Evaluation of the Loop Mediated Isothermal DNA Amplification (LAMP) Kit for Malaria Diagnosis in P. vivax Endemic Settings of Colombia

Background

Most commonly used malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections which are frequent in low transmission settings. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too laborious for field deployment. In this study, the applicability of a malaria diagnosis kit based on loop-mediated isothermal amplification (mLAMP) was assessed in malaria endemic areas of Colombia with Plasmodium vivax predominance.

Methodology/Principal Findings

First, a passive case detection (PCD) study on 278 febrile patients recruited in Tierralta (department of Cordoba) was conducted to assess the diagnostic performance of the mLAMP method. Second, an active case detection (ACD) study on 980 volunteers was conducted in 10 sentinel sites with different epidemiological profiles. Whole blood samples were processed for microscopic and mLAMP diagnosis. Additionally RT-PCR and nested RT-PCR were used as reference tests. In the PCD study, P. falciparum accounted for 23.9% and P. vivax for 76.1% of the infections and no cases of mixed-infections were identified. Microscopy sensitivity for P. falciparum and P. vivax were 100% and 86.1%, respectively. mLAMP sensitivity for P. falciparum and P. vivax was 100% and 91.4%, respectively. In the ACD study, mLAMP detected 65 times more cases than microscopy. A high proportion (98.0%) of the infections detected by mLAMP was from volunteers without symptoms.

Conclusions/Significance

mLAMP sensitivity and specificity were comparable to RT-PCR. LAMP was significantly superior to microscopy and in P. vivax low-endemicity settings and under minimum infrastructure conditions, it displayed sensitivity and specificity similar to that of single-well RT-PCR for detection of both P. falciparum and P. vivax infections. Here, the dramatically increased detection of asymptomatic malaria infections by mLAMP demonstrates the usefulness of this new tool for diagnosis, surveillance, and screening in elimination strategies.     

Prospects for Malaria Elimination in Mesoamerica and Hispaniola

Malaria remains endemic in 21 countries of the American continent with an estimated 427,000 cases per year. Approximately 10% of these occur in the Mesoamerican and Caribbean regions. During the last decade, malaria transmission in Mesoamerica showed a decrease of ~85%; whereas, in the Caribbean region, Hispaniola (comprising the Dominican Republic [DR] and Haiti) presented an overall rise in malaria transmission, primarily due to a steady increase in Haiti, while DR experienced a significant transmission decrease in this period.The significant malaria reduction observed recently in the region prompted the launch of an initiative for Malaria Elimination in Mesoamerica and Hispaniola (EMMIE) with the active involvement of the National Malaria Control Programs (NMCPs) of nine countries, the Regional Coordination Mechanism (RCM) for Mesoamerica, and the Council of Health Ministries of Central America and Dominican Republic (COMISCA). The EMMIE initiative is supported by the Global Fund for Aids, Tuberculosis and Malaria (GFATM) with active participation of multiple partners including Ministries of Health, bilateral and multilateral agencies, as well as research centers. EMMIE’s main goal is to achieve elimination of malaria transmission in the region by 2020. Here we discuss the prospects, challenges, and research needs associated with this initiative that, if successful, could represent a paradigm for other malaria-affected regions.     

Transient Changes in Bacterioplankton Communities Induced by the Submarine Volcanic Eruption of El Hierro (Canary Islands)

The submarine volcanic eruption occurring near El Hierro (Canary Islands) in October 2011 provided a unique opportunity to determine the effects of such events on the microbial populations of the surrounding waters. The birth of a new underwater volcano produced a large plume of vent material detectable from space that led to abrupt changes in the physical-chemical properties of the water column. We combined flow cytometry and 454-pyrosequencing of 16S rRNA gene amplicons (V1–V3 regions for Bacteria and V3–V5 for Archaea) to monitor the area around the volcano through the eruptive and post-eruptive phases (November 2011 to April 2012). Flow cytometric analyses revealed higher abundance and relative activity (expressed as a percentage of high-nucleic acid content cells) of heterotrophic prokaryotes during the eruptive process as compared to post-eruptive stages. Changes observed in populations detectable by flow cytometry were more evident at depths closer to the volcano (~70–200 m), coinciding also with oxygen depletion. Alpha-diversity analyses revealed that species richness (Chao1 index) decreased during the eruptive phase; however, no dramatic changes in community composition were observed. The most abundant taxa during the eruptive phase were similar to those in the post-eruptive stages and to those typically prevalent in oceanic bacterioplankton communities (i.e. the alphaproteobacterial SAR11 group, the Flavobacteriia class of the Bacteroidetes and certain groups of Gammaproteobacteria). Yet, although at low abundance, we also detected the presence of taxa not typically found in bacterioplankton communities such as the Epsilonproteobacteria and members of the candidate division ZB3, particularly during the eruptive stage. These groups are often associated with deep-sea hydrothermal vents or sulfur-rich springs. Both cytometric and sequence analyses showed that once the eruption ceased, evidences of the volcano-induced changes were no longer observed.     

