6320 research outputs found
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Real-time PCR/MCA assay using fluorescence resonance energy transfer for the genotyping of resistance related DHPS-540 mutations in Plasmodium falciparum
No full textBACKGROUND: Sulphadoxine-pyrimethamine has been abandoned as first- or second-line treatment by most African malaria endemic countries in favour of artemisinin-based combination treatments, but the drug is still used as intermittent preventive treatment during pregnancy. However, resistance to sulphadoxine-pyrimethamine has been increasing in the past few years and, although the link between molecular markers and treatment failure has not been firmly established, at least for pregnant women, it is important to monitor such markers. METHODS: This paper reports a novel sensitive, semi-quantitative and specific real-time PCR and melting curve analysis (MCA) assay using fluorescence resonance energy transfer (FRET) for the detection of DHPS-540, an important predictor for SP resistance. FRET/MCA was evaluated using 78 clinical samples from malaria patients and compared to PCR-RFLP. RESULTS: Sixty-two samples were in perfect agreement between both assays. One sample showed a small wild type signal with FRET/MCA that indicates a polyclonal infection. Four samples were not able to generate enough material in both assays to distinguish mutant from wild-type infection, six samples gave no signal in PCR-RFLP and five samples gave no amplification in FRET/MCA. CONCLUSION: FRET/MCA is an effective tool for the identification of SNPs in drug studies and epidemiological surveys on resistance markers in general and DHPS-540 mutation in particular
Production of human immunodeficiency virus type 1 (HIV-1) pseudoviruses using linear HIV-1 envelope expression cassettes
No full textHIV-1 pseudoviruses constitute an important tool in HIV-1 vaccine and entry inhibitor research. Single-cycle pseudoviruses carrying functional envelopes are generated by co-transfecting HEK293T cells with pNL4-3.LucR(-)E(-) and Env expression plasmids. However, cloning of Env genes is time consuming and single Env clones are not representative of the diversity of HIV-1 in a patient's blood sample. A new method to construct Env expression cassettes is proposed which can be used for the rapid generation of heterogeneous HIV-1 pseudoviruses without a cloning step. The linear Env expression cassettes are constructed by ligating PCR amplified Env genes between a 5' CMV promoter and 3' SV40 polyadenylation element. The resulting cassettes generate pseudoviruses carrying heterogeneous Env variants of a primary HIV-1 isolate derived from viral RNA or proviral DNA. The influence of cis-acting sequences upstream of the Env gene on infectivity was compared between pseudoviruses generated from plasmids and linear expression cassettes. The results suggest that the presence of these upstream sequences tends to result in higher infectivity of pseudoviruses when present in heterogeneous Env expression cassettes, but they do not enhance infectivity of pseudoviruses generated with homogeneous Env expression constructs. Using linear expression cassettes allows for the rapid production of heterogeneous patient-derived functional Env genes
Application of PCR-RFLP for the exploration of the molecular diversity of Leishmania infantum in Algeria
No full textIn recent years, new methods have been developed for the molecular typing of Leishmania that need to be extensively validated by studies of clinical isolates in a well defined epidemiological context. The present study is a contribution to this effort. Using PCR-RFLP of gp63 and cpb genes, we analysed 59 isolates of L. (L.) infantum obtained from different regions of Algeria and originating from different clinical forms, hosts and zymodemes. PCR-RFLP identified 15 different genotypes among the four zymodemes analysed, thereby demonstrating a higher discriminatory power than multilocus enzyme electrophoresis. We did not see any significant relationships between PCR-RFLP patterns and host origin. However, cpb polymorphism showed two interesting trends: a possible relationship with the cutaneous origin of the isolates and an association with a West-East cline. We verified the proof of evidence of the direct applicability of gp63 and cpb PCR-RFLP in blood samples from dogs. Further work is needed to compare the sensitivity of pattern detection with cpb and gp63 PCR-RFLP but our results pave the way to future multilocus PCR-RFLP studies of L. (L.) infantum populations
Evaluation of the GIDEON expert computer program for the diagnosis of imported febrile illnesses
