Differential Diagnosis of Malaria on Truelab Uno?, a Portable,Real-Time,MicroPCR Device for Point-Of-Care Applications

Background

Sensitive and specific detection of malarial parasites is crucial in controlling the significant malaria burden in the developing world. Also important is being able to identify life threatening Plasmodium falciparum malaria quickly and accurately to reduce malaria related mortality. Existing methods such as microscopy and rapid diagnostic tests (RDTs) have major shortcomings. Here, we describe a new real-time PCR-based diagnostic test device at point-of-care service for resource-limited settings.

Methods

Truenat^® Malaria, a chip-based microPCR test, was developed by bigtec Labs, Bangalore, India, for differential identification of Plasmodium falciparum and Plasmodium vivax parasites. The Truenat Malaria tests runs on bigtec’s Truelab Uno^® microPCR device, a handheld, battery operated, and easy-to-use real-time microPCR device. The performance of Truenat^® Malaria was evaluated versus the WHO nested PCR protocol. The Truenat^® Malaria was further evaluated in a triple-blinded study design using a sample panel of 281 specimens created from the clinical samples characterized by expert microscopy and a rapid diagnostic test kit by the National Institute of Malaria Research (NIMR). A comparative evaluation was done on the Truelab Uno^® and a commercial real-time PCR system.

Results

The limit of detection of the Truenat Malaria assay was found to be <5 parasites/μl for both P. falciparum and P. vivax. The Truenat^® Malaria test was found to have sensitivity and specificity of 100% each, compared to the WHO nested PCR protocol based on the evaluation of 100 samples. The sensitivity using expert microscopy as the reference standard was determined to be around 99.3% (95% CI: 95.5–99.9) at the species level. Mixed infections were identified more accurately by Truenat Malaria (32 samples identified as mixed) versus expert microscopy and RDTs which detected 4 and 5 mixed samples, respectively.

Conclusion

The Truenat^® Malaria microPCR test is a valuable diagnostic tool and implementation should be considered not only for malaria diagnosis but also for active surveillance and epidemiological intervention.     

Genomic organization and chromosomal location of the human gene encoding the B-lymphocyte activation antigen B7

The human B lymphocyte activation antigen B7 provides regulatory signals for T lymphocytes as a consequence of binding to its ligands CD28 and CTLA-4. The cDNA for B7 has previously been isolated and predicted to encode a type I membrane protein. The predicted polypeptide has a secretory signal peptide followed by two contiguous Ig-like domains, a hydrophobic transmembrane region and a short cytoplasmic tail. Here we report the exon-intron genomic organization of human B7 and the chromosomal location. The gene has six exons that span approximately 32 kilobases of DNA. Exon 1 is not translated and the second exon contains the initiation ATG codon and encodes a predicted signal peptide. This gene structure is characteristic for several eukaryotic genes with tissue-specific expression. The third and fourth exons correspond to two Ig-like domains whereas the fifth and sixth exons encode respectively the trans-membrane portion and the cytoplasmic tail. This close relationship between exons and functional domains is a characteristic feature of genes of the Ig superfamily. Cell surface expression of the B7 gene product has previously been mapped to human chromosome 12 by antibody reactivity with the B7-specific monoclonal antibody BB-1. We here demonstrate that theB7 gene is located to theq21-qter region of chromosome 3 by DNA blot analysis of human × rodent somatic cell hybrids.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M83071-M83075, M83077. Address correspondence and offprint requests to: B. Dupont, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue (Room S709), New York, NY 10021, USA.     

Purification of nucleosidyl peptides by chromatography on dihydroxyboryl-substituted polyacrylamide and cellulose

A new column chromatographic method is presented for the purification of peptides which are covalently bound to nucleoside analogs (nucleosidyl peptides). The procedure involves complex formation between the cis-diol moiety of the nucleosidyl peptide and the dihydroxyborylphenyl group which is linked either to polyacrylamide or to cellulose as a support; thus, the nucleosidyl peptides can be reversibly bound to the column while all other peptides are eluted in the void volume. This approach is exemplified by the purification of two peptides of rabbit muscle pyruvate kinase labeled with 5′-p-fluorosulfonylbenzoyl adenosine and one peptide of bovine liver glutamate dehydrogenase modified with 5′-p-fluorosulfonylbenzoyl guanosine. The method may be generally applicable to the purification of peptides resulting from the affinity labeling of nucleotide sites in proteins.     

