Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites

Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1–8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed.     

Gastric Juice Polymerase Chain Reaction: An Alternative to Histology in the Diagnosis of Helicobacter pylori Infection

Background. Infection from Helicobacter pylori plays a role in several gastroduodenal diseases. The recent availability of molecular techniques, particularly the polymerase chain reaction (PCR), allows us to detect small amounts of this bacterium. The aims of this study were to compare PCR and histological findings and to ascertain the clinical usefulness of H. pylori PCR identification in different biological samples.
Materials and Methods. We studied 94 consecutive patients. Saliva, gastric juice, and four antral and four body biopsies were obtained from each patients. H. pylori was evaluated histologically in two antral and two body biopsies (Giemsa or Warthin-Starry stain). After extraction, DNA was submitted for PCR amplification using the two primers HPU1 and HPU2, which amplified a 411-bp product from the urease gene A.
Results. Forty-nine patients were H. pylori -positive at histological workup. The sensitivity of PCR was 92% for gastric juice, 73% for antral biopsies, 61% for body biopsies, and 13% for saliva. Of the 45 H. pylori -negative patients at histological assessment, 7 (16%) had positive findings on PCR, mainly when gastric juice was examined.
Conclusions. These results indicate that PCR is as sensitive as histological assessment. We suggest that PCR H. pylori detection in gastric juice is a sensitive method for diagnosing this infection.     

The GURKE gene is required for normal organization of the apical region in the Arabidopsis embryo

Dicot plant embryos undergo a transition from radial to bilateral symmetry. In Arabidopsis, this change reflects patterning within the apical region, resulting in the formation of the cotyledon and shoot meristem primordia. Mutations in the GURKE gene give seedlings with highly reduced or no cotyledons. Both strong and weak gurke alleles confer this phenotypic variability although strong alleles often eliminate the entire apex and sometimes also part of the hypocotyl. The root and the root meristem as well as the radial pattern of concentric tissue layers are essentially normal. The mutant seedling phenotype can be traced back to the triangular/early-heart stage of embryogenesis when abnormal cell divisions occur within the apical region such that no or only rudimentary cotyledon primordia are established. The postembryonic development of gurke seedlings was examined in culture. In weak alleles, apical growth gave rise to abnormal leaves and stem-like structures and, eventually, abnormal flowers. In strong alleles, the apical region often failed to grow but occasionally produced fused leaf-like structures with no dorso-ventral polarity and a totally unorganized vascular system while no stems developed. The observations suggest that the GURKE gene is involved primarily in the organization of the apical region in the embryo and may also play a role during postembryonic development.     

Cytosolic and chloroplastic glutamine synthetase of sugarbeet (Beta vulgaris) respond differently to organ ontogeny and nitrogen source

Changes in the activity and subunit composition of cytosolic glutamine synthetase (GS 1; EC 6.3.1.2) and chloroplastic GS (GS 2) were studied in response to an internal (organ ontogeny) and external signal (N source: NO₃⁻ or NH₄⁺). Maximum GS 1 activity of all organs examined was measured in the fibre roots, irrespective of the N source. The response of GS 1 to the N source was, however, organ specific. In the fibre roots, NH₄⁺ nutrition resulted in a 2- to 7-fold (based on protein or freshweight, respectively) increase of GS 1 activity compared to NO₃⁻-grown plants. In contrast to the roots, GS 1 activity in the leaf blades was 2-fold lower with NH₄⁺ nutrition, whereas only minor changes occurred in the petioles. GS 2 activity was highest in the mature and senescing leaf blade; activity was 2-fold higher with NH₄⁺ than with NO₃⁻ nutrition. Not only activity, but also subunit composition of GS 1 changed during organ ontogeny as well as in response to the N source. In contrast to GS 1, only minor changes were evident in GS 2 subunit composition, despite significant changes in GS 2 activity. Up to 5 different GS 1 subunits of ≈41–43 kDa were separated; they were identical in all organs examined. GS 2 was composed of 4 different subunits of ≈48 kDa.     

