Ultrastructural Characterization of Serially Passaged Amastigote Like Forms of Leishmania (Leishmania) Donovani

The present study was done to establish an in vitro axenic culture of amastigote like forms of Leishmania (Leishmania) donovani (Dd-8 strain), the causative agent of Indian kala-azar. Transformation of promastigotes to amastigote like forms was induced by temperature shift from 26±1℃ to 34±1℃ at pH 7.0 in NNN medium. These forms were dividing as evidenced by flow cytometry. Scanning and transmission electron microscopic studies revealed a remarkable ultrastructural similarity of these in vitro cultured amastigotes with intracellular amastigotes. These forms have been successfully maintained for a period of more than one year, during which they have remained infective. On subjecting these forms to temperature of 26±1℃, they reverted back to the promastigote forms. Thus a simple NNN medium, free from foetal calf serum has been developed to generate large amounts of amastigote like forms which can be used for further biochemical, immunological and chemotherapeutic studies

AmBisome Monotherapy and Combination AmBisome-Miltefosine Therapy for the Treatment of Visceral Leishmaniasis in Patients Coinfected With Human Immunodeficiency Virus in India: A Randomized Open-Label, Parallel-Arm, Phase 3 Trial.

BACKGROUND: Visceral leishmaniasis (VL) in patients with human immunodeficiency virus (HIV) presents an increasingly important patient cohort in areas where both infections are endemic. Evidence for treatment is sparce, with no high-quality studies from the Indian subcontinent. METHODS: This is a randomized, open-label, parallel-arm, phase 3 trial conducted within a single hospital in Patna, India. One hundred and fifty patients aged ≥18 years with serologically confirmed HIV and parasitologically confirmed VL were randomly allocated to 1 of 2 treatment arms, either a total 40 mg/kg intravenous liposomal amphotericin B (AmBisome; Gilead Pharmaceuticals) administered in 8 equal doses over 24 days or a total 30 mg/kg intravenous AmBisome administered in 6 equal doses given concomitantly with a total 1.4 g oral miltefosine administered through 2 daily doses of 50 mg over 14 days. The primary outcome was intention-to-treat relapse-free survival at day 210, defined as absence of signs and symptoms of VL or, if symptomatic, negative parasitological investigations. RESULTS: Among 243 patients assessed for eligibility, 150 were recruited between 2 January 2017 and 5 April 2018, with no loss to follow-up. Relapse-free survival at day 210 was 85% (64/75; 95% CI, 77-100%) in the monotherapy arm, and 96%, (72/75; 90-100%) in the combination arm. Nineteen percent (28/150) were infected with concurrent tuberculosis, divided equally between arms. Excluding those with concurrent tuberculosis, relapse-free survival at day 210 was 90% (55/61; 82-100%) in the monotherapy and 97% (59/61; 91-100%) in the combination therapy arm. Serious adverse events were uncommon and similar in each arm. CONCLUSIONS: Combination therapy appears to be safe, well tolerated, and effective, and halves treatment duration of current recommendations. CLINICAL TRIALS REGISTRATION: Clinical Trial Registry India (CTRI/2015/05/005807; the protocol is available online at https://osf.io/avz7r)

Simultaneous determination of guanosine and 8-hydroxy-guanosine by differential pulse voltammetry

1222-1226Differential pulse voltammetry has been used for the simultaneous determination of guanosine and 8-hydroxyguanosine in phosphate buffers. Well-separated differential pulse voltammetric peaks for guanosine and 8-hydroxyguanosine are observed at pyrolytic graphite electrode. The proposed method is simple, rapid and provides a wide determination range of 0.5 μM to 0.1 mM with good reproducibility. Interference of ascorbic acid and some guanine related compounds has also been studied and it is found that they do not interfere at very low concentration level