No full textOBJECTIVE: The authors evaluate the performance of the expert system Global Infectious Diseases and Epidemiology Network (GIDEON) in diagnosing febrile illnesses occurring after a stay in the tropics. METHODS: One investigator (E.B.) entered into the program the collected characteristics of 161 febrile travelers randomly extracted from a database of 1842 cases prospectively included during a study on imported fever. Accuracy was considered acceptable if the correct diagnosis appeared in the top 5 GIDEON ranking list. Interuser agreement was assessed by J.V.d.E. and J.M., who also entered the data of the first 50 sample cases with an established diagnosis. RESULTS: The sample was epidemiologically and clinically representative of the whole cohort. An infectious etiology had been established in 129 cases; diagnosis was unknown in 31 cases and non-infectious in 1 case. GIDEON generated a median of 29 diagnoses per case, including 23 with a probability lower than 1%. Accuracy was acceptable in 64% of the 129 fevers with infectious etiology. It tended to decrease when more than 3 findings were entered per case. Eleven (8%) severe conditions were rejected by GIDEON because non-disease-related characteristics had been introduced. In other cases, the posttest probability was inadequately affected by the insufficient weight of absent relevant findings. Interuser agreement was good for acceptable accuracy and final ranking (kappa=0.83 and 0.72, respectively). CONCLUSION: The performance of GIDEON in diagnosing imported fever is relatively good and reproducible but is impaired by some conceptual weaknesses. Its use might be hazardous for inexperienced physicians
A bayesian evaluation of four immunological assays for the diagnosis of clinical cryptosporidiosis in calves
No full textA Bayesian approach was used to evaluate four immunological assays for the clinical diagnosis of cryptosporidiosis in calves: an immunofluorescence assay (IFA), two ELISA tests and an immunochromatographic (dipstick) assay. Faecal samples from 287 calves aged less than 6 weeks with clinical signs of gastrointestinal disease were examined for the presence of Cryptosporidium spp. The high prevalence (63%) of Cryptosporidium spp. indicated the relevance of this agent in the aetiology of diarrhoea in calves. All diagnostic assays were found to be relatively specific (IFA: 94.8%; Tetra ELISA: 95.9%; Techlab ELISA: 92.7%; dipstick assay: 91.5%) and sensitive (IFA: 97.4%; Tetra ELISA: 93.6%; Techlab ELISA: 95.4%; dipstick: 87.8%). Despite a lower sensitivity, the dipstick assay provided a practical alternative to laboratory diagnosis of clinical cryptosporidiosis in calves
Development of a competitive ELISA for detection of Theileria annulata infection
No full textIn previous studies, Theileria annulata surface protein (TaSP) was identified as an immunodominant antigen and successfully used to develop and validate a recombinant-protein-based ELISA for the detection of circulating antibodies in serum of T. annulata–infected animals. In this study, the same antigen was used to develop a competitive ELISA (cELISA) using a monoclonal antibody that was found to bind to TaSP. The cELISA accurately differentiated T. annulata-infected from uninfected animals and demonstrated a satisfactory performance with a calculated sensitivity and specificity of 77.4% and 100%, respectively. Thus the test proved its suitability for the diagnosis of tropical theileriosis and has application for use in serological surveys to monitor the prevalence of the disease or identify carrier animals with high specificity
Bayesian latent class models with conditionally dependent diagnostic tests: a case study
No full textIn the assessment of the accuracy of diagnostic tests for infectious diseases, the true disease status of the subjects is often unknown due to the lack of a gold standard test. Latent class models with two latent classes, representing diseased and non-diseased subjects, are often used to analyze this type of data. In its basic format, latent class analysis requires the observed outcomes to be statistically independent conditional on the disease status. In most diagnostic settings, this assumption is highly questionable. During the last decade, several methods have been proposed to estimate latent class models with conditional dependence between the test results. A class of flexible fixed and random effects models were described by Dendukuri and Joseph in a Bayesian framework. We illustrate these models using the analysis of a diagnostic study of three field tests and an imperfect reference test for the diagnosis of visceral leishmaniasis. We show that, as observed earlier by Albert and Dodd, different dependence models may result in similar fits to the data while resulting in different inferences. Given this problem, selection of appropriate latent class models should be based on substantive subject matter knowledge. If several clinically plausible models are supported by the data, a sensitivity analysis should be performed by describing the results obtained from different models and using different priors. Copyright © 2008 John Wiley & Sons, Ltd
A frontal mucocele caused by an immune reconstitution inflammatory syndrome in a patient with HIV infection
No full textWe describe a 55-year-old bisexual Belgian man with a multi-drug resistant HIV infection who developed an Immune Reconstitution Inflammatory Syndrome (IRIS) presenting as a mucocele of the frontal sinus, one year after starting a new effective darunavir containing antiretroviral treatment regimen. His CD4+ lymphocyte count had increased from 3 cells/mm3 prior to the start of the latter treatment to 196 cells/mm3 just before he developed the IRIS phenomenon. IRIS is a paradoxical clinical deterioration during highly active antiretroviral treatment (HAART), due to an exaggerated immune-inflammatory reaction. With the increasing numbers of persons living with HIV infection and the increased use of HAART it is expected that in the future more otolalyngological manifestations of IRIS will be detected