Resource partitioning of the host fungus Coriolus versicolor by two ciid beetles: the role of odour compounds and host ageing

The ciid beetles Octotemnus glabriculus and Cis boleti exploit different developmental stages of fruit bodies of their preferred host fungus Coriolus versicolor . Larvae of the smaller beetle, O. glabriculus , mainly use young, expanding, fruit bodies; adults of O. glabriculus are predominantly found in young fruit bodies. By contrast, adults and larvae of the larger beetle, C. boleti , are prevalent in fully developed fruit bodies of C. versicolor . Because fruit bodies of most genets emerge during spring and early summer and mature by autumn, O. glabriculus and C. boleti breed in separated seasons. Adults and larvae of O. glabriculus are abundant in spring and early summer. By contrast, the number of adults and larvae of C. boleti increases gradually from late spring to summer and peaks in autumn. We conducted a field experiment that suggests that the phenological dynamics of C. versicolor fruit bodies drive the separation of breeding seasons between O. glabriculus and C. boleti . Additionally, laboratory experiments revealed that O. glabriculus and C. boleti have differential behavioural responses to odour compounds from young and mature fruit bodies of C. versicolor . We conclude that age-related changes in the chemical composition of fruit bodies may allow O. glabriculus and C. boleti to discriminate among C. versicolor , thus providing a mechanism for the partitioning of the resource.     

Still Searching for a Suitable Molecular Test to Detect Hidden Plasmodium Infection: A Proposal for Blood Donor Screening in Brazil

Background

Efforts have been made to establish sensitive diagnostic tools for malaria screening in blood banks in order to detect malaria asymptomatic carriers. Microscopy, the malaria reference test in Brazil, is time consuming and its sensitivity depends on microscopist experience. Although molecular tools are available, some aspects need to be considered for large-scale screening: accuracy and robustness for detecting low parasitemia, affordability for application to large number of samples and flexibility to perform on individual or pooled samples.

Methodology

In this retrospective study, we evaluated four molecular assays for detection of malaria parasites in a set of 56 samples previously evaluated by expert microscopy. In addition, we evaluated the effect of pooling samples on the sensitivity and specificity of the molecular assays. A well-characterized cultured sample with 1 parasite/μL was included in all the tests evaluated. DNA was extracted with QIAamp DNA Blood Mini Kit and eluted in 50 μL to concentrate the DNA. Pools were assembled with 10 samples each. Molecular protocols targeting 18S rRNA, included one qPCR genus specific (Lima-genus), one duplex qPCR genus/Pf (PET-genus, PET-Pf) and one duplex qPCR specie-specific (Rougemont: Roug-Pf/Pv and Roug-Pm/Po). Additionally a nested PCR protocol specie-specific was used (Snou-Pf, Snou-Pv, Snou-Pm and Snou-Po).

Results

The limit of detection was 3.5 p/μL and 0.35p/μl for the PET-genus and Lima-genus assays, respectively. Considering the positive (n = 13) and negative (n = 39) unpooled individual samples according to microscopy, the sensitivity of the two genus qPCR assays was 76.9% (Lima-genus) and 72.7% (PET-genus). The Lima-genus and PET-genus showed both sensitivity of 86.7% in the pooled samples. The genus protocols yielded similar results (Kappa value of 1.000) in both individual and pooled samples.

Conclusions

Efforts should be made to improve performance of molecular tests to enable the detection of low-density parasitemia if these tests are to be utilized for blood transfusion screening.     

