Development of Real-Time PCR Methods for the Detection of Bacterial Meningitis Pathogens without DNA Extraction

Neisseria meningitidis (Nm), Haemophilus influenzae (Hi), and Streptococcus pneumoniae (Sp) are the lead causes of bacterial meningitis. Detection of these pathogens from clinical specimens using traditional real-time PCR (rt-PCR) requires DNA extraction to remove the PCR inhibitors prior to testing, which is time consuming and labor intensive. In this study, five species-specific (Nm-sodC and -ctrA, Hi-hpd#1 and -hpd#3 and Sp-lytA) and six serogroup-specific rt-PCR tests (A, B, C, W, X, Y) targeting Nm capsular genes were evaluated in the two direct rt-PCR methods using PerfeCTa and 5x Omni that do not require DNA extraction. The sensitivity and specify of the two direct rt-PCR methods were compared to TaqMan traditional rt-PCR, the current standard rt-PCR method for the detection of meningitis pathogens. The LLD for all 11 rt-PCR tests ranged from 6,227 to 272,229 CFU/ml for TaqMan, 1,824–135,982 for 5x Omni, and 168–6,836 CFU/ml for PerfeCTa. The diagnostic sensitivity using TaqMan ranged from 89.2%-99.6%, except for NmB-csb, which was 69.7%. For 5x Omni, the sensitivity varied from 67.1% to 99.8%, with three tests below 90%. The sensitivity of these tests using PerfeCTa varied from 89.4% to 99.8%. The specificity ranges of the 11 tests were 98.0–99.9%, 97.5–99.9%, and 92.9–99.9% for TaqMan, 5x Omni, and PerfeCTa, respectively. PerfeCTa direct rt-PCR demonstrated similar or better sensitivity compared to 5x Omni direct rt-PCR or TaqMan traditional rt-PCR. Since the direct rt-PCR method does not require DNA extraction, it reduces the time and cost for processing CSF specimens, increases testing throughput, decreases the risk of cross-contamination, and conserves precious CSF. The direct rt-PCR method will be beneficial to laboratories with high testing volume.     

Repellent activity of Cymbopogon citratus and Tagetes minuta and their specific volatiles against Megalurothrips sjostedti

Cowpea is an important source of protein for people in Africa. However, the crop suffers major damage and yield losses due to bean flower thrips, Megalurothrips sjostedti Trybom (Thysanoptera: Thripidae). Although companion plants are known to reduce the damage caused by insect pests, the role of their volatiles in repelling pests from target plants has been the subject of few investigations. Here, we used the Y‐tube olfactometer experiments and chemical analyses to investigate the effect of volatiles from cowpea flowers and two companion plants; lemongrass, Cymbopogon citratus and Mexican marigold, Tagetes minuta on the olfactory responses of M. sjostedti. The results revealed that M. sjostedti males and females were repelled by the volatiles from freshly cut leaves of C. citratus. The combination of freshly cut leaves of C. citratus and cowpea flower was repellent to females but not to males. The female thrips, but not males, were repelled by the volatiles from the vegetative stage of T. minuta. Fifty‐four compounds were identified in the volatiles from two herbal plants. Among the major compounds, citral and a 4‐component blend comprised of dihydrotagetone, (Z)‐3‐hexenyl acetate, limonene and (Z)‐β‐ocimene repelled females but dihydrotagetone alone attracted females. While myrcene combined with cowpea flower volatiles enhanced the attraction of females M. sjostedti, when tested alone was not attractive. These results highlight the potential of volatiles from C. citratus and T. minuta to repel M. sjostedti females. The use of these plants as companion plants in a cowpea cropping system could reduce M. sjostedti infestation.     

Discrepant Prevalence and Incidence of Leishmania Infection between Two Neighboring Villages in Central Mali Based on Leishmanin Skin Test Surveys

Apart from a single report, the last publication of cutaneous leishmaniasis (CL) in Mali dates back more than 20 years. The absence of information on the current status of CL in Mali led us to conduct a cohort study in Kemena and Sougoula, two villages in Central Mali from which cases of CL have been recently diagnosed by Mali''s reference dermatology center in Bamako. In May 2006, we determined the baseline prevalence of Leishmania infection in the two villages using the leishmanin skin test (LST). LST-negative individuals were then re-tested over two consecutive years to estimate the annual incidence of Leishmania infection. The prevalence of Leishmania infection was significantly higher in Kemena than in Sougoula (45.4% vs. 19.9%; OR: 3.36, CI: 2.66–4.18). The annual incidence of Leishmania infection was also significantly higher in Kemena (18.5% and 17% for 2007 and 2008, respectively) than in Sougoula (5.7% for both years). These data demonstrate that the risk of Leishmania infection was stable in both villages and confirm the initial observation of a significantly higher risk of infection in Kemena (OR: 3.78; CI: 2.45–6.18 in 2007; and OR: 3.36; CI: 1.95–5.8 in 2008; P<0.005). The absence of spatial clustering of LST-positive individuals in both villages indicated that transmission may be occurring anywhere within the villages. Although Kemena and Sougoula are only 5 km apart and share epidemiologic characteristics such as stable transmission and random distribution of LST-positive individuals, they differ markedly in the prevalence and annual incidence of Leishmania infection. Here we establish ongoing transmission of Leishmania in Kemena and Sougoula, Central Mali, and are currently investigating the underlying factors that may be responsible for the discrepant infection rates we observed between them.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00344084","term_id":"NCT00344084"}}NCT00344084     

