Polymerase chain reaction of peripheral blood as a tool for the diagnosis of visceral leishmaniasis in children

The diagnosis of visceral leishmaniasis (VL) generally requires the use of invasive tests for the collection of infected tissue (aspirates of bone marrow, spleen, liver or lymph nodes). This difficulty has led to the search for safer and less painful techniques to confirm the occurrence of the disease in children. Polymerase chain reaction (PCR) is a method that is advantageous in that it allows the use of peripheral blood samples for diagnosis. This paper reports the utilisation of PCR on peripheral blood samples to diagnose VL in 45 children in Mato Grosso do Sul, Brazil. This technique is compared with methods carried out using tissue collected by invasive procedures, including direct microscopy, culture and detection of Leishmania DNA by PCR in bone marrow aspirates. The results show that PCR of peripheral blood provides great sensitivity (95.6%) that is similar to that from the PCR of bone marrow aspirates (91.1%) and higher than that achieved with microscopy (80%) or culture (26.7%) methods. PCR of peripheral blood proved to be a suitable tool for the diagnosis of VL in children because it is highly sensitive and safe, with tissue collection being less invasive than in traditional tests.

8-weeks training program attenuates mitochondrial oxidative stress in the liver of emotionally stressed rats

In recent years it has been shown that emotional stress induced by immobilization may change the balance between pro-oxidant and antioxidant factors inducing oxidative damage. On the other hand, contradictory views exist concerning the role of physical activity on redox metabolism. Consequently, the present work was designed to assess the influence of an 8-week moderate swimming training program in emotionally stressed rats. Sixty 1-month-old male albino Wistar rats weighing 125-135 g were used in this experimental study. They were divided into three groups, as Control (lot A; n=20), Stressed (lot B; n=20) and Stressed & Exercised (lot C; n=20). Rats were stressed by placing the animals in a 25 x 7 cm plastic bottle 1 h/day, 5 days a week for 8 weeks. Protein carbonyl content values in liver homogenates were significantly increased in stressed animals (0.58+/-0.02 vs 0.86+/-0.03; p=0.018) which clearly indicated that emotional stress was associated with oxidative stress. Ultrastructural alterations, predominantly mitochondrial swelling and the decrease of cristae number observed by electron microscopy represented direct evidence of membrane injury. The most striking feature of our study was that we also found differences between stressed rats and stressed rats that performed our 8 week training program. Consequently our results highlight the potential benefit of a moderate training program to reduce oxidative damage induced by emotional stress since it attenuated protein oxidation and mitochondrial alterations.

Prevalent role of TCR alpha-chain in the selection of the preimmune repertoire specific for a human tumor-associated self-antigen.

The specificity of recognition of pMHC complexes by T lymphocytes is determined by the V regions of the TCR alpha- and beta-chains. Recent experimental evidence has suggested that Ag-specific TCR repertoires may exhibit a more V alpha- than V beta-restricted usage. Whether V alpha usage is narrowed during immune responses to Ag or if, on the contrary, restricted V alpha usage is already defined at the early stages of TCR repertoire selection, however, has remained unexplored. Here, we analyzed V and CDR3 TCR regions of single circulating naive T cells specifically detected ex vivo and isolated with HLA-A2/melan-A peptide multimers. Similarly to what was previously observed for melan-A-specific Ag-experienced T cells, we found a relatively wide V beta usage, but a preferential V alpha 2.1 usage. Restricted V alpha 2.1 usage was also found among single CD8(+) A2/melan-A multimer(+) thymocytes, indicating that V alpha-restricted selection takes place in the thymus. V alpha 2.1 usage, however, was independent from functional avidity of Ag recognition. Thus, interaction of the pMHC complex with selected V alpha-chains contributes to set the broad Ag specificity, as underlined by preferential binding of A2/melan-A multimers to V alpha 2.1-bearing TCRs, whereas functional outcomes result from the sum of these with other interactions between pMHC complex and TCR.

Acute respiratory and cardiovascular admissions after a public smoking ban in geneva, Switzerland.

