Manuel Segovia

Comparison of Diagnostic Sensitivities of Three Assays (Bartels Enzyme Immunoassay [EIA], Biotest EIA, and Binax NOW Immunochromatographic Test) for Detection of Legionella pneumophila Serogroup 1 Antigen in Urine

ABSTRACT The Bartels enzyme immunoassay (EIA), Biotest EIA, and Binax NOW immunochromatographic test (ICT) urinary antigen kits for the detection of Legionella pneumophila serogroup 1 were compared using 178 frozen urine samples. When nonconcentrated urine samples were used, the sensitivity levels of both enzyme EIAs were significantly higher than the sensitivity level of the ICT (Bartels EIA, 71.3%; Biotest EIA, 65.1%; Binax NOW ICT, 37% [P < 0.001]). After concentration of the urine samples, no significant differences in sensitivity were found among the three tests.

Risk Factors and Primary Prevention of Congenital Chagas Disease in a Nonendemic Country

BACKGROUND In this longitudinal cohort study we evaluated the congenital transmission of Chagas disease (CD) in a nonendemic area. The aim of this work was to analyze the predictive value of a Trypanosoma cruzi-positive polymerase chain reaction (PCR) result in pregnant women for the diagnosis of vertical transmission and to evaluate the use of PCR as a tool for early detection of infection. METHODS The offspring of 59 seropositive pregnant mothers were followed up. The parasitological status of mothers was studied by PCR in a total of 64 pregnancies; 10 of these women had received treatment before pregnancy. Sixty-five infants (including a pair of twins) were monitored at 0, 6, 9, and 12 months of age by PCR and serology. In cases of congenital transmission, hemoculture and parasite lineage typing were performed. RESULTS Nine infants had acquired CD congenitally. This represents a transmission rate of 13.8% among seropositive mothers (9 infected newborns of 65 total live births). All infants were infected with T. cruzi discrete typing unit V strain. A statistically significant correlation was found between T. cruzi vertical transmission and a positive PCR result during pregnancy (31%; 9 infected newborns in 29 live births). No infected infants were detected among 10 mothers who were treated before they became pregnant, compared with 16.4% (9 of 55 live births) among untreated mothers. CONCLUSIONS PCR is a useful tool for the detection of congenital CD, and the treatment of infected women of childbearing age seems to be useful for preventing vertical transmission.

The urgent need to develop new drugs and tools for the treatment of Chagas disease

Chagas disease Chagas disease, also known as American trypanosomiasis, is caused by the protozoan parasite Trypanosoma cruzi, which was discovered by the Brazilian Carlos Chagas in 1909, and affects people predominantly from poor rural areas of Latin America. Approximately 10–12 million people are infected in endemic areas and the disease kills more than 15,000 people each year [1]. T. cruzi is composed of hetero geneous strains that have been recently classified into six groups, TcI–VI, which have different geographical distributions and biological properties [2]. The parasite is transmitted in the feces of a triatomine insect after feeding on the blood of diverse mammals, including humans and other vertebrates. The parasite is able to penetrate the new host through broken skin or through the mucous membranes of the eyes and mouth. Vertical transmission is the second source of infection. In endemic countries, the vertical transmission rate is between less than 1 and 17% depending on geographic area [3]. Oral transmission to humans and animals has also been described after the ingestion of food and drink contaminated with the feces of an infected triatomine [4]. In nonendemic areas, vertical transmission and blood and organ donations from immigrants coming from an endemic area are the main pathways of Chagas disease transmission. In Europe (especially Spain and Portugal), the USA, Canada, Japan and Australia there are large numbers of South American immigrants who are often unaware of their infection, which has serious public health implications for the management of blood banks and healthcare provision [5]. Therefore, the compulsory screening of T. cruzi in hemoderivatives and solid organs donated by patients from Latin America should be considered. The infection goes through two clinical periods: an acute and a consecutive chronic phase [6]. The first symptoms of acute Chagas disease appear 1 week after entry of the parasite and include fever, discomfort and swelling of the lymph nodes and tissues. The most common manifestation of acute Chagas disease is Romaña’s sign, which is simply the bite mark of the vector when it is located in the eyelid. In most patients, acute Chagas disease symptoms resolve spontaneously. Nevertheless, without specific treatment during the acute phase, the infection progresses to a long asymptomatic period, which may last for years, and is known as indeterminate or asymptomatic chronic Chagas disease. It is estimated that in around one-third of those infected, clinical manifestations of different degrees of Maria J Muñoz

Dynamics and size polymorphisms of minichromosomes in Leishmania major LV-561 cloned lines