Molecular mechanisms of antimony resistance in Leishmania

Leishmaniasis causes significant morbidity and mortality worldwide. The disease is endemic in developing countries of tropical regions, and in recent years economic globalization and increased travel have extended its reach to people in developed countries. In the absence of effective vaccines and vector-control measures, the main line of defence against the disease is chemotherapy. Organic pentavalent antimonials [Sb(V)] have been the first-line drugs for the treatment of leishmaniasis for the last six decades, and clinical resistance to these drugs has emerged as a primary obstacle to successful treatment and control. A multiplicity of resistance mechanisms have been described in resistant Leishmania mutants developed in vitro by stepwise increases of the concentration of either antimony [Sb(III)] or the related metal arsenic [As(III)], the most prevalent mechanism being upregulated Sb(III) detoxification and sequestration. With the availability of resistant field isolates, it has now become possible to elucidate mechanisms of clinical resistance. The present review describes the mechanisms of antimony resistance in Leishmania and highlights the links between previous hypotheses and current developments in field studies. Unravelling the molecular mechanisms of clinical resistance could allow the prevention and circumvention of resistance, as well as rational drug design for the treatment of drug-resistant Leishmania

Use of Leishmania donovani Field Isolates Expressing the Luciferase Reporter Gene in In Vitro Drug Screening

Currently available primary screens for the selection of candidate antileishmanial compounds are not ideal. These techniques are time-consuming, laborious, and difficult to scale and require macrophages, which limit their use for high-throughput screening. We have developed Leishmania donovani field isolates that constitutively express the firefly luciferase reporter gene (luc) as a part of an episomal vector. An excellent correlation between parasite number and luciferase activity was observed. luc expression was stable, even in the absence of drug selection, for 4 weeks. The transfectants were infective to macrophages, and intracellular amastigotes exhibited luciferase activity. The suitability of these recombinant field isolates for in vitro screening of antileishmanial drugs was established. The luciferase-expressing sodium stibogluconate-resistant cell lines offer a model for the screening of compounds for resistance. The system is in routine use at the Central Drug Research Institute, Lucknow, India, for high-throughput screening of newly synthesized compounds

Electrochemical oxidation of 6-hydroxy-2,4,5- triaminopyrimidine at pyrolytic graphite electrode

1015-1023The electrochemical oxidation of 6-hydroxy-2,4,5-triaminopyrimidine (<b style="mso-bidi-font-weight: normal">I) at pyrolytic  graphite electrode has been studied in phosphate buffers of <i style="mso-bidi-font-style: normal">pH range 2.5-10.8. Under cyclic voltammetric conditions, the 2e, 2H+ oxidation of this compound is found to give alloxan, which has been characterized on the basis of IR, mass and NMR spectral data. The kinetics of the decay of the UV -absorbing intermediate generated during electrooxidation of compound (I) has been studied and first order rate constants for the disappearance of UV -absorbing intermediate have been calculated. A tentative mechanism in which electrode reaction is followed by chemical steps (EC) for the oxidation of <b style="mso-bidi-font-weight: normal">I has also been suggested.</span

Predictive value of early serum beta-human chorionic gonadotrophin for the successful outcome in women undergoing in vitro fertilization

Aims: Pregnancies achieved by in vitro fertilization (IVF) are at increased risk of adverse outcome. The main objective of this study was to evaluate the predictive value of β-human chorionic gonadotrophin (β-HCG) and age of the patient for the successful outcome in IVF. Materials and Methods: A retrospective study was done in 139 pregnancies after IVF at single IVF center from June 2007 to July 2012. The age of the patient and initial serum values of β-HCG on day 14 of embryo transfer were correlated with ongoing pregnancy (>12 weeks gestation). Results: The β-HCG level on day 14 of more than 347 mIU/ml has a sensitivity of 72.2% and specificity of 73.6% in prediction of pregnancy beyond 12 weeks period of gestation. Positive likelihood ratio (LR) is 2.74 and negative LR is 0.37, (receiver operating characteristic area = 0.79). Discussion: In IVF cycles, there is a lot of stress on the couples while the cycle is going on. There was a positive correlation between the higher values of early serum β-HCG levels and ongoing pregnancy. Hence, it can be used as an independent predictor of a successful outcome of IVF cycle. Conclusion: We concluded from our study that early serum β-HCG can be used as a predictor of a successful outcome in IVF