Trimethylamine-N-oxide (TMAO) is a product of dietary, gut microbiome, and tissues metabolism. Elevated blood TMAO levels are associated with heart attack, stroke and chronic kidney disease (CKD). The purpose of our study was to investigate the gut microbiota associated with trimethylamine (TMA) production, the precursor of TMAO, and the serum levels of TMAO and inflammatory biomarkers associated with type 2 diabetes mellitus (T2DM) and CKD. Twenty adults with T2DM and advanced CKD and 20 healthy adults participated in the study. Analyses included anthropometric and metabolic parameters, characterization of TMA producing gut microbiota, and concentrations of TMAO, lipopolysaccharides (LPS) endotoxin, zonulin (Zo) gut permeability marker, and serum inflammatory and endothelial dysfunction biomarkers. Diversity of the gut microbiota was identified by amplification of V3–V4 regions of the 16S ribosomal RNA genes and DNA sequencing. TMAO was quantified by Mass Spectrometry and serum biomarkers by ELISA. The significance of measurements justified by statistical analysis. The gut microbiome in T2DM-CKD patients exhibited a higher incidence of TMA-producing bacteria than control, p < 0.05. The serum levels of TMAO in T2DM-CKD patients were significantly higher than controls, p < 0.05. TMAO showed a positive correlation with Zo and LPS, inflammatory and endothelial dysfunction biomarkers. A positive correlation was observed between Zo and LPS in T2DM-CKD subjects. An increased abundance of TMA-producing bacteria in the gut microbiota of T2DM-CKD patients together with excessive TMAO and increased gut permeability might impact their risk for cardiovascular disease through elevation of chronic inflammation and endothelial dysfunction.
Lipopolysaccharide (LPS), a potent endotoxin present in the outer membrane of Gram-negative bacteria, causes chronic immune responses associated with inflammation. In the present study, the association between LPS and the dysbiosis of Gram-negative bacteria in the gut microbiome was determined in patients with type 2 diabetes mellitus (T2DM) and chronic kidney disease (T2DM-CKD; stages 4 and 5, not on dialysis) compared with healthy individuals. Microbiome diversity was analyzed in patients with T2DM-CKD and healthy controls by sequencing the hypervariable sub-regions of the 16S ribosomal RNA gene from stool samples. Serum samples were assayed by ELISA for LPS, C-reactive protein (CRP), tumor necrosis factor-α (TNFα), interleukin-6 (IL6) and endothelin-1. A total of four gut Gram-negative phyla (Bacteroidetes, Proteobacteria, Fusobacteria and Verrucomicrobia) were identified in the gut microbiome of the T2DM-CKD and control groups. Proteobacteria, Verrucomicrobia and Fusobacteria exhibited significantly increased relative abundance in patients with T2DM-CKD when compared with controls (P<0.05). The levels of serum LPS were significantly increased in patients with T2DM-CKD compared with controls (P<0.05). Elevated serum LPS was significantly correlated with increased levels of TNFα, IL6 and CRP. The dysbiosis of Gram-negative bacteria in the gut microbiome of patients with T2DM-CKD may contribute to the elevated serum levels of LPS and the consequential effects on the inflammatory biomarkers in these patients. The association between the dysbiosis of Gram-negative bacteria in the gut microbiome of patients with T2DM-CKD, increased LPS levels and the effects on inflammatory biomarkers may provide insight into potential diagnostic and therapeutic approaches in the treatment of T2DM-CKD.
Diagnostic parameters for visceral leishmaniasis (VL), a potentially fatal parasitic disease caused by Leishmania donovani, have been redefined in the last decade with the development of serological and molecular tests, though a definitive diagnosis still banks on the century-old parasitological methods in many areas. Recombinant antigens have improved performance of serodiagnostic methods. Serology-based tests, rk39 antigen dipstick, and direct agglutination test commonly employed in the field are highly sensitive methods, however, fail to distinguish past infections. Molecular approaches have become increasingly relevant due to remarkable sensitivity, specificity, and flexibility in choice of samples. Quantitative polymerase chain reaction is a highly sensitive and specific tool used in referral labs for detection/assessment of parasite load in VL patients and subsequently in monitoring treatment response to antileishmanial agents. The method displays potential to provide threshold for distinguishing asymptomatics in endemic areas. Currently, improvement in VL diagnostics is required for successful decentralized (point-of-care) testing in field conditions and to detect VL-HIV co-infection. Techniques such as loop-mediated isothermal amplification offer a reliable molecular diagnostic method for field application. The diagnosis based on bioanalytics/biosensors promise frontiers for point-of-care VL detection after adequate standardization. This review summarizes the recent developments in VL diagnostics, drawing attention towards the need for standardization of the diagnostics across the affected regions.