BACKGROUND: Many countries have introduced legislations for public smoking bans to reduce the harmful effects of exposure to tobacco smoke. Smoking bans cause significant reductions in admissions for acute coronary syndromes but their impact on respiratory diseases is unclear. In Geneva, Switzerland, two popular votes led to a stepwise implementation of a state smoking ban in public places, with a temporary suspension. This study evaluated the effect of this smoking ban on hospitalisations for acute respiratory and cardiovascular diseases. METHODS: This before and after intervention study was conducted at the University Hospitals of Geneva, Switzerland, across 4 periods with different smoking legislations. It included 5,345 patients with a first hospitalisation for acute coronary syndrome, ischemic stroke, acute exacerbation of chronic obstructive pulmonary disease, pneumonia and acute asthma. The main outcomes were the incidence rate ratios (IRR) of admissions for each diagnosis after the final ban compared to the pre-ban period and adjusted for age, gender, season, influenza epidemic and secular trend. RESULTS: Hospitalisations for acute exacerbation of chronic obstructive pulmonary disease significantly decreased over the 4 periods and were lowest after the final ban (IRR = 0.54 [95%CI: 0.42-0.68]). We observed a trend in reduced admissions for acute coronary syndromes (IRR = 0.90 [95%CI: 0.80-1.00]). Admissions for ischemic stroke, asthma and pneumonia did not significantly change. CONCLUSIONS: A legislative smoking ban was followed by a strong decrease in hospitalisations for acute exacerbation of chronic obstructive pulmonary disease and a trend for reduced admissions for acute coronary syndrome. Smoking bans are likely to be very beneficial for patients with chronic obstructive pulmonary disease.

Aseptic meningitis caused by Leptospira spp diagnosed by polymerase chain reaction

Leptospirosis is a zoonotic disease caused by the pathogenic Leptospira spp. The clinical presentations are diverse, ranging from undifferentiated fever to fulminant disease including meningeal forms. The neurological leptospirosis forms are usually neglected. The aim of this study was to investigate leptospirosis as the cause of aseptic meningitis using different diagnostic techniques including the polymerase chain reaction (PCR). Thirty-nine cerebrospinal fluid (CSF) samples from patients presenting with meningeal abnormalities, predominance of lymphocytes and negative results by traditional microbiological tests were processed by leptospiral culture, anti-leptospiral antibody response and PCR. Leptospira spp DNA was detected in 23 (58.97%) of the CSF samples. Anti-leptospiral antibodies were found in 13 (33.33%) CSF samples. Twelve CSF samples were positive by PCR assay and negative by microscopic agglutination test (MAT) assay. Two CSF samples were positive by MAT and negative by PCR. The positive and negative agreement between both tests was 11 and 14, respectively. CSF samples from six cases of unknown diagnosis were positive by PCR assay. Eight cases showed positive results using PCR and MAT. Leptospirosis could be detected by PCR assay from the 3rd-26th day after illness onset. The sensitivity of the PCR was assessed with confirmed cases of leptospirosis (by MAT) and found to be 89.5%. All CSFs were negative by culture. PCR was found to be a powerful tool for diagnosing meningitis cases of leptospirosis. We recommend that it may be used as a supplementary diagnostic tool, especially in the early stages of the disease, when other diagnostic techniques such as serology are not sensitive.

Impact of human metapneumovirus infection on in and outpatients for the years 2006-2008 in Southern Brazil

The human metapneumovirus (hMPV), member of the Paramyxoviridae family, has been reported as an important agent involved with acute respiratory infections (ARIs). The aim of this study is to identify hMPV as the etiological agent of ARIs on in and outpatients in the city of Curitiba, Southern Brazil, and describe clinical data of hMPV subtyping. A retrospective study was performed in 1,572 respiratory samples over a period of three years. hMPV was detected by reverse transcription-polymerase chain reaction and subtyping was performed by nucleotide sequencing. hMPV was present in 61 (3.9%) samples and subtypes A1, A2a, B1 and B2 were detected. The incidence of hMPV was higher in outpatients (5.9%), whose mean age was 19.7 years (range 6 months-75 years old), than in inpatients (3%), whose mean age was 7.6 months (range 1 month-26 years old). The outpatients had upper respiratory tract infections with flu-like symptoms and all hospitalized children had lower respiratory tract infections. A pediatric patient died from complications associated with hMPV A2a infection. hMPV has been reported as a respiratory pathogen in all age groups. No correlation was observed between viral subtype and disease severity in the samples of this study.

Survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction

This work reports a survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction (PCR). Seventy pampas deer were captured in the dry season and surveyed using PCR, microscopic agglutination test (MAT) (n = 51) and by both techniques (n = 47). PCR detected infections in two pampas deer and MAT detected infections in three. Through sequencing and phylogenetic analyses, the PCR-amplified fragment detected in deer was identified as Leptospira interrogans. Serovars Pomona and Butembo were detected using MAT and the highest titre was 200 for serovar Pomona. Epidemiological aspects of the findings are discussed.

Mitochondrial haplogroups and polymorphisms reveal no association with sporadic prostate cancer in a southern European population.

