum.

Brote de fiebre Q y seroprevalencia en una población rural de la provincia de Soria

Fundamento El proposito de este estudio es describir un brote de fiebre Q aguda en una poblacion rural de Soria durante la primavera de 1998 y estudiar la prevalencia de anticuerpos IgG frente a Coxiella burnetii en dicha poblacion. MEtodos 1. Brote de fiebre Q: los datos epidemiologicos,clinicos y analiticos se obtuvieron de la historia clinica y por encuesta estandarizada de todos los casos clinicos.Estos se confirmaron por fijacion del complemento. 2. Seroprevalencia: 253 sueros fueron seleccionados por muestreo no probabilistico de conveniencia a partir de muestras de sueros extraidas entre el 1 septiembre de 1996 y el 28 de febrero de 1999. Se consideraron positivos aquellos que por inmunofluorescencia indirecta tenian titulos de anticuerpos IgG frente a C. burnetii fase II iguales o superiores a 1/80. Resultados 1. Se confirmaron 14 casos de fiebre Q con una media de edad de 21,5 ±3,1 anos. El 64% de los pacientes presentaron neumonia y el 36% unaclinica inespecifica. No se encontraron antecedentes de contacto directo con animales, pero en los alrededores del pueblo habia 4 rebanos con un total de 2.614 ovejas. 2. La seroprevalencia fue del 60% (intervalo de confianzadel 95%: 54÷66). La seroprevalencia no se incremento a raiz del brote descrito en este estudio (p > 0,05). ConclusiOn. La alta seroprevalencia de anticuerpos frente a C. burnetii en esta poblacion indica que esta area es hiperendemica para dicha infeccion y al no haberse declarado ningun caso de infeccion en los anos anteriores parece indicar que o bien cursa de forma asintomatica o los signos clinicos son extremadamente leves.Probablemente los rebanos de ovejas fueron el foco de infeccion y la via aerea el mecanismo de transmision.

Evaluación de dos métodos inmunocromatográficos comerciales para el diagnóstico rápido de Giardia duodenalis y Cryptosporidium spp. en muestras de heces

INTRODUCTION To assess and compare the performance of two immunochromatographic tests for the simultaneous detection of Giardia duodenalis and Cryptosporidium spp. in faeces. MATERIALS AND METHODS In this study 254 faeces samples were tested using the two immunochromatography strips Cryto-Giardia (CerTest Biotec) and Stick Crypto-Giardia (Operon). RESULTS In the diagnosis of G. duodenalis, the sensitivity and specificity of the kits were 97% and 100%, respectively for the CerTest; and 97% and 95% for Operon. In the diagnosis of Cryptosporidium spp. Certest strip rendering a sensitivity of 100%, compared to with a sensitivity of 92% using Operon. There were no false positives using either technique. CONCLUSIONS Both methods yielded good sensitivity and specificity values and are thus useful tools for a rapid diagnosis of G. duodenalis and Cryptosporidium spp. The benefits of immunochromatography methods are that there is no requirement for expert microscopists or special equipment.

Infección por el VIH en el adulto: cambios epidemiológicos durante 25 años (1983–2008) en un área de la Comunidad de Madrid

OBJECTIVE To describe the epidemiological characteristics and evolution of a cohort of HIV-infected patients in Madrid (Spain) over a period of 25 years. METHODS Longitudinal, prospective, cohort study of all patients diagnosed with HIV infection seen at the Hospital de Móstoles (1983-2008). RESULTS Of the 2156 patients attended, 73% were men. In 68%, the route of infection was illicit drug use. There was a peak of new diagnoses in 1991 (188 patients) and a subsequent gradual decline. Sexual transmission increased over the time period studied, with a higher rise in heterosexual transmission. The percentage of immigrants with the infection also increased (more than 60% in the last 3 years). Sub-Saharan Africa remains the predominant region of origin of our immigrant patients. Over the period studied, 5% of patients were diagnosed during the acute infection, and 20% to 30% were diagnosed with AIDS within 1 year after detection of HIV infection. The estimated rate of new diagnoses of HIV infection in 2006 was 195 per million population (pmp), 269 pmp in men and 121 pmp in women. CONCLUSIONS Many changes have occurred in the epidemiology of HIV infection in our area in the last 25 years. Routine serologic testing without waiting for warning signs would have an impact on early diagnosis and improving the prognosis of these patients.

Original breveEvaluación de dos métodos inmunocromatográficos comerciales para el diagnóstico rápido de Giardia duodenalis y Cryptosporidium spp. en muestras de hecesAssessment of two commercially available immunochromatographic assays for a rapid diagnosis of Giardia duodenalis and Cryptosporidium spp. in human fecal specimens

INTRODUCTION To assess and compare the performance of two immunochromatographic tests for the simultaneous detection of Giardia duodenalis and Cryptosporidium spp. in faeces. MATERIALS AND METHODS In this study 254 faeces samples were tested using the two immunochromatography strips Cryto-Giardia (CerTest Biotec) and Stick Crypto-Giardia (Operon). RESULTS In the diagnosis of G. duodenalis, the sensitivity and specificity of the kits were 97% and 100%, respectively for the CerTest; and 97% and 95% for Operon. In the diagnosis of Cryptosporidium spp. Certest strip rendering a sensitivity of 100%, compared to with a sensitivity of 92% using Operon. There were no false positives using either technique. CONCLUSIONS Both methods yielded good sensitivity and specificity values and are thus useful tools for a rapid diagnosis of G. duodenalis and Cryptosporidium spp. The benefits of immunochromatography methods are that there is no requirement for expert microscopists or special equipment.