A Novel Expansin Protein from the White-Rot Fungus Schizophyllum commune

A novel expansin protein (ScExlx1) was found, cloned and expressed from the Basidiomycete fungus Schizophylum commune. This protein showed the canonical features of plant expansins. ScExlx1 showed the ability to form “bubbles” in cotton fibers, reduce the size of avicel particles and enhance reducing sugar liberation from cotton fibers pretreated with the protein and then treated with cellulases. ScExlx1 was able to bind cellulose, birchwood xylan and chitin and this property was not affected by different sodium chloride concentrations. A novel property of ScExlx1 is its capacity to enhance reducing sugars (N-acetyl glucosamine) liberation from pretreated chitin and further added with chitinase, which has not been reported for any expansin or expansin-like protein. To the best of our knowledge, this is the first report of a bona fide fungal expansin found in a basidiomycete and we could express the bioactive protein in Pichia pastoris.     

Development of diagnostic markers for selection of the subacid trait in peach

Peaches with low acidity are preferred in the market and this trait is usually selected in commercial breeding programs. A major gene (D/d) has been described for this character located on linkage group 5 of peach, where the low acid character is determined by the dominant D allele. In this paper, we analyze a collection of 231 varieties and 542 offspring to identify diagnostic markers for this character. The CPPCT040 single sequence repeat (SSR) is known to be tightly linked to D. We found that one of its alleles (193) is diagnostic for the subacid character and identified with high probability individuals with low acidity (titratable acidity <5.5 mg/l). The region around CPPCT040 was explored using 13 DNA fragments for a total of 5,297 bp, covering a length of 70.4 kbp of the peach genome. The sequenced fragments detected 19 single nucleotide polymorphisms (SNPs) and five indels. All subacid individuals shared a large haplotype (>24 kb) around CPPCT040, a region with higher than average SNPs between acid and subacid varieties. The CPPCT040 marker plus one of the SNPs identified (DH875) were used to genotype a collection of 542 seedlings, from different crosses expected to segregate for this character, which were phenotyped by tasting the fruit in the field. Data provided by both markers were always consistent and only 24 plants (4 %) did not fit the expectations. These markers and others that can be obtained from the haplotype identified can be readily used for marker-assisted selection in peach breeding.     

Muscle Fiber Viability,a Novel Method for the Fast Detection of Ischemic Muscle Injury in Rats

Acute lower extremity ischemia is a limb- and life-threatening clinical problem. Rapid detection of the degree of injury is crucial, however at present there are no exact diagnostic tests available to achieve this purpose. Our goal was to examine a novel technique - which has the potential to accurately assess the degree of ischemic muscle injury within a short period of time - in a clinically relevant rodent model. Male Wistar rats were exposed to 4, 6, 8 and 9 hours of bilateral lower limb ischemia induced by the occlusion of the infrarenal aorta. Additional animals underwent 8 and 9 hours of ischemia followed by 2 hours of reperfusion to examine the effects of revascularization. Muscle samples were collected from the left anterior tibial muscle for viability assessment. The degree of muscle damage (muscle fiber viability) was assessed by morphometric evaluation of NADH-tetrazolium reductase reaction on frozen sections. Right hind limbs were perfusion-fixed with paraformaldehyde and glutaraldehyde for light and electron microscopic examinations. Muscle fiber viability decreased progressively over the time of ischemia, with significant differences found between the consecutive times. High correlation was detected between the length of ischemia and the values of muscle fiber viability. After reperfusion, viability showed significant reduction in the 8-hour-ischemia and 2-hour-reperfusion group compared to the 8-hour-ischemia-only group, and decreased further after 9 hours of ischemia and 2 hours of reperfusion. Light- and electron microscopic findings correlated strongly with the values of muscle fiber viability: lesser viability values represented higher degree of ultrastructural injury while similar viability results corresponded to similar morphological injury. Muscle fiber viability was capable of accurately determining the degree of muscle injury in our rat model. Our method might therefore be useful in clinical settings in the diagnostics of acute ischemic muscle injury.     

Changes in plant species composition and litter production in response to roads and trails in the laurel forest of Tenerife (Canary Islands)

Abstract

Road density has increased in the Canary Islands' forests during the last century, affecting an unknown amount of forested area. We studied road effects on vegetation in the relict laurel forest of Tenerife. We assessed edge effects on plant species richness, plant composition and litter production. Effects of anthropogenic corridors on vegetation differed between paved roads and unpaved trails. Opportunistic species (shade intolerant) dominated road edges, but composition differed among all sites. Multivariate analysis revealed convergence in species composition along the corridor-interior gradient. For trails, both species richness and litter production did not differ significantly between edge and interior. Road edge effects on vegetation were detectable only within the first 10 m towards the interior. This suggests that the main effects of roads and trails on species richness are limited to the immediate edge of the laurel forest. Litter fall along road edges was half that of the interior. However, no significant differences were detected due to the high variability of the data. A buffer of approximately 10 m would result in the reduction of the total area of the remaining undisturbed laurel forest. Based on these results, the building of new paved roads should not be considered. Low human population inflow into the Anaga Rural Park needs to be maintained on a sustainable basis. Forest managers should take these road/trail effects into account when planning new road openings in this ecosystem.