BACKGROUND It is known that mitochondria play an important role in certain cancers (prostate, renal, breast, or colorectal) and coronary disease. These organelles play an essential role in apoptosis and the production of reactive oxygen species; in addition, mtDNA also reveals the history of populations and ancient human migration. All these events and variations in the mitochondrial genome are thought to cause some cancers, including prostate cancer, and also help us to group individuals into common origin groups. The aim of the present study is to analyze the different haplogroups and variations in the sequence in the mitochondrial genome of a southern European population consisting of subjects affected (n = 239) and non-affected (n = 150) by sporadic prostate cancer. METHODOLOGY AND PRINCIPAL FINDINGS Using primer extension analysis and DNA sequencing, we identified the nine major European haplogroups and CR polymorphisms. The frequencies of the haplogroups did not differ between patients and control cohorts, whereas the CR polymorphism T16356C was significantly higher in patients with PC compared to the controls (p = 0.029). PSA, staging, and Gleason score were associated with none of the nine major European haplogroups. The CR polymorphisms G16129A (p = 0.007) and T16224C (p = 0.022) were significantly associated with Gleason score, whereas T16311C (p = 0.046) was linked with T-stage. CONCLUSIONS AND SIGNIFICANCE Our results do not suggest that mtDNA haplogroups could be involved in sporadic prostate cancer etiology and pathogenesis as previous studies performed in middle Europe population. Although some significant associations have been obtained in studying CR polymorphisms, further studies should be performed to validate these results.

Dermatophyte identification in skin and hair samples using a simple and reliable nested polymerase chain reaction assay.

BACKGROUND: Dermatophyte identification in tinea capitis is essential for choosing the appropriate treatment and in tinea infections to identify the possible source. The failure of fungi to grow in cultures frequently occurs, especially in cases of previous antifungal therapy. OBJECTIVES: To develop a rapid polymerase chain reaction (PCR) sequencing assay for dermatophyte identification in tinea capitis and tinea corporis. MATERIAL AND METHODS: Fungal DNA was extracted from hair and skin samples that were confirmed to be positive by direct mycological examination. Dermatophytes were identified by the sequence of a 28S ribosomal DNA subunit amplicon generated by nested PCR. RESULTS: Nested PCR was found to be necessary to obtain amplicons in substantial amounts for dermatophyte identification by sequencing. The results agreed with those of classical mycological identification in 14 of 23, 6 of 10, and 20 of 23 cases of tinea capitis, tinea corporis and tinea pedis, respectively, from which a dermatophyte was obtained in culture. In seven of the 56 cases, another dermatophyte was identified, revealing previous misidentification. A dermatophyte was identified in 12 of 18, three of five, and four of nine cases of tinea capitis, tinea corporis and tinea pedis, respectively, in cases in which no dermatophyte grew in culture. CONCLUSIONS: Although the gold standard dermatophyte identification from clinical samples remains fungal cultures, the assay developed in the present study is especially suitable for tinea capitis. Improved sensitivity for the identification of dermatophyte species was obtained as it is possible to identify the dermatophyte when the fungus fails to grow in cultures.

Echinococcus granulosus genotypes circulating in alpacas (Lama pacos) and pigs (Sus scrofa) from an endemic region in Peru

The identification of the genotypes of Echinococcus granulosus present in livestock and wild animals within regions endemic for cystic echinococcosis (CE) is epidemiologically important. Individual strains display different biological characteristics that contribute to outbreaks of CE and that must be taken into account in the design of intervention programs. In this study, samples of hydatid cysts due to E. granulosus were collected from alpacas (4) in Puno and pigs (8) in Ayacucho in Peru, an endemic region for CE. Polymerase chain reaction amplification and DNA sequencing of specific regions of the mitochondrial cytochrome C oxidase subunit 1 and NADH dehydrogenase subunit 1 genes confirmed the presence of a strain common to sheep, the G1 genotype, in alpacas. Two different strains of E. granulosus were identified in pigs: the G1 and the G7 genotypes. This is the first report of the G1 genotype of E. granulosus in alpacas in endemic regions of CE in Peru.

Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR). A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.

Infections with human coronaviruses NL63 and OC43 among hospitalised and outpatient individuals in São Paulo, Brazil

The incidence and clinical features of human coronaviruses (HCoVs) among Brazilian patients with respiratory illness are not well known. We investigated the prevalence of HCoVs among Brazilian outpatients and hospitalised patients with respiratory illnesses during 2009 and 2010. To identify the HCoVs, pancoronavirus and species-specific reverse-transcriptase polymerase chain reaction assays were performed. Five of 394 samples were positive for HCoVs (1.2%): 1/182 (0.5%) outpatients and 4/212 (1.8%) hospitalised patients. The OC43 and NL63 HCoVs were identified. Two patients were admitted to the intensive care unit. Underlying chronic disease was reported in cases and one diabetic adult died. HCoVs can cause lower respiratory infections and hospitalisation. Patients with pre-existing conditions and respiratory infections should be evaluated for HCoV infections.

