Evaluation of chest radiography, lytA real-time PCR, and other routine tests for diagnosis of community-acquired pneumonia and estimation of possible attributable fraction of pneumococcus in northern Togo.

Streptococcus pneumoniae (Spn) is a leading cause of community-acquired pneumonia (CAP), yet existing diagnostic tools remain inadequate. We aimed to evaluate laboratory and radiological methods for detecting pneumococcal aetiology in CAP patients and to estimate Spn prevalence in this group. All-aged patients hospitalized with clinically defined CAP in northern Togo were enrolled during 2010-2013. Latent class analysis pooled results of semi-automated blood culture (SABC), whole blood lytA real-time polymerase chain reaction (rt-PCR), serum C-reactive protein (CRP), and chest radiography (CXR) and categorized patients as likely pneumococcal or non-pneumococcal CAP. We enrolled 1684 patients; 1501 had results for all tests. CXR, SABC, lytA rt-PCR and CRP >71·2 mg/l had sensitivities of 94% [95% confidence interval (CI) 87-100], 13% (95% CI 10-16), 17% (95% CI 14-21) and 78% (95% CI 75-80), and specificities of 88% (95% CI 84-93), 100% (95% CI 99-100), 97% (95% CI 96-99) and 77% (95% CI 75-79), respectively. Pneumococcal attributable proportion was 34% (95% CI 32-37), increasing with age and in men. We estimated that Spn caused one third of CAP. Whole blood lytA rt-PCR was more sensitive than SABC; both had low sensitivity and high specificity. Conversely CXR was highly sensitive and reasonably specific; it could be a useful tool for epidemiological studies aiming to define Spn pneumonia incidence across all ages.

Development of a one step real time RT-PCR assay to detect and quantify Dugbe virus.

A one-step real time quantitative RT-PCR (qRT-PCR) assay was developed to detect all published Dugbe virus (DUGV) genomes of the Nairovirus genus. Primers and probes were designed to detect specific sequences on the most conserved regions of the S segment. The limit of detection of the assay was 10 copies per reaction which is an improvement of 3 log(10)FFU/mL over the sensitivity of conventional RT-PCR. The specificity of the primers and probe was confirmed with the closely related Nairoviruses CCHFV and Hazara virus, and on the non-related viruses Coronavirus and Influenza A virus. This qRT-PCR assay was used to screen nucleic acids extracted from 498 ticks collected in the Republic of Chad. One sample was found positive suggesting that DUGV is present in this part of the world. The molecular assay developed in this study is sensitive, specific and rapid and can be used for research and epidemiological studies.

Evaluation of real-time nucleic acid sequence-based amplification for detection of Chikungunya virus in clinical samples.

The Chikungunya virus (CHIKV) is a member of the genus Alphavirus that is transmitted to humans by Aedes mosquitoes. In 2005 and 2006, the Indian Ocean island of La Réunion was hit with an unprecedented CHIKV fever outbreak that infected 300 000 people. In the present study, we describe the evaluation of real-time nucleic acid sequence-based amplification (RT-NASBA) for the detection of CHIKV in clinical samples. A co-extracted and co-amplified chimerical CHIKV RNA sequence was used as an internal control to eliminate false-negative results. The detection threshold of the assay was determined from quantified CHIKV-positive plasma, and estimated to be 200 copies per NASBA reaction. The specificity of the assay was determined using blast analyses and non-cross-reactivity using an O'nyong-nyong virus culture and 250 CHIKV RT-PCR-negative plasma samples. A 100 % specificity was found and no invalid result was obtained, showing the good quality of the nucleic acid extraction. The assay was then evaluated using 252 CHIKV-positive RT-PCR plasma samples. The samples were all tested positive, including those with low viral load. This evaluation showed that the RT-NASBA is a rapid (5 h from sample nucleic acid extraction to detection), sensitive, specific and reliable method for the routine diagnosis of CHIKV in clinical samples.

Detection of human immunodeficiency virus type 1 antiretroviral resistance mutations by high-density DNA probe arrays.

Genotypic resistance testing has become an important tool in the clinical management of patients infected with human immunodeficiency virus type 1 (HIV-1). Standard sequencing methodology and hybridization-based technology are the two principal methods used for HIV-1 genotyping. This report describes an evaluation of a new hybridization-based HIV-1 genotypic test of 99 clinical samples from patients infected mostly with HIV-1 subtype B and receiving treatment. This test combines RNA extraction with magnetic silica particles, amplification by nested reverse transcriptase PCR, and detection with high-density probe arrays designed to detect 204 antiretroviral resistance mutations simultaneously in Gag cleavage sites, protease, reverse transcriptase, integrase, and gp41. The nested reverse transcriptase PCR success rates at viral loads exceeding 1,000 copies/ml were 98% for the 2.1-kb amplicon that covers the Gag cleavage sites and the protease and reverse transcriptase genes, 92% for the gp41 amplicon, and 100% for the integrase amplicon. We analyzed 4,465 relevant codons with the HIV-1 DNA chip genotyping assay and the classic sequence-based method. Key resistance mutations in protease and reverse transcriptase were identified correctly 95 and 92% of the time, respectively. This test should be a valuable alternative to the standard sequence-based system for HIV-1 drug resistance monitoring and a useful diagnostic tool for simultaneous multiple genetic analyses.