Abstract. An otherwise healthy 20-year-old woman in Goa, India, received antibiotics after a diagnosis of upper respiratory tract infection. One week later, vivax malaria was diagnosed at a health center, but the patient developed respiratory distress and lost consciousness. She arrived at emergency department in shock, breathless, and comatose. She died within minutes. Two independent laboratories later confirmed Plasmodium vivax by microscopy (140,000/μL) and by nested and real-time polymerase chain reaction methods. Post-mortem examination showed congestion of alveolar capillaries by heavy monocytic infiltrates, along with diffuse damage to alveolar membranes consistent with acute respiratory distress syndrome. Parasites seen in lung tissue were roughly proportionate to both peripheral hyperparasitemia and those seen in other organs without lesions. In this patient, vivax malaria caused a rapidly fatal respiratory distress.
Indian Leishmania donovani isolates (n ؍ 19) from regional zones representing various levels of antimony resistance displayed significantly (P < 0.01) correlated results with respect to in vitro susceptibility to the antileishmanial drugs sodium antimony gluconate, amphotericin B, and Miltefosine, raising the possibility of cross-resistance mechanisms operating in the field isolates. The results of gene expression analysis of LdMT and LdRos3 were suggestive of alternate mechanisms of Miltefosine susceptibility in the isolates.A high (Ͼ60%) proportion of non-antimony-responsive cases of Kala azar in India and the anthroponotic mode of transmission of the parasite causing the disease increase the chances of the generation and spreading of drug-resistant parasites (15, 17). The second-line antileishmanials amphotericin B (AmB) and Miltefosine (MIL) are highly effective for treatment of antimony-resistant patients but are of limited utility because of adverse reactions and high cost. A recent report of unresponsiveness to Ambisome in Sudanese patients of VL is worrisome and indicates the emergence of AmB-resistant parasites (9). Preliminary data from a phase IV trial with MIL suggested a doubling of the relapse rate, indicating lower drug efficacy than in phase II and III trials and providing a warning about the emergence of resistance (3,18,19).Earlier studies using isolates from responsive and nonresponsive patients indicated that resistance to antimonials is an intrinsic property of the parasite (4,8,15,16). Antimony resistance varies among zones representing differing levels of endemicity, emphasizing the acquired nature of resistance in the region (15). Sodium antimony gluconate (SAG)-resistant isolates exhibited cross-resistance to AmB and MIL, with HSP83 and a calpain-related protein being implicated in resistance by modulating drug-induced programmed cell death (21). Since the use of MIL for VL treatment has been introduced only recently, resistance has not yet been reported in the field; however, a wide range of 50% effective doses (ED 50 ) of MIL has been observed for parasite isolates from Nepal and Peru (23). The results of earlier studies revealed a role in MIL uptake and susceptibility for the LdMT-LdRos3-dependent flippase machinery at the plasma membrane (10-12). The present study was aimed at (i) evaluating the in vitro natural susceptibility of field isolates of Leishmania donovani to SAG, AmB, and MIL and (ii) correlating MIL susceptibility with the mRNA expression of LdMT and LdRos3 to explore their role in MIL resistance and potential as markers of MIL resistance in field isolates.The present study considered 19 L. donovani isolates from VL patients representing regional zones with various degrees of disease endemicity. In vitro susceptibility of parasites from SAG-treated patients (responsive and nonresponsive) and AmB-treated patients (all responded to treatment, and no clinical resistance was observed) was studied. Informed consent based on the guidelines of the Ethical Committee, Safdarjun...