Desert dust impacts on human health: an alarming worldwide reality and a need for studies in West Africa

High desert dust concentrations raise concerns about adverse health effects on human populations. Based on a systematic literature review, this paper aims to learn more about the relationship between desert dust and human health in the world and to analyse the place of West Africa as a study area of interest. Papers focussing on the potential relationship between dust and health and showing quantitative analyses, published between January 1999 and September 2011, were identified using the ISI Web of Knowledge database (N?=?50). A number of adverse health effects, including respiratory, cardiovascular and cardiopulmonary diseases, are associated with dust. This survey highlights obvious dust impacts on human health independently of the study area, health outcomes and method. Moreover, it reveals an imbalance between the areas most exposed to dust and the areas most studied in terms of health effects. None of these studies has been conducted in West Africa, despite the proximity of the Sahara, which produces about half of the yearly global mineral dust. In view of the alarming results in many parts of the world (Asia, Europe, America), this paper concludes by stressing the importance of carrying out impact studies of Saharan dust in West Africa, where dust events are more frequent and intense than anywhere else.     

Experimental ATR device for real-time FTIR imaging of living cells using brilliant synchrotron radiation sources

In this contribution we present the design of an original Attenuated Total Reflection (ATR)-based device designed for an IR microscope coupled to a FPA detector and optimized for in-vivo cell imaging. The optical element has been designed to perform real time experiments of cell biochemical processes. The device includes a manually removable Ge-crystal that guarantees an ease manipulation during the cell culture and a large flat surface to support the cell growth and the required change of the culture wells. This layout will allow performing sequential ATR IR imaging with the crystal immersed in the culture wells, minimizing contributions due to water vapors in the optical system. Using existing brilliant synchrotron radiation sources this ATR device may collect images at the surface of the Ge crystal at a sub-cellular spatial resolution with a penetration depth of the evanescent wave inside the sample of ~ 500 nm within few seconds. A brief summary of the cellular components that should be detected with such optical device is also presented.     

Effects of Age,Hemoglobin Type and Parasite Strain on IgG Recognition of Plasmodium falciparum–Infected Erythrocytes in Malian Children

Background

Naturally-acquired antibody responses to antigens on the surface of Plasmodium falciparum-infected red blood cells (iRBCs) have been implicated in antimalarial immunity. To profile the development of this immunity, we have been studying a cohort of Malian children living in an area with intense seasonal malaria transmission.

Methodology/Principal Findings

We collected plasma from a sub-cohort of 176 Malian children aged 3-11 years, before (May) and after (December) the 2009 transmission season. To measure the effect of hemoglobin (Hb) type on antibody responses, we enrolled age-matched HbAA, HbAS and HbAC children. To quantify antibody recognition of iRBCs, we designed a high-throughput flow cytometry assay to rapidly test numerous plasma samples against multiple parasite strains. We evaluated antibody reactivity of each plasma sample to 3 laboratory-adapted parasite lines (FCR3, D10, PC26) and 4 short-term-cultured parasite isolates (2 Malian and 2 Cambodian). 97% of children recognized ≥1 parasite strain and the proportion of IgG responders increased significantly during the transmission season for most parasite strains. Both strain-specific and strain-transcending IgG responses were detected, and varied by age, Hb type and parasite strain. In addition, the breadth of IgG responses to parasite strains increased with age in HbAA, but not in HbAS or HbAC, children.

Conclusions/Significance

Our assay detects both strain-specific and strain-transcending IgG responses to iRBCs. The magnitude and breadth of these responses varied not only by age, but also by Hb type and parasite strain used. These findings indicate that studies of acquired humoral immunity should account for Hb type and test large numbers of diverse parasite strains.     

Docking proteins and peptides under evolutionary constraints in Critical Assessment of PRediction of Interactions rounds 38 to 45

Computational structural prediction of macromolecular interactions is a fundamental tool toward the global understanding of cellular processes. The Critical Assessment of PRediction of Interactions (CAPRI) community-wide experiment provides excellent opportunities for blind testing computational docking methods and includes original targets, thus widening the range of docking applications. Our participation in CAPRI rounds 38 to 45 enabled us to expand the way we include evolutionary information in structural predictions beyond our standard free docking InterEvDock pipeline. InterEvDock integrates a coarse-grained potential that accounts for interface coevolution based on joint multiple sequence alignments of two protein partners (co-alignments). However, even though such co-alignments could be built for none of the CAPRI targets in rounds 38 to 45, including host-pathogen and protein-oligosaccharide complexes and a redesigned interface, we identified multiple strategies that can be used to incorporate evolutionary constraints, which helped us to identify the most likely macromolecular binding modes. These strategies include template-based modeling where only local adjustments should be applied when query-template sequence identity is above 30% and larger perturbations are needed below this threshold; covariation-based structure prediction for individual protein partners; and the identification of evolutionarily conserved and structurally recurrent anchoring interface motifs. Overall, we submitted correct predictions among the top 5 models for 12 out of 19 interface challenges, including four High- and five Medium-quality predictions. Our top 20 models included correct predictions for three out of the five targets we missed in the top 5, including two targets for which misleading biological data led us to downgrade correct free docking models.     

