Real-Time Fluorimetry Loop-Mediated Isothermal Amplification for Diagnosis of Leishmaniasis and as a Tool for Assessment of Cure for Post–Kala-Azar Dermal Leishmaniasis

Despite the dwindling number of visceral leishmaniasis (VL) cases in India, there is an urgent need for early and unequivocal diagnostics for controlling and preventing the reemergence of VL. Post–kala-azar dermal leishmaniasis (PKDL), a dermal sequela of VL, serves as a reservoir of the parasite. Diagnosis of PKDL, especially the macular variant, is challenging and poses impediment toward attainment of VL elimination. In this study, a real-time fluorimetry loop-mediated isothermal amplification (RealAmp) assay has been established for the detection of different clinical manifestations of leishmaniasis. The study included 150 leishmaniasis patients (25 VL, 25 cutaneous leishmaniasis [CL], and 100-PKDL) along with 120 controls. The assay demonstrated sensitivity of 100% (95% CI: 86.68–100) for diagnosis of VL and PKDL (95% CI: 79.61–100) and 96% (95% CI: 86.68–100) for CL with 100% specificity. Moreover, considering the cardinal role of PKDL, diagnosis using minimally invasive slit aspirate was explored, which demonstrated remarkable sensitivity of 96% (95% CI: 87.64–98.47). As a test of cure for PKDL, RealAmp successfully detected parasite in two of posttreatment cases who later reported relapse on follow-up. Also, direct sample lysis using slit aspirate was attempted in a small group that yielded sensitivity of 89% (95% CI: 67.20–96.90). RealAmp depicted excellent diagnostic accuracy in the diagnosis of leishmaniasis in concordance with the established SYBR Green I–based visual loop-mediated isothermal amplification (LAMP) and the reference comparator real-time PCR. The study endorsed the employment of LAMP either as visual-LAMP or RealAmp for an accurate and expeditious diagnosis of PKDL and as a tool for assessment of cure.

Synthesis of 5-Phenyl-(5,6-Diphenyl-)-2,3-Dihydro-1,2,4-Triazine-3-Thions and Investigation of Their Reactions with 1,2-Dibromoethane

5-Phenyl-(5,6-diphenyl-)-1,2,4-triazine-3(2H)-thions (1,2) were synthesized by condensation of phenylglyoxal monohydrate with thiosemicarbazide and benzyl with thiosemicarbazide hydrochloride, respectively. Phenylglyoxal monohydrate was obtained by oxidation of acetophenone with selenous acid by the Riley reaction, while benzyl (1,2-diphenylethane-1,2-dione) waw obtained by oxidation of (2-hydroxy-1,2-diphenylethanone) benzoin with nitric acid by the known procedure. The compounds 1 and 2 were studied in reactions with 1,2-dibromoethane in various ratios. The interaction of triazinethione 1 with 1,2-dibromoethane in molar ratios of 1:1 and 1:2 led to the formation of a previously unknown 3-[(2-bromoethyl)sulfanyl]-5-phenyl-1,2,4-triazine (3а). Using a double excess of 1,2-dibromoethane increased the yield of compound 3а by 11 %. The interaction of triazinethione 2 with 1,2-dibromoethane in molar ratios 1:1 and 2:1 led to the formation of 3-[(2-bromoethyl)sulfanyl]-5,6-diphenyl-1,2,4-triazine (3b). In the 1H NMR spectra of compounds 3a,b there are signals of aromatic ring protons in the region of 7.23-8.81 ppm, a triplet of protons of the SCH2 group at 3.22 and 4.06 ppm, and a triplet of protons of the CH2Br group at 4.39 and 5.28 ppm. It is of interest that1,2-bis-(5-phenyl-[1,2,4]triazinyl-3-sulfanyl)-ethane is formed in the case of the reaction of compound 1 with 1,2-dibromoethane in the 2:1 ratio of reagents. In the 1H NMR spectra of the latter, in contrast to the spectrum of compound 3a, there is a signal of the S-CH2 group protons, which are equivalent in this structure and form a singlet at 3.79 ppm. Compound 3a was also studied by chromatography-mass spectrometry (direct sample insertion). It should be noted that under the conditions of mass spectrum imaging, the bromine atom is split off and the mass spectrum shows a peak with m/z 216 with an intensity of 38% and no peak of the molecular ion. The peak with the maximum intensity (m/z 116, 100 %) seems to correspond to 4-thia-6,7-diaza-1-azoniumbicyclo[3.2.0]hept-1(5)-ene. Its high intensity is due to the fact that the structure of the 1,2-dihydrotriazetium cation is aromatic.

Direct sample introduction GC-MS/MS for quantification of organic chemicals in mammalian tissues and blood extracted with polymers without clean-up

Solvent extracts of mammalian tissues and blood contain a large amount of co-extracted matrix components, in particular lipids, which can adversely affect instrumental analysis. Clean-up typically degrades non-persistent chemicals. Alternatively, passive sampling with the polymer polydimethylsiloxane (PDMS) has been used for a comprehensive extraction from tissue without altering the mixture composition. Despite a smaller fraction of matrix being co-extracted by PDMS than by solvent extraction, direct analysis of PDMS extracts was only possible with direct sample introduction (DSI) GC-MS/MS, which prevented co-extracted matrix components entering the system. Limits of quantitation (LOQ) ranged from 4 to 20 pg μL−1 ethyl acetate (PDMS extract) for pesticides and persistent organic pollutants (POPs). The group of organophosphorus flame retardants showed higher LOQs up to 107 pg μL−1 due to sorption to active sites at the injection system. Intraday precision ranged between 1 and 10%, while the range of interday precision was between 1 and 18% depending on the analyte. The method was developed using pork liver, brain, and fat as well as blood and was then applied to analyze human post-mortem tissues where polychlorinated biphenyls (PCBs) as well as dichlorodiphenyltrichloroethane (DDT) and DDT metabolites were detected.

Integrated microsystems for the in situ genetic detection of dengue virus in whole blood using direct sample preparation and isothermal amplification

An in situ detection system compatible with LAMP that can detect the dengue virus and discriminate between its serotypes in the whole blood.

Molecular imaging of bacterial infections: Overcoming the barriers to clinical translation

Clinical diagnostic tools requiring direct sample testing cannot be applied to infections deep within the body, and clinically available imaging tools lack specificity. New approaches are needed for early diagnosis and monitoring of bacterial infections and rapid detection of drug-resistant organisms. Molecular imaging allows for longitudinal, noninvasive assessments and can provide key information about infectious processes deep within the body.