BackgroundMiltefosine (MIL) is an oral antileishmanial drug used for treatment of visceral leishmaniasis (VL) in the Indian subcontinent. Recent reports indicate a significant decline in its efficacy with a high rate of relapse in VL as well as post kala-azar dermal leishmaniasis (PKDL). We investigated the parasitic factors apparently involved in miltefosine unresponsiveness in clinical isolates of Leishmania donovani.MethodologyL. donovani isolated from patients of VL and PKDL at pretreatment stage (LdPreTx, n = 9), patients that relapsed after MIL treatment (LdRelapse, n = 7) and parasites made experimentally resistant to MIL (LdM30) were included in this study. MIL uptake was estimated using liquid chromatography coupled mass spectrometry. Reactive oxygen species and intracellular thiol content were measured fluorometrically. Q-PCR was used to assess the differential expression of genes associated with MIL resistance.ResultsLdRelapse parasites exhibited higher IC50 both at promastigote level (7.92 ± 1.30 μM) and at intracellular amastigote level (11.35 ± 6.48 μM) when compared with LdPreTx parasites (3.27 ± 1.52 μM) and (3.85 ± 3.11 μM), respectively. The percent infectivity (72 hrs post infection) of LdRelapse parasites was significantly higher (80.71 ± 5.67%, P<0.001) in comparison to LdPreTx (60.44 ± 2.80%). MIL accumulation was significantly lower in LdRelapse parasites (1.7 fold, P<0.001) and in LdM30 parasites (2.4 fold, P<0.001) when compared with LdPreTx parasites. MIL induced ROS levels were significantly lower (p<0.05) in macrophages infected with LdRelapse while intracellular thiol content were significantly higher in LdRelapse compared to LdPreTx, indicating a better tolerance for oxidative stress in LdRelapse isolates. Genes associated with oxidative stress, metabolic processes and transporters showed modulated expression in LdRelapse and LdM30 parasites in comparison with LdPreTx parasites.ConclusionThe present study highlights the parasitic factors and pathways responsible for miltefosine unresponsiveness in VL and PKDL.
BackgroundLeishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. A robust, rapid and cost effective diagnostic test is warranted for on-time diagnosis and field application.MethodsWe have developed a loop mediated isothermal amplification (LAMP) assay with primers (n = 6) based on Leishmania donovani kDNA for detection of Leishmania infection, using a closed tube to prevent cross-contamination. The assay was used to detect Leishmania infection in biological samples obtained from patients of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL).ResultsThe assay was positive for L. donovani, L. tropica and L. major parasites, with the highest sensitivity towards L. donovani (1 fg DNA). The high sensitivity of the assay for detection of L. donovani was reflected in its ability to detect parasite DNA within 30 min of amplification time with a threshold detection limit of ≥25 copies per reaction. The assay detected parasite in 64 of 66 VL blood samples (sensitivity, 96.9%; 95% CI: 89.6-99.2%), 15 of 15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI:79.6-100%), 65 of 67 PKDL tissue biopsy samples (sensitivity, 97%; 95% CI:89.7-99.2%). The assay was evaluated in a few cases of CL wherein it was found positive in 8 of 10 tissue biopsies (sensitivity, 80%; 95% CI: 49-94.3%). The assay was negative in all control blood (n = 76) and tissue biopsy (n = 24) samples (specificity, 100%; 95% CI: 96.3-100%). Further, the assay was evaluated for its utility in assessment of cure in treated VL and PKDL patients. The assay detected parasite DNA in 2 of 20VL blood samples and 2 of 21 PKDL tissue samples. Out of 4 cases that were positive for parasite DNA at post treatment stage, 2 patients (1VL and 1 PKDL) returned with relapse.ConclusionsThe study demonstrated a Leishmania genus specific closed tube LAMP assay for reliable and rapid molecular diagnosis of VL and PKDL with potential for application in assessment of cure.
BackgroundPatients with Post kala-azar dermal leishmaniasis (PKDL) are considered a reservoir of Leishmania donovani. It is imperative to identify and treat them early for control of visceral leishmaniasis (VL), a current priority in the Indian subcontinent. We explored trends in clinico-epidemiological features of PKDL cases over last two decades, for improving management of the disease.MethodsClinically suspected cases were diagnosed with rK39 strip test followed by parasitological confirmation by microscopy and/or PCR/qPCR in skin tissue/slit aspirates. Patients were treated with antimonials till 2008 and subsequently with miltefosine.ResultsThe study indicated higher incidence of PKDL cases in areas of high endemicity for VL, with 20 % cases reporting no history of VL. Approximately 26 % cases of PKDL were initially misdiagnosed at primary health centers. Duration between onset of PKDL and diagnosis was above 12 months in 80 % cases. Diagnostic sensitivity was 32-36 % with microscopy and 96–100 % with PCR/qPCR. Compliance to treatment was over 85 % with miltefosine while 15 % with antimonials. Relapse rate with miltefosine was up to 13.2 %.ConclusionsPKDL patients tend to delay reporting and are often misdiagnosed. Confirmatory diagnosis using minimally invasive skin slit aspirate samples would help overcome such issues. There was a paradigm shift in compliance with miltefosine; however, increasing relapse rate indicated the need for newer therapies with oral formulations.
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