Liposomal amphotericin B is more effective in polymorphic lesions of post kala-azar dermal leishmaniasis

Background: Post kala-azar dermal leishmaniasis (PKDL) is thought to be the reservoir of infection for visceral leishmaniasis in South Asia. The development of strategies for the diagnosis and treatment of PKDL are important for the implementation of the visceral leishmaniasis elimination program. Aims: Liposomal amphotericin B (L-AMB) has been an overwhelming success in the treatment of visceral leishmaniasis. However, the empirical three-week regimen of L-AMB proposed for PKDL was shown to be inadequate, especially in the macular variant. This study aimed to delineate response of the different variants of PKDL to L-AMB. Methods: Skin biopsies were collected from PKDL cases at disease presentation and upon completion of treatment with L-AMB. Parasite DNA was detected by Internal Transcribed Spacer-1 PCR (ITS-1 PCR) and quantified by amplification of parasite kDNA. CD68 + macrophages were estimated in tissue sections by immunohistochemistry. Results: Treatment with L-AMB decreased the parasite load by 97% in polymorphic cases but only by 45% in macular cases. The median parasite load (89965 vs 5445 parasites/μg of genomic DNA) as well as infiltration by CD68+ cells before treatment was much greater in the polymorphic cases. Limitations: Although monitoring of the parasite load for 12 months post-treatment would have been ideal, this was not possible owing to logistical issues as well as the invasive nature of biopsy collection procedure. Conclusion: A dramatic decrease in the parasite burden was noted in patients with polymorphic lesions. Although patients with macular disease also had a decrease in parasite burden, this was not as marked as in the polymorphic cases. There was also a significantly greater infiltration of CD68 + macrophages in polymorphic PKDL before therapy

A Study on Burden of Carbapenem-Resistant Pseudomonas aeruginosa and Acinetobacter baumannii Infections in a Tertiary Care Hospital of Eastern India

Introduction: Healthcare associated infections(HAI) bymulti-drug resistant organisms(MDRO) are major cause ofmortality and morbidity having significant impact on qualityof life and economic burden. HAI by carbapenem-resistantPseudomonas aeruginosa (CRPsA) and Acinetobacterbaumannii (CRAB) are emerging threat for their highantibiotic resistance and spread via mobile genetic elements.Objectives of this study were to detect prevalence of CRPsAand CRAB infections in a tertiary care hospital of EasternIndia and to determine their antimicrobial resistance profile.Material and methods: This observational study was done inDepartment of Microbiology from January 2018-June 2019.From HAI patients, different clinical samples were collected.Culture and identification by standard conventional methodsand antimicrobial susceptibility tests by modified KirbyBauer disc-diffusion method following CLSI guidelines wereperformed. CRPsA and CRAB cases were identified whenisolates were resistant to ≥1 carbapenem, 10µg imipenemdisc(zone diameter ≤15mm for P. aeruginosa or ≤18mm forA. baumanii) or meropenem disc (≤15mm for P. aeruginosa or≤14mm for A. baumanii).Result: From 27,043 clinical samples, 1785(6.6%)Acinetobacter baumannii and 777(2.87%) Pseudomonasaeruginosa were isolated. CRAB and CRPsA prevalencewere 74.17% and 62.29% respectively. Carbapenemresistance were further categorised into imipenem-resistantmeropenem-resistant (IRMR) (A. baumanii-63.19%, P.aeruginosa-51.61%), imipenem-resistant-meropenemsensitive (IRMS) (A. baumanii-10.48%, P. aeruginosa-9.13%), meropenem-resistant-imipenem-sensitive (MRIS)(A. baumanii -0.51%, P. aeruginosa -1.54%) phenotypes.Fourth category was imipenem-sensitive-meropenemsensitive (ISMS) (A. baumanii -25.82%, P. aeruginosa-37.71%). Carbapenem-resistant groups showed significantlyhigh resistance for all antibiotics excepting colistin.Conclusion: Carbapenems are often used for treatingMDRO. But high carbapenem-resistance in HAI is alarming,warranting judicious use of antibiotics.