Combined effect of alcohol and cigarette smoke on lipid peroxidation and antioxidant status in rats

The effect of long-term administration of alcohol and cigarette smoke independently and both in combination on lipid peroxidation and antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) was studied in liver, kidney, heart and lungs of albino rats. The levels of peroxidation products viz., malondialdehyde, hydroperoxides and conjugated dienes were increased in all the tissues of alcohol administered and smoke-exposed rats. Activities of SOD and CAT were decreased in alcohol-treated and alcohol and smoke combination groups, but increased in smoke-exposed group. Activities of GPx and GST have shown an increase, while concentration of reduced glutathione was found decreased in all the three groups.     

Allosteric site-mediated active site inhibition of PBP2a using Quercetin 3-O-rutinoside and its combination

Recent crystallographic study revealed the involvement of allosteric site in active site inhibition of penicillin binding protein (PBP2a), where one molecule of Ceftaroline (Cef) binds to the allosteric site of PBP2a and paved way for the other molecule (Cef) to bind at the active site. Though Cef has the potency to inhibit the PBP2a, its adverse side effects are of major concern. Previous studies have reported the antibacterial property of Quercetin derivatives, a group of natural compounds. Hence, the present study aims to evaluate the effect of Quercetin 3-o-rutinoside (Rut) in allosteric site-mediated active site inhibition of PBP2a. The molecular docking studies between allosteric site and ligands (Rut, Que, and Cef) revealed a better binding efficiency (G-score) of Rut (?7.790318) and Cef (?6.194946) with respect to Que (?5.079284). Molecular dynamic (MD) simulation studies showed significant changes at the active site in the presence of ligands (Rut and Cef) at allosteric site. Four different combinations of Rut and Cef were docked and their G-scores ranged between ?6.320 and ?8.623. MD studies revealed the stability of the key residue (Ser403) with Rut being at both sites, compared to other complexes. Morphological analysis through electron microscopy confirmed that combination of Rut and Cefixime was able to disturb the bacterial cell membrane in a similar fashion to that of Rut and Cefixime alone. The results of this study indicate that the affinity of Rut at both sites were equally good, with further validations Rut could be considered as an alternative for inhibiting MRSA growth.     

Population fluctuations of the diamondback moth,Plutella xylostella (L.) on cabbages inBacillus thuringiensis sprayed and non sprayed plots and factors affecting within-generation survival of immatures

Summary The population fluctuations and within-generation survival of immatures stages of the diamondback moth,Plutella xylostella on summer cabbages, were examined in unsprayed plots in 1984 and 1985 and in plots sprayed with two formulations ofBacillus thuringiensis in 1985. There was two distincy generations per crop and no noticeable difference in population fluctuations were observed in unsprayed plots between years. There was distinct difference between unsprayed and sprayed plots in 1985, from the larval period onwards. However, the survivorship patterns in all plots in both years were a Type 2 based onDeevey's (1947) classification. Life table studies showed that there was essentially no difference in the mortality agents acting on each of the stages except for numerical differences in the within-generation mortality rates. The major mortality rates during the egg stage were the parasitoid,Trichogramma spp. and unknown factors including rainfall; in the larval 1 stage was unknown mortality; in the larval 2 stage was the parasitoid,Apanteles plutellae and during the pupal stage was unknown mortality and parasitoid,Diadromus subtilicornis. Adult mortality was determined for generation 2. It was relatively higher in the unsprayed plots compared to theB. thuringiensis sprayed plots. The contribution of abiotic factors such as rainfall and temperature, and biotic factors such as parasitoids and predators in determining within-generation population levels and the fluctuation of populations on cabbage were discussed.     

An Efficient Protection-Free One-Pot Chemical Synthesis of Modified Nucleoside-5′-Triphosphates

A simple, reliable, and an efficient “one-pot, three step” chemical method for the synthesis of modified nucleoside triphosphates such as 5-methylcytidine-5′-triphosphate (5-MeCTP), pseudouridine-5′-triphosphate (pseudoUTP) and N¹-methylpseudouridine-5′-triphosphate (N¹-methylpseudoUTP) starting from the corresponding nucleoside is described. The overall reaction involves the monophosphorylation of nucleoside, followed by the reaction with pyrophosphate and subsequent hydrolysis of the cyclic intermediate to furnish the corresponding NTP in moderate yields with high purity (>99.5%).