Quantification of proviral load of human immunodeficiency virus type 2 subtypes A and B using real-time PCR.

We have developed and evaluated a new method to quantify human immunodeficiency virus type 2 (HIV-2) proviral DNA based on LightCycler real-time PCR. The assay has a detection limit of 5 copies/10(5) peripheral blood mononuclear cells (PBMC) and is insensitive to HIV-2 strain variability: HIV-2 subtypes A and B are both recognized and quantified. The intra- and interassay coefficients of variation range from 16 to 40% for high provirus concentrations (5 x 10(5) copies) and from 41 to 39% for low concentrations (5 copies). We used this method to compare the proviral DNA load and viral RNA load in plasma with clinical and immunological status for 29 patients infected by HIV-2 (subtype A in 17 and subtype B in 12). The proviral load (median, 201 copies/10(5) PBMC) was similar to that reported for HIV-1 infection. The median proviral loads did not correlate with the CD4(+) cell count categories and were as follows for CD4(+) cell counts of >400, 200 to 400, and <200 cells/mm(3), respectively: 121 copies/10(5) PBMC (n = 8; range, <5 to 712 copies/10(5) PBMC); 114 copies/10(5) PBMC (n = 9; range, <5 to 1,907 copies/10(5) PBMC); and 285 copies/10(5) PBMC (n = 12; range, 53 to 2,524 copies/10(5) PBMC). Proviral load did not correlate with plasma HIV-2 RNA positivity. As HIV-2 is considered to replicate less efficiently than HIV-1, these high proviral loads might be explained by the proliferation of infected cells.

Selection of HIV-specific immunogenic epitopes by screening random peptide libraries with HIV-1-positive sera.

Efforts to develop a protective HIV-1 vaccine have been hindered by difficulties in identifying epitopes capable of inducing broad neutralizing Ab responses. In fact, the high mutation rate occurring in HIV-1 envelope proteins and the complex structure of gp120 as an oligomer associated with gp41 result in a high degree of antigenic polymorphism. To overcome these obstacles, we screened random peptide libraries using sera from HIV-infected subjects to identify antigenic and immunogenic mimics of HIV-1 epitopes. After extensive counterscreening with HIV-negative sera, we isolated peptides specifically recognized by Abs from HIV-1-infected individuals. These peptides behaved as antigenic mimics of linear or conformational HIV-1 epitopes generated in vivo in infected subjects. Consistent with these findings, sera of simian HIV-infected monkeys also recognized the HIV-specific epitopes. The selected peptides were immunogenic in mice, where they elicited HIV-specific Abs that effectively neutralized HIV-1 isolates. These results demonstrate that pools of HIV-1 mimotopes can be selected from combinatorial peptide libraries by taking advantage of the HIV-specific Ab repertoire induced by the natural infection.

Quantitative and discriminative detection of individual HIV-1 mRNA subspecies by an RNAse mapping assay.

HIV-1 genes are expressed through the complex splicing of a single mRNA precursor leading to three mRNA classes: unspliced, singly-spliced and multiply-spliced. Each class may include several mRNA species specifically encoding one or two HIV-1 proteins. Northern blotting and RT-PCR are the techniques currently used to analyse HIV-1 mRNA expression. Northern blotting allows quantitative detection of these three classes of viral RNA but does not discriminate between individual RNA species. RT-PCR allows discrimination between different species but does not provide a quantitative analysis. Here, we describe an application of an RNAse mapping assay which gives both quantitative and discriminative HIV-1 RNA detection. A radiolabeled probe overlapping the major splicing sites of HIV-1 used for the generation of HIV-1 mRNA subspecies was synthesized. This probe protects differential sizes of these species, allowing discrimination between them. We investigated the RNA expression pattern in high titer HIV-1 producing cells. The HIV-1-specific probe allowed the detection of multiply-spliced vpr, rev and nef mRNAs, singly-spliced env mRNA and unspliced genomic RNA. With its discriminative and quantitative properties, this application is particularly convenient for the investigation of HIV-1 mRNA expression during the course of HIV-1 infections.