A Study on Demographic Profile and Risk Factor Association in Pityriasis Versicolor Cases Attending at a Tertiary Care Hospital in Kolkata

Pityriasis versicolor (PV), also known as tinea versicolor, is a chronic, mild, superficial and recurrent infection of the stratum corneum, caused by different Malassezia spp and seen predominantly in young age group and primarily in hot and humid climates. The aim of this study was to analyze epidemiological parameters and risk factors association in clinically diagnosed PV cases and also the mycological evaluation of those PV cases. Methods: A total of 116 patients attending the OPD of Dermatology were included and analysed for detailed history, clinical examination, epidemiological parameters, risk factors and investigations. Skin scrapings collected were processed by direct microscopy with 10% KOH and culture in modified Dixon agar (mDA). Isolates were identified by colony morphology, gram staining, biochemical characteristics & tween assimilation test. Results: Females were more affected (56.03%) than the males (43.97%) with F: M ratio 1.27:1. PV affected most commonly (36.21%) in 11-20 years of age group. Students (32.29%) were affected in maximum. Majority of affected patients (65.52%) used oily body creams, whereas 34.48% cases shared their body towels with others. 10.34% cases were associated with seborrheic dermatitis. Seasonal occurrence mostly seen in May - August. Patients with type III (Medium) complexion (56.03%) with normal skin texture (49.14%) were mostly affected. Maximum patients (74.14%) were associated with excessive sweating. 18.96% patients were associated with Type II DM. Most of the cases presented with macular, scaly hypopigmented, bilaterally asymmetrically distributed and having well defined margin. Neck was the most affected site (28.45%) followed by back (20.69%). Conclusion: M.furfur was the most common isolate (47.06%) followed by M. globosa (24.71%) and M. sympodialis (15.29%).

Effectiveness of malabaricone-A in P-glycoprotein over-expressing cancer cell lines

Background: A major impediment in treatment for cancers is resistance to chemotherapy and is primarily attributed to over-expression of efflux pumps. This study aimed to establish the cytotoxicity of malabaricone-A (MAL-A) in P-glycoprotein/multidrug resistance (P-gp/MDR) over-expressing hematopoietic cancer cell lines.Methods: Leukemia and multiple myeloma cell lines were indirectly evaluated for their P-gp/MDR status by examining Calcein-AM fluorescence and cell viability was assessed by the MTS-PMS assay.Results: The fluorescence of calcein was significantly decreased in three cell lines LP-1, RPMI-8226 and CEM-ADR 5000 and reversal with verapamil endorsed their P-gp/MDR activity. The mean IC50 of MAL-A in these MDR+ cell lines (5.40±1.41 to 12.33±0.78 µg/ml) was comparable with the MDR- leukemic (9.72±1.08 to 19.26±0.75 µg/ml) and multiple myeloma cell lines (9.65±0.39 to 18.05±0.17 ?g/ml).Conclusions: Irrespective of their P-gp activity, the cytotoxicity of MAL-A was comparable, making it worthy of future pharmacological consideration in multidrug resistance.

Anti-tumor effect of fruit rind of Myristica malabarica in an Ehrlich ascites carcinoma model

Background: Among the various modalities of anti-cancer treatment, cancer chemotherapy plays a very vital role. The alarming side effects being its main drawback leads to relentless research for newer agents. A new natural agent with promising anti-cancer properties from in-vitro studies leads to this study. Here we have evaluated the anti-tumor activity of a crude extract of fruit rind of Myristica malabarica in an Ehrlich ascites carcinoma model in mice.Methods: A murine model of cancer was established with i.p. inoculation of Ehrlich Ascites carcinoma (EAC) cells; animals were divided into five groups (including normal control) to observe the inhibitory effect of a crude extract of the fruit rind of Myristica malabarica/rampatri (0-100mg/kg b.w. i.p.) as compared with methotrexate (0.4mg/kg bw., i.p.). Blood and ascitic fluid were collected on the 10th day for analysis.Results: In the EAC model, there was an increase in tumor volume, tumor weight, and tumor packed cell volume, which was decreased by rampatri (50 and 100mg/kg bw) along with an increase in the mean survival time (MST). Rampatri caused minimal alterations in hematological parameters, renal functions remained unchanged but an increase in hepatic SGOT was demonstrated.Conclusions: The crude extract of rampatri (containing Malabaricones) exhibited significant anti-tumor activity with minimal effect on hematological and renal functions.