Protective immunity conferred by porcine circovirus 2 ORF2‐based DNA vaccine in mice

Post‐weaning multisystemic wasting syndrome (PMWS) associated with porcine circovirus type 2 (PCV2) has caused the swine industry significant health challenges and economic damage. Although inactivated and subunit vaccines against PMWS have been used widely, so far no DNA vaccine is available. In this study, with the aim of exploring a new route for developing a vaccine against PCV2, the immunogenicity of a DNA vaccine was evaluated in mice. The pEGFP‐N1 vector was used to construct a PCV2 Cap gene recombinant vaccine. To assess the immunogenicity of pEGFP‐Cap, 80 BALB/c mice were immunized three times at 2 weekly intervals with pEGFP‐Cap, LG‐strain vaccine, pEGFP‐N1 vector or PBS and then challenged with PCV2. IgG and cytokines were assessed by indirect ELISA and ELISA, respectively. Specimens stained with hematoxylin and eosin (HE) and immunohistochemistry (IHC) techniques were examined histopathologically. It was found that vaccination of the mice with the pEGFP‐Cap induced solid protection against PCV2 infection through induction of highly specific serum IgG antibodies and cytokines (IFN‐γ and IL‐10), and a small PCV2 viral load. The mice treated with the pEGFP‐Cap and LG‐strain developed no histopathologically detectable lesions (HE stain) and IHC techniques revealed only a few positive cells. Thus, this study demonstrated that recombinant pEGFP‐Cap substantially alleviates PCV2 infection in mice and provides evidence that a DNA vaccine could be an alternative to PCV2 vaccines against PMWS.     

Filariasis Attenuates Anemia and Proinflammatory Responses Associated with Clinical Malaria: A Matched Prospective Study in Children and Young Adults

Background

Wuchereria bancrofti (Wb) and Mansonella perstans (Mp) are blood-borne filarial parasites that are endemic in many countries of Africa, including Mali. The geographic distribution of Wb and Mp overlaps considerably with that of malaria, and coinfection is common. Although chronic filarial infection has been shown to alter immune responses to malaria parasites, its effect on clinical and immunologic responses in acute malaria is unknown.

Methodology/Principal Findings

To address this question, 31 filaria-positive (FIL+) and 31 filaria-negative (FIL−) children and young adults, matched for age, gender and hemoglobin type, were followed prospectively through a malaria transmission season. Filarial infection was defined by the presence of Wb or Mp microfilariae on calibrated thick smears performed between 10 pm and 2 am and/or by the presence of circulating filarial antigen in serum. Clinical malaria was defined as axillary temperature ≥37.5°C or another symptom or sign compatible with malaria infection plus the presence of asexual malaria parasites on a thick blood smear. Although the incidence of clinical malaria, time to first episode, clinical signs and symptoms, and malaria parasitemia were comparable between the two groups, geometric mean hemoglobin levels were significantly decreased in FIL− subjects at the height of the transmission season compared to FIL+ subjects (11.4 g/dL vs. 12.5 g/dL, p<0.01). Plasma levels of IL-1ra, IP-10 and IL-8 were significantly decreased in FIL+ subjects at the time of presentation with clinical malaria (99, 2145 and 49 pg/ml, respectively as compared to 474, 5522 and 247 pg/ml in FIL− subjects).

Conclusions/Significance

These data suggest that pre-existent filarial infection attenuates immune responses associated with severe malaria and protects against anemia, but has little effect on susceptibility to or severity of acute malaria infection. The apparent protective effect of filarial infection against anemia is intriguing and warrants further study in a larger cohort.     

Ethnopharmacological survey of different uses of seven medicinal plants from Mali, (West Africa) in the regions Doila, Kolokani and Siby

An ethnopharmacological survey was carried out to collect information on the use of seven medicinal plants in rural areas in the nearby regions of Bamako, Mali. The plants were Opilia celtidifolia, Anthocleista djalonensis, Erythrina senegalensis, Heliotropium indicum, Trichilia emetica, Piliostigma thonningii and Cochlospermum tinctorium About 50 medical indications were reported for the use of these plants in traditional medicine. The most frequent ailments reported were malaria, abdominal pain and dermatitis. The highest number of usages was reported for the treatment of malaria (22%). The majority of the remedies were prepared from freshly collected plant material from the wild and from a single species only. They were mainly taken orally, but some applications were prepared with a mixture of plants or ingredients such as honey, sugar, salt, ginger and pepper. Decoction of the leaves was the main form of preparation (65%) and leaf powder was mostly used for the preparation of infusions (13%). The part of the plants most frequently used was the leaves. There was a high degree of informant consensus for the species and their medicinal indications between the healers interviewed. The results of this study showed that people are still dependent on medicinal plants in these rural areas of Mali.