Various lines and cloned lines of Leishmania major of varying degrees of virulence in BALB/c mice possessed size polymorphic multicopy minichromosomes related to previously described LD1/CD1 and 715-class DNAs of Leishmania. The minichromosomes were not necessary for virulence. Two of these DNAs (M180 and M210), coexisting in a single cloned line, showed remarkable dynamics in terms of loss or gain when followed through multiple transfers during in vitro culture and in vivo passage in BALB/c mice. Although there was significant sequence heterogeneity among minichromosomes, M180 sequences were present within large (megabase) and in intermediate (550-760 kb) chromosomes in the L. major lines analysed. M180 related small DNAs were also detected in Leishmania mexicana and Leishmania donovani isolates, suggesting that the generation of these molecules involves a common, probably functional basic mechanism widespread in Leishmania.

Trypanosoma cruzi paraflagellar rod proteins 2 and 3 contain immunodominant CD8+ T-cell epitopes that are recognized by cytotoxic T cells from Chagas disease patients

The protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas disease. To date, no vaccine is available for protection against T. cruzi infection. The CD8(+) T cells immune response against specific antigens has shown to efficiently control the spread of the parasite in murine experimental infection. However, data concerning CD8(+) response in Chagas patients are still restricted to a few epitopes. We have studied the existence of immunodominant CD8(+) T cell epitopes in the paraflagellar rod proteins 2 and 3 (PFR2 and PFR3) from T. cruzi in a mouse model, and analyzed their recognition by cytotoxic T lymphocytes from Chagas disease patients. Immunization of C57BL/6-A2/K(b) transgenic mice with plasmids coding for the fusion proteins PFR2-HSP70 and PFR3-HSP70 induced a specific CTL response against two PFRs epitopes (PFR2(449-457) and PFR3(481-489)), and showed specific lysis percentages of 24 and 12, respectively. Moreover, the PFR2(19-28), PFR2(156-163), PFR2(449-457), PFR3(428-436), PFR3(475-482) and PFR3(481-489) peptides were observed to have a high binding affinity to the HLA-A*02:01 molecule. Remarkably, these HLA-A*02:01-binding peptides are successfully processed and presented during natural infection by T. cruzi in the context of MHC class I as evidenced by using peptide-pulsed K562-A2 cells as antigen presenting cells. The T cells from Chagas disease chronic patients specific for PFR2/PFR3 selected CD8(+) epitopes showed a pro-inflammatory cytokine secretion profile (IFN-γ, TNF-α and IL-6). A positive Granzime B secretion was observed in three out of 16 patients in response to PFR2(156-163) and PFR2(449-457) peptides, two out of 11 patients in response to PFR2(19-28) peptide and one out of 14 and 11 patients in response to PFR3(428-436) and PFR3(481-489) peptides, respectively. The PFRs-specific cytotoxic activity in purified PBMC was only detected in patients in the indeterminate phase of the disease.

Assessment of multiple coagulase-negative staphylococci isolated in blood cultures using pulsed-field gel electrophoresis

One of the criteria used to determine the clinical importance of coagulase-negative staphylococci (CMS) is the isolation of the bacteria from sequential blood cultures. Sequential isolates of CNS obtained from five immunocompromised patients over three months were genetically characterized by pulsed-field gel electrophoresis (PFGE). This typing method was compared to two first-line typing methods: determination of the species and of antibiotic susceptibility. In four patients the initial clinical evaluation changed because of the PFGE results; several episodes of bacteremia would have been wrongly assessed if only the biotype and the antibiotype had been determined. Pulsed-field gel electrophoresis should therefore be used for CNS strains from immunocompromised patients or those suffering from chronic diseases with non-concordant biotype and antibiotype.

Characterisation of the novel junctions of two minichromosomes of Leishmania major

The minichromosomes M210 and M230 of Leishmania major are linear amplicons which have an inverted repeat structure. We report here the molecular cloning of the megabasic chromosome region where M210 and M230 recombination points are located. The analysis of this chromosomal region revealed a 374 bp fragment directly repeated 3 times, which was also repeated elsewhere in the genome. DNA fragments containing the novel junctions of the minichromosomes M210 and M230 have been likewise cloned. The nucleotide sequence of these fragments was determined and compared with their corresponding DNAs in the source chromosome. It revealed that M210 contains a central 226 bp non-repeated fragment, whereas the central fragment of M230 was 5 kb long. A 9 bp sequence which was repeated in inverted orientation around the recombination points of M230 was found. M210 showed instead an A + T rich DNA which could be implicated in its generation.