Phagocytes and the Leishmania Parasite: A Marriage of Convenience

The current drive by the Government of India to eliminate Leishmaniasis has pinpointed post-kala azar dermal leishmaniasis (PKDL) as the strongest contender for the disease reservoir. This emphasizes the necessity to consider the eradication of PKDL as top priority, and hinges on its early diagnosis and management. We undertook this challenge and have provided insights into Leishmania biology, by focusing our efforts in (i) delineating the immunopathogenesis of PKDL, a disease unique to South Asia (ii) developing diagnostic/prognostic tools for monitoring antileishmanial treatment in patients with visceral leishmaniasis and PKDL. In order to delineate the immunopathogenesis of PKDL, it was established that the parasite adopts multiple approaches to deviously manipulate host monocytes/macrophages, and thus facilitate parasite survival and disease progression. The parasite adopts a multipronged approach that includes attenuation of the oxidative burst within phagocytes, polarization of monocytes/ macrophages towards alternate activation, enhancement of CD8 T-cell exhaustion and a decreased presence of Langerhans cells. Identification of these immunological changes have allowed for development of biomarkers that have been exploited to develop diagnostic and prognostic markers for monitoring the disease progression, either in terms of antibody based markers, or quantification of the parasite load, the latter being the most definitive approach. Measurement of parasite load has proved to be an effective tool for monitoring the effectiveness of chemotherapy. Taken together, the identification of biomarkers and new chemotherapeutic modalities has helped in improved management and potential elimination of leishmaniasis.

A study of the free radical scavenging effects of Piper betle leaf extract in patients with vitiligo.

Background: Vitiligo is an idiopathic skin disease manifested by depigmented macules. It is characterised by melanocyte destruction, and redox imbalance is proposed to play a contributory role. Aim: The aim of this study was to analyze the effects of an ethanolic extract of Piper betle leaves on the generation of reactive oxygen species in erythrocytes sourced from vitiligo patients. Methods: The effect of Piper betle on the generation of reactive oxygen species in erythrocytes was measured by fl ow cytometry in patients with active and stable vitiligo versus healthy controls, using 5-(and-6)-chloromethyl-2’-7’-dichlorodihydrofl uorescein diacetate. Results: The generation of reactive oxygen species in erythrocytes was higher in patients with vitiligo (n = 23) compared to healthy controls (n = 18). The geometrical mean fl uorescence channel was 23.05 ± 2.11 in patients versus 17.77 ± 1.79 in controls, P = 0.039. The levels of reactive oxygen species were higher in patients with active vitiligo. Treatment of erythrocytes with Piper betle in concentrations of 0.5 and 1.0 μg/ml signifi cantly decreased the baseline levels of reactive oxygen species by 31.7% in healthy controls, and 47.6% and 44.3% in patients with active vitiligo, respectively. Piper betle effectively scavenged hydrogen peroxide, which was evident by a decrease in the geometrical mean fl uorescence channel by 52.4% and 62.9% in healthy controls, and 45.0% and 57.0% in patients with active vitiligo. Limitations: The study had a small sample size. Future studies should focus on evaluation of the antioxidant role of Piper betle at the lesional site. Conclusion: This pilot study indicates that patients with active vitiligo demonstrate enhanced generation of reactive oxygen species in erythrocytes, which was signifi cantly reduced following ex vivo treatment with Piper betle.