Polymerase chain reaction for the evaluation of Schistosoma mansoni infection in two low endemicity areas of Minas Gerais, Brazil

This study aimed to evaluate the occurrence of schistosomiasis in areas with low endemicity using polymerase chain reaction (PCR) as a diagnostic method. We analysed faecal samples from 219 individuals residing in Piau and Coronel Pacheco, state of Minas Gerais, Brazil, using a single faecal sample from each individual and two slides of the Kato-Katz technique as a gold standard. Fifteen out of the 219 samples were positive with both methods of diagnosis. One sample was diagnosed as positive by the Kato-Katz technique only and 61 were diagnosed only by PCR. The positivity rates were 7.3% with the Kato-Katz method and 34.7% with PCR. When both techniques were assumed to have 100% specificity and positive individuals were identified by both methods, the sensitivity of the Kato-Katz method was 20.8% and the PCR sensitivity was 98.7%. The Kappa index between the two techniques was 0.234, suggesting weak agreement. The assessment of a single faecal sample by PCR detected more cases of infection than the analysis of one sample with two slides using the Kato-Katz technique, suggesting that PCR can be a useful diagnostic tool, particularly in areas with low endemicity.

Estudi de la relació estructura-funció de les proteïnes CPT1

En aquest treball s’ha analitzat la relació estructura-funció dels enzims CPT1, o Carnitina palmitoïltransferasa 1, que catalitza la reacció de transesterificació dels àcids grassos de cadena llarga a acil-carnitines, per tal que puguin accedir a la matriu mitocondrial i ser oxidats. Aquest enzim es troba estrictament regulat per malonil-CoA, primer intermediari de la síntesi d’àcids grassos, establint-se així una regulació coordinada entre la formació i la degradació de grasses. S’han estudiat els tres isotips de CPT1 descrits fins al moment: CPT1A, CPT1B i CPT1C. Mitjançant l’expressió heteròloga de mutants de CPT1A de rata i CPT1B de porc en el llevat P. pastoris, s’ha estudiat l’efecte sobre la inhibició per malonil-CoA de petits canvis en la seva estructura, per tal de trobar una relació entre la seva funció enzimàtica i la disposició conformacional de la proteïna. Segons els resultats obtinguts, el residu Glu590 de CPT1A de rata estaria impedint la unió de l’inhibidor, mentre que el residu Met593 estaria afavorint aquesta unió. Els estudis amb l’enzim CPT1B de porc demostraren l’existència d’un determinant positiu per la sensibilitat al malonil-CoA en els primers 18 residus de la proteïna, i definiren la posició Glu17 com la responsable de l’alta afinitat a la carnitina i la baixa sensibilitat a la inhibició per malonil-CoA (8). Es clonà i caracteritzà la regió promotora del gen de CPT1C humana, amb la intenció d’analitzar la funcionalitat de putatius elements de resposta identificats in silico. Cap dels elements estudiats resultà ser funcional in vivo. A més, es demostrà que la manca d’activitat catalítica de la proteïna no és deguda a l’extensió C-terminal que presenta respecte els isotips A i B, tot i presentar un alt percentatge d’identitat de seqüència. S’ha amplificat una isoforma humana de CPT1C (Pubmed Acc. Num. AK299866), corresponent a la regió carboxiterminal de la proteïna, que es pretén utilitzar per obtenir el primer cristall de la part soluble d’una proteïna CPT1.     

Unveiling healthy carriers and subclinical infections among household contacts of leprosy patients who play potential roles in the disease chain of transmission

Leprosy transmission still occurs despite the availability of highly effective treatment. The next step towards successfully eliminating leprosy is interrupting the chain of transmission of the aetiological agent, Mycobacterium leprae. In this investigation, we provide evidence that household contacts (HHCs) of leprosy patients might not only have subclinical infections, but may also be actively involved in bacilli transmission. We studied 444 patients and 1,352 contacts using anti-phenolic glycolipid-I (PGL-I) serology and quantitative polymerase chain reaction (qPCR) to test for M. leprae DNA in nasal swabs. We classified the patients according to the clinical form of their disease and the contacts according to the characteristics of their index case. Overall, 63.3% and 34.2% of patients tested positive by ELISA and PCR, respectively. For HHCs, 13.3% had a positive ELISA test result and 4.7% had a positive PCR test result. The presence of circulating anti-PGL-I among healthy contacts (with or without a positive PCR test result from nasal swabs) was considered to indicate a subclinical infection. DNA detected in nasal swabs also indicates the presence of bacilli at the site of transmission and bacterial entrance. We suggest that the concomitant use of both assays may allow us to detect subclinical infection in HHCs and to identify possible bacilli carriers who may transmit and disseminate disease in endemic regions. Chemoprophylaxis of these contacts is suggested.