Most common clinical presentation of Q fever in a province in the north of Spain

Q fever is a world-wide zoonosis, produced by Coxiella burnetti, a microorganism belonging to the Riskettsiaceae family. Q fever can appear as a non localized feverish disease, as a feverish syndrome with hepatic impairment or as an atypic pneumonia [1, 2]. The form of presentation varies between different geografic areas, thus, feverish form is more prevalent in Australia [3], while the hepatic one is prevalent in France [4] and the pneumonic one in Nova Scotia (Canada) [5]. In order to know which is the most common presentation pattern of Q fever in our area, we have checked all cases diagnosed between 1 January, 1984 and 31 December, 1996, in the province of Soria, situated in northern Spain. A suggestive clinical feature plus antibody detection against Coxiella burnetti were used as diagnostic criteria. The techniques used for serum diagnosis were complement fixation and/or indirect immunofluorescence. Two samples of sera were obtained in all cases, one during the acute phase of the disease, another 2-4 weeks after. Those cases in which seroconversion or a 4-fold antibody titer rise were found, were considered positive. Thirteen cases of Q fever were diagnosed. Patient age at the time of diagnosis ranged from 10 to 41 years old, with a mean age of 26.72 ? 8.05. Of all the Q fever cases, eleven were men and two women. Pneumonia was the most common presentation pattern, counting 10 cases. Another three were a feverish syndrome without any focus and with an intense headache, a feverish form with hepatitis, and a chronic endocarditis. Like in other areas of northern Spain, pneumonia was the most common form of Q fever. Thus, in a group of 60 patients, 75% were pneumonia [6], in a family outbreak of 5 cases, 3 (60%) presented pneumonia [7] and of 492 Q fever cases diagnosed between 1984 and 1991, 265 (54%) had pneumonia [8]. On the other hand, in central and southern regions of Spain, relative prevalence of pneumonia went down, the hepatic form being the most frequent [9, 10]. All our cases presented as an atypical pneumonia with alveolar infiltrate, with cough in four cases and headache in three. Four patients also had chest pain and none had hemoptysis. Only in two cases, contact with animals or derived products could be found as epidemiologic data explaining how the disease was transmitted. Animals infected with C. burnetti are usually the most important source of infection. Man can be infected by aerosol inhalation, or milk or fresh contaminated cheese ingestion [1, 2, 11]. This variability in the form of clinical presentation of Q fever has been related with the presence of different strains of C. burnetti in the different geographic areas and with plasmids that would regulate the virulence of the strains [2]. It has also been postulated that different clinical patterns would be related to different ways of disease transmission. So, in those cases in which C. burnetii is transmitted by milk or dairy products ingestion, both hepatitis and pneumonia can occur, and in those cases in which the organism is acquired by air inhalation, only pneumonia would occur [12].

Atypical Mansonella ozzardi Microfilariae from an Endemic Area of Brazilian Amazonia

Mansonellosis is endemic in several regions of Africa, the Caribbean, and Latin America. Mansonella ozzardi and Mansonella perstans have been reported in Latin America, including the Amazon region. A morphological and molecular microfilariae study was performed in Pauini (Brazil). Blood samples were collected from 40 individuals, and were analyzed by Giemsa-stained blood film and by two different nested polymerase chain reactions which detect internal transcribed spacer-1 and the major sperm protein gene. By microscopy, 14 of 40 were positive: 11 as M. ozzardi and three as M. perstans-like infections. Both molecular methods detected 19 positive cases as M. ozzardi, including those 14 individuals detected by microscopy, without detectable genetic differences among any of the 19 positive samples. Molecular techniques showed an improvement of mansonellosis diagnosis and may become an effective tool to evaluate the present status of M. ozzardi and M. perstans in Latin America.

Cutaneous phaeohyphomycosis due to Alternaria sp. treated successfully with itraconazole

Combined therapy using tetracycline and nicotinamide may be more effective than tetracycline alone [8,9]. Nycomed-DAK kindly supplied the oxytetracycline and placebo tablets. Henny Bang Jakobsen of Nycomed-DAK provided valuable assistance in planning the study. Financial assistance was provided by Aalborg Stifts Julelotteri. Addendum: Due to the unusually cold weather in Denmark in the summer of 1993, we suggested to the patients that they receive oxytetracycline on an open basis during the summer of 1994. In October, 1994, 25 patients were interviewed by telephone. Fifteen of these patients were treated with 500 mg to 2 g oxytetracycline for up to several months during the hot summer of 1994. Three did not think there had been any improvement in their disease during treatment, six felt there had been moderate improvement, and six felt there had been marked improvement during the treatment period.