Evaluation of Analgesic Activity of Methanolic Extract of Bougainvillea spectabilis Leaves in Experimental Animal Models.

Background: Anti-inflammatory activity of leaves of Bougainvillea spectabilis (family Nyctaginaceae) has already been demonstrated in experimental animals. As pain is one of the important components of inflammation, we had set forward a study this find out possible analgesic activity of the same in animal models Objective: Evaluation of analgesic effects of, Bougainvillea spectabilis in mice models. Methods: 215 gm of fresh dried leaves of Bougainvillea spectabilis (BS) were collected from the local area during the flowering season and air dried. Following Methanol extraction, under reduced pressure solvent was removed on a rotary evaporator. The lyophilized extract was collected and the yield was 8 gm. That was used as an emulsion prepared in propylene glycol and orally administered (20 and 50 mg/kg). Central and peripheral analgesic activities of Bougainvillea spectabilis (BS) were evaluated by tail flick, tail immersion test and writhing test (acetic acid induced) respectively. Study Design: This is an experimental study designed on animal models. Results: Bougainvillea spectabilis (BS) had shown no analgesic action in central analgesic model at different hours as the reaction time was less than 10 seconds at all time interval. With regard to peripheral analgesic activity, maximal activity was observed at 50 mg/kg b.w. The mean writhes ± standard deviation were 42.7±0.9 and 40±0.5 respectively in BS (20 mg/kg) and BS (50 mg/kg) in comparison to standard drug aspirin (33.3±0.4), control mice being 55.3±0.4. Conclusion: Our data indicates that Bougainvillea spectabilis (50 mg/kg) has a significant peripheral analgesic activity. Without isolating the active principles it’s extremely difficult to pinpoint the mechanisms contributing to the observed analgesic activities of Bougainvillea spectabilis and extrapolate that in clinical practice.

Evaluation of anti-leishmanial activity of artemisinin combined with amphotericin B or miltefosine in Leishmania donovani promastigotes.

Background: The increasing incidence of drug resistance in Leishmaniasis necessitates evaluation of combination chemotherapy. Miltefosine and amphotericin B are established anti-leishmanial drugs, while artemisinin has shown significant leishmanicidal activity in experimental models. In this study, we have evaluated the additive/synergistic effect of artemisinin with amphotericin B or miltefosine. Methods: Leishmania parasites were isolated from the bone marrow aspirate of a patient with visceral leishmaniasis. Parasites were typed as Leishmania donovani by restriction fragment length polymorphism of internal transcribed spacer 1 region of Leishmania genome. Promastigotes were incubated in a fixed ratio combination of artemisinin (0-500 μM) and amphotericin B (0-100 nM) or miltefosine (0-100 μM) and cell viability was assessed. An isobologram was constructed to evaluate the additive/synergistic effect, wherein it was considered additive if the mean sum fractional inhibitory concentration (mean ΣFIC) at the IC50 level was <2, but ≥1 and synergism, if the mean ΣFIC was <1. Results: The isobologram showed an additive effect for three combinations of artemisinin-amphotericin B and artemisinin-miltefosine, the mean ΣFICs ranging from 1.02 to 1.44 and 1.08 to 1.33 along with a synergistic effect with one combination, the mean ΣFICs being 0.58 and 0.81 respectively. Conclusions: This study supports the combination use of artemisinin-amphotericin B and artemisinin-miltefosine, worthy of future pharmacological consideration.

Effects of different methods of decontamination for successful cultivation of Mycobacterium tuberculosis.