Changes in the fatty acid profiles of red blood cell membrane phospholipids in human neonates during the first month of life

We have studied the changes in the fatty acid profiles of red blood cell membrane phospholipids in 47 infants who were exclusively fed human milk from birth to 1 month of life. Twenty blood samples were obtained from cord, 15 at 7 days and 12 at 30 days after birth. Membrane phospholipids were obtained from erythrocyte ghosts by thin-layer chromatography and fatty acid composition was determined by gas liquid chromatography. Phosphatidylcholine showed the most important changes during early life; stearic, w6 eicosatrienoic and arachidonic acids decreased whereas oleic and linoleic acids increased. In phosphatidylethanolamine, palmitic and stearic acid declined and oleic, linoleic and docosahexenoic acids increased with advancing age. Small changes were noted for individual fatty acids in phosphatidylserine. In sphingomyelin stearic acid increased from birth to 1 month and linoleic, arachidonic and nervonic acids decreased. Total polyunsaturated fatty acids of the w6 series greater than 18 carbon atoms increased with advancing age in phosphatidylethanolamine and decreased in choline and serine phosphoglycerides and in sphingomyelin. Long chain fatty acids derived from linoleic acid decreased in phosphatidylcholine but increased in ethanolamine and serine phosphoglycerides. The different behavior in the changes observed in fatty acid patterns for each erythrocyte membrane phospholipid may be a consequence of its different location in the cell membrane bilayer and specific exchange with plasma lipid fractions.     

Influence of the Ionic Environment on the Conformation of Aspartic Acid and Possible Relevance to Its Neurotransmitter Action

The conformational changes of aspartic acid, a possible neurotransmitter, induced by increasing ionic strength were investigated by means of NMR spectroscopy. The effects of Li, Na, K, and Cs chlorides in basic and neutral conditions were characterized. The effects of the presence of both Ca2+ and Na+ or K+ were also examined. The results always showed a relative stabilization of the two conformers with gauche carboxylates at the expense of the anticonformer. The significant quantitative difference found between Na+ and K+ in basic solutions, as well as between calcium-free and calcium-containing solutions, may bear some relevance for the role of aspartate as a CNS neurotransmitter.     

Hollow fiber-based liquid phase microextraction with factorial design optimization and gas chromatography–tandem mass spectrometry for determination of cannabinoids in human hair

A new method, based on hollow fiber liquid-phase microextraction (HF-LPME) and gas chromatography–tandem mass spectrometry (GC–MSMS), was developed for determination of Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in samples of human hair. Since hair is a solid matrix, the samples were subjected to alkaline digestion using NaOH. The aqueous solutions obtained were extracted using a 6 cm polypropylene fiber (600 μm i.d., 200 μm wall thickness, 0.2 μm pore size) for each extraction. A 2⁵⁻¹ fractional factorial design for screening, and a central composite design for optimization of significant variables, was applied during development of the extraction method. The variables evaluated were the type of extraction solvent, pH, stirring speed, extraction time, and acceptor phase volume. The optimized conditions for the proposed extraction procedure were 10 mg of hair sample; 20 μL of butyl acetate; aqueous (pH 14) donor phase containing 6.8% NaCl; 600 rpm stirring speed; 20 min extraction time. A linear response was obtained in the ranges 1–500 pg mg⁻¹ (CBD and CBN) and 20–500 pg mg⁻¹ (THC), with regression coefficients >0.99. Precision, determined as the relative standard deviation, was 3.3–8.9% (intra-day) and 4.4–13.7% (inter-day). Absolute recoveries varied in the ranges 4.4–4.8% (CBD), 7.6–8.9% (THC) and 7.7–8.2% (CBN). Limits of detection (LOD, S/N = 3) and quantification (LOQ, S/N = 10) were 0.5–15 pg mg⁻¹ and 1–20 pg mg⁻¹, respectively. The method was successfully used to determine CBD, THC and CBN in hair samples from patients in a drug dependency rehabilitation center. Concentrations varied in the ranges 1–18 pg mg⁻¹ (CBD), 20–232 pg mg⁻¹ (THC) and 9–107 pg mg⁻¹ (CBN), confirming the suitability of the method for monitoring studies.