Diagnóstico y tratamiento de la enfermedad de Chagas

Resumen La infeccion por Tripanosoma cruzi o enfermedad de Chagas fue descubierta por Carlos Chagas hace mas de 100 anos. Esta considerada como una enfermedad tropical olvidada, aunque esta infeccion causa mas de 15.000 muertes anuales. Actualmente afecta a 8 millones de personas en 21 paises de America Latina. Sin embargo, debido a los movimientos migratorios, esta enfermedad tambien esta presente en zona no endemica. El numero de poblacion inmigrante con enfermedad de Chagas cronica ha aumentado en Espana en los ultimos anos. Ademas se han informado varios casos de transmision congenita. Algunos de los pacientes presentan manifestaciones clinicas graves y requieren tratamiento especializado, como la implantacion de marcapasos e incluso trasplante de corazon. Por tanto, esta infeccion tiene unos impactos clinico, social y economico considerables, principalmente en areas con una alta tasa de inmigracion. Benznidazol y nifurtimox son los unicos medicamentos disponibles para el tratamiento etiologico desde 1960. El tratamiento con ambos farmacos esta recomendado en la fase aguda y cronica de la enfermedad; no obstante, es urgente la busqueda de nuevos farmacos que presenten mayor eficacia y sean mejor tolerados por los pacientes. Recientemente se han desarrollado nuevas estrategias de diagnostico y control de la cura de la infeccion en fase cronica, permitiendo analizar la eficacia de los farmacos.Trypanosoma cruzi infection, or Chagas disease, was discovered more than 100 years ago by Carlos Chagas. Although the infection kills more than 15,000 people each year, it is still classified as a neglected tropical disease. Today, this disease affects eight million people in 21 Latin American countries and, due to immigration, is also present in non-endemic countries. In recent years, the size of the immigrant population with chronic imported forms of Chagas disease has increased in Spain. In addition, several cases of congenital transmission have been reported. Some patients have severe infection and require specialized treatment such as pacemaker implantation or even heart transplantation, representing a considerable clinical, social and economic burden, particularly in areas with a large immigrant population. Since the 1960s, the only drugs available for the etiological treatment of this infection have been benznidazole and nifurtimox. Although new, more effective and better tolerated compounds are urgently needed, treatment with these trypanocidal drugs is recommended in both the acute and chronic stages of Chagas disease. New strategies for diagnosis and infection control in chronically infected patients have recently been reported, allowing the effectiveness of treatments to be assessed.

Inverted repeat structure and homologous sequences in the LD1 amplicons of Leishmania spp.

In the parasitic trypanosomatids of the genus Leishmania, novel circular (CD) and linear (LD) multicopy genetic elements arise de novo either spontaneously or as a result of drug selection. We report that the LD1 minichromosomes of L. donovani, L. major and L. mexicana (ranging in size from 180 to 230 kb) have an inverted repeat structure and contain homologous sequences located at similar distances from the telomere; one half of the chromosome being the mirror image of the other. They must therefore have originated from a unique conserved source chromosome; the size polymorphism being generated by the point at which inversion occurs. The circular CD1 elements appear to be circularised segments of the LD1 elements. These observations lead to a unified concept of how minichromosomes LD1 and circular CD1 genetic elements emerge within the Leishmania and contribute to evolution of karyotype.

Spatial distribution of human asymptomatic Leishmania infantum infection in southeast Spain: A study of environmental, demographic and social risk factors

Recent PCR studies indicate that asymptomatic L. infantum infection is common in people in southern Europe. Understanding its spatial distribution is a requisite to evaluate the public health implications and to design disease control schemes. We investigated infection in blood samples from 657 donors in southeast Spain using PCR and antibody ELISA. They came from 19 blood centers and were interviewed about their residence, occupation, dog ownership and Leishmaniosis awareness. The percentage of PCR and ELISA positives were 8% (49/618) and 2% (13/657). Donors residences were spatially clustered around blood donning centers and PCR prevalence was 18% in rural municipalities with 20-1330 inhabitants, 12% in those with 1467-5088 inhabitants and 3% in larger communities, and was associated with dog ownership (p<0.05). Further analysis of data from rural donors indicated that PCR status was strongly related to the climate, altitude and soil type in the donors residence area and not to other demographic or sociologic variables. Mixed logistic regression analysis predicted PCR prevalence to be greatest in the 200-300m altitude range with a mean spring-summer (time of highest vector activity) temperature of 18.4-19.0°C. A temperature and altitude risk map was generated that will provide the basis for elaborating evidence-based vector surveillance studies.