Background & objectives: There has been an extensive invasion of tuberculosis at the global level by multidrug resistant as well as extensively drug resistant organisms. Attempts to recover the pathogen in pure culture have frequently failed since the specimens are often highly contaminated and also due to use of insufficient or over-active decontamination procedures. Hence in the present study different methods of decontamination were tested to evaluate their independent efficacies for culture of Mycobacterium tuberculosis. Methods: A total of 359 samples (241 sputum, 59 urine, 50 endometrium biopsy, 9 pus samples) from clinically suspected cases of tuberculosis were subjected to four different methods of decontamination followed by inoculation in Lowenstein-Jensen medium (LJM), and bilayered medium (BLM) and Kirchner’s liquid medium (KLM) to determine the influence of differential decontamination processes. Sputum scanty and positive specimens were graded and each sample was subjected to decontamination by four different techniques. Results: Treatment of specimens with 4 per cent NaOH yielded minimum recovery of pure cultures, while use of 2 per cent NaOH produced higher number of contaminants compared to other methods of decontamination. Addition of N-acetyl L-cystein (NALC) coupled with 2 per cent NaOH to the samples for decontamination provided fairly reasonable recovery, but the highest number of M. tuberculosis cultures could be obtained when the specimens were treated with tri-sodium phosphate and benzalkonium (TSPB). Among the sputum positive cases recovery of growth of M. tuberculosis was higher with greater number of bacilli present in the specimens. Regarding the influence of culture media, BLM produced not only rapid growth, but reasonably higher rate of isolation of M. tuberculosis. Interpretation & conclusions: Although use of TSPB was found to be an efficient method of decontamination for successful isolation of M. tuberculosis from contaminated samples, both NALC+ 2 per cent NaOH and TSPB also showed significant recovery of M. tuberculosis cultures in BLM that can facilitate early diagnosis and initiation of treatment.

Antioxidant effect of ethanolic extract of Piper betle Linn. (Paan) on erythrocytes from patients with HbE-beta thalassemia.

HbE-beta thalassemia is caused by an interaction between HbE and defective b globin gene of thalassemia. Repeated blood transfusions cause an iron overload, triggering an enhanced generation of free radicals. In the present study, the anti-oxidant property of ethanolic extract of the leaves of Piper betle Linn. (PB) was evaluated in the erythrocytes from patients with HbE-beta thalassemia. In patients with HbE-beta thalassemia (n = 30) and age- and sex-matched healthy individuals (n = 30), the baseline level of reactive oxygen species (ROS) and free radical scavenging activity in the erythrocytes was measured by flow cytometry using dihydrodichlorofluorescein diacetate (H2DCFDA), in terms of the geometric mean fluorescence channel (GMFC). The baseline generation of ROS was significantly higher in the erythrocytes from patients with HbE-beta thalassemia, as compared to healthy volunteers, the GMFC being 67.20 ± 4.64 vs. 23.03 ± 1.88 (p<0.001), which was effectively decreased by PB. Similarly, H2O2 (0.5-1.0 mM) induced a higher increase in the GMFC in the erythrocytes from patients with HbE-beta thalassemia, as compared to controls which was effectively reduced by PB. Taken together, PB showed promising anti-oxidant activity against the erythrocytes from patients with HbE-beta thalassemia.

Functionality of drug efflux pumps in antimonial resistant Leishmania donovani field isolates.

The recent upsurge of antimony (Sb) resistance is a major impediment to successful chemotherapy of visceral leishmaniasis (VL). Mechanisms involved in antimony resistance have demonstrated an upregulation of drug efflux pumps; however, the biological role drug efflux pumps in clinical isolates remains to be substantiated. Thus, in this study, the functionality of drug efflux pumps was measured in promastigotes and axenic amastigotes isolated from VL patients, who were either Sb-sensitive (AG83, 2001 and MC9) or resistant (NS2, 41 and GE1) using rhodamine123 as a substrate for multidrug resistant (MDR) pumps and calcein as a substrate for multidrug resistance-associated proteins (MRP) respectively; their specificity was confirmed using established blockers. Sb-resistant (Sb-R) isolates accumulated higher amounts of R123, as compared to Sb-sensitive (Sb-S) isolates. Verapamil, a MDR inhibitor failed to alter R123 accumulation, suggesting absence of classical MDR activity. In Sb-R isolates, both promastigotes and axenic amastigotes accumulated significantly lower amounts of calcein than Sb-S isolates and probenecid, an established pan MRP blocker, marginally increased calcein accumulation. Depletion of ATP dramatically increased calcein accumulation primarily in Sb-R isolates, indicating existence of a MRP-like pump, which was more active in Sb-R isolates. In conclusion, our data suggested that overfunctioning of a MRP-like pump contributed towards generation of Sb-R phenotype in L. donovani field isolates.

Visceral leishmaniasis - current therapeutic modalities.

Major therapeutic obstacles in the treatment of visceral leishmaniasis (VL) include the alarming increase in antimonial unresponsiveness especially in Bihar, India and relapses in HIV-Leishmania co-infected patients. The therapeutic armamentarium for VL is currently plagued with several limitations as the available drugs are toxic, majority are effective only parenterally and need to be administered for extended periods. The first orally effective drug, miltefosine has been approved for treating VL. In antimony refractory zones, pentavalent antimony has been largely replaced by amphotericin B deoxycholate, but prolonged hospitalization, toxic effects, and requirement for monitoring greatly hamper its widespread application in endemic regions. Lipid formulations of amphotericin B, a remarkable advance in amphotericin B therapy, have greatly reduced toxicity enabling large doses to be delivered over a short period. Even a single dose treatment with liposomal amphotericin B cures > 90 per cent patients; however, the stumbling block is its prohibitive cost that precludes its widespread accessibility in endemic countries. Studies using paromomycin in VL are encouraging, and judging by the preliminary results of a recently concluded phase III trial, it could be an extremely useful and affordable antileishmanial drug. Other orally effective drugs include the azoles and allopurinol but these have met with limited success owing to either poor efficacy or unacceptable toxicity. Sitamaquine has undergone limited evaluation, and the data suggest effective antileishmanial activity; its role has to be delineated for which additional developmental studies are proposed. This review highlights the progress made in the treatment of VL, including the multiple mechanisms of action of antileishmanial drugs with a view to enable the researcher to undertake the challenge of providing affordable and effective chemotherapy.

Antibodies against 9-O-acetylated sialic acids in childhood acute lymphoblastic leukemia: a two-year study with 186 samples following protocol MCP 943.

Initial studies have revealed an enhanced surface expression of 9-O-acetylated sialoglycoconjugates (9-OAcSGs) on lymphoblasts concomitant with high titers of antibodies (anti-9-OAcSGs) in childhood acute lymphoblastic leukemia (ALL). This study was undertaken in 186 coded samples from 69 ALL patients to evaluate if antibodies against these sialoglycans could monitor response to the treatment. An ELISA was developed using bovine submaxillary mucin (BSM) containing high % of 9-O-acetylated sialic acids (9-OAcSA) as the capture antigen, to investigate serum levels of anti 9-OAcSGs in a single-center series of pediatric, clinically-diagnosed and immunophenotypically confirmed ALL patients, as compared to 130 healthy controls. At presentation, a 3.8-fold increase in anti-9-OAcSGs levels was detected in 63/69 ALL patients (mean +/- SEM was 102.8 +/- 6.3 microg/ml) as compared to normal controls (27.17 +/- 0.76 microg/ml), assay sensitivity being 91.3%. On an individual basis (n = 25) in patients who were longitudinally monitored for two years, a significant decline in their mean +/- SEM of OD405 was observed from 0.85 +/- 0.06 to 0.28 +/- 0.03. Additionally, a dot-blot was developed to evaluate the proportion of immune-complexed 9-OAcSGs in these patients employing achatinin-H, a 9-OAcSA-binding lectin. Our data indicate that these economically viable ELISA-based approaches allow for reliable, sensitive and rapid diagnosis of ALL. We contend that these disease-specific antibodies could be considered as potential markers both for the initial diagnosis of ALL and possibly for longitudinal monitoring of the disease.