Vaccine potential of HLA-A2 epitopes from Leishmania Cysteine Protease Type III (CPC).

Although the precise host-defence mechanisms are not completely understood, T-cell-mediated immune responses are believed to play a pivotal role in controlling parasite infection. In this study, the potential HLA*A2 restricted peptides were predicted and the ability of peptides to bind HLA-A*02 was confirmed by a MHC stabilization assay. Two of the peptides tested stabilized HLA-A*02: (a) LLATTVSGL (P1) and (b) LMTNGPLEV (P3). The potential of the peptides to generate protective immune response was evaluated in patients with treated visceral leishmaniasis as well as in healthy control subjects. Our data suggest that CD8+ T-cell proliferation against the selected peptide was significantly higher compared to unstimulated culture conditions. The stimulation of peripheral blood mononuclear cells with epitopes individually or as a cocktail upregulated IFN-γ production, which indicates its pivotal role in protective immune response. The IFN-γ production was mainly in a CD8+ T-cells-dependent manner, which suggested that these epitopes had an immunoprophylactic potential in a MHC class I-dependent manner. Moreover, no role of the CD3+ T cell was observed in the IL-10 production against the selected peptides, and no role was found in disease pathogenesis. Further studies on the role of these synthetic peptides may contribute significantly to developing a polytope vaccine idea towards leishmaniasis.

Development of SCAR markers and UP-PCR cross-hybridization method for specific detection of four major subgroups of Rhizoctonia from infected turfgrasses.

A rapid identification assay for Waitea circinata (anamorph: Rhizoctonia spp.) varieties zeae and circinata causing patch diseases on turfgrasses was developed based on the universally primed PCR (UP-PCR) products cross-blot hybridization. Tester isolates belonging to the two varieties of W. circinata were amplified with a single UP primer L21, which generated multiple DNA fragments for each variety. Probes were prepared with UP-PCR products of each tester isolate by labeling with digoxigenin. Fieldcollected W. circinata isolates and representative isolates of different R. solani anastomosis groups (AG) and AG subgroups were amplified with L21, immobilized on nylon membrane and cross hybridized with the two probes. Isolates within a W. circinata variety cross-hybridized strongly, while non-homologous isolates did not cross-hybridize or did so weakly. Closely related W. circinata varieties zeae and circinata were clearly distinguished with this assay. Sequence-characterized amplified region (SCAR) markers also were developed from UP-PCR products to identify isolates of Thanatephorus cucumeris (anamorph: R. solani) AG 1-IB and AG 2-2IIIB. These two AGs are commonly isolated from diseased, cool-season turfgrasses. The specific SCAR markers that were developed could differentiate isolates of AG 1-IB or AG 2-2IIIB groups. These SCAR markers did not amplify a product from genomic DNA of nontarget isolates of Rhizoctonia. The specificities and sensitivities of the SCAR primers were tested on total DNA extracted from several field-grown, cool-season turf species having severe brown-patch symptoms. First, the leaf samples from diseased turf species were tested for the anastomosis groups of the causal pathogen, and thereafter the total DNA was amplified with the specific primers. The specific primers were sensitive and unique enough to produce a band from total DNA of diseased turfgrasses infected with either AG 1-IB or AG 2-2IIIB.

Porocercospora seminalis gen. et comb. nov., the causal organism of buffalograss false smut.

False smut caused by Cercospora seminalis is an important disease of buffalograss (Buchloë dactyloides) affecting seed production. The pathogen prevents normal caryopsis development and causes considerable yield loss and reduced seed germination. The current taxonomic placement of the false-smut causal pathogen in the genus Cercospora is incorrect based on its morphological characteristics and DNA phylogeny. In the present study the phylogenetic position of C. seminalis is clarified based on DNA sequence analysis of three loci namely the internal transcribed spacer (ITS) region, partial nuclear ribosomal large subunit (LSU) and partial sequences of the RNA polymerase II second largest subunit (RPB2). A collection of C. seminalis isolates was made from buffalograss sites near Lincoln, Nebraska. DNA sequence data indicated that Cercospora seminalis is phylogenetically close to but distinct from species of Bipolaris and Curvularia (Pleosporaceae, Pleosporales). Cercospora seminalis morphologically had unique characteristics, namely densely aggregated and repeatedly branched conidiophores arising from a brown stroma, monotretic conidiogenous cells with inconspicuous loci, and scolecosporous conidia with distosepta, and thickened, darkened hila. Porocercospora is introduced as a new genus to accommodate the buffalograss false-smut pathogen.

Emergency unit assessment of seven tertiary hospitals in Nepal using the WHO tool: a cross-sectional study.

BACKGROUND: In 2021, the Nepal national emergency care system's assessment (ECSA) identified 39 activities and 11 facility-specific goals to improve care. To support implementation of the ECSA facility-based goals, this pilot study used the World Health Organization's (WHO) Hospital Emergency Unit Assessment Tool (HEAT) to evaluate key functions of emergency care at tertiary hospitals in Kathmandu, Nepal. METHODS: This cross-sectional study used the standardized HEAT assessment tool. Data on facility characteristics, human resources, clinical services, and signal functions were gathered via key informant interviews conducted by trained study personnel. Seven tertiary referral centers in the Kathmandu valley were selected for pilot evaluation including governmental, academic, and private hospitals. Descriptive statistics were generated, and comparative analyses were conducted. RESULTS: All facilities had continuous emergency care services but differed in the extent of availability of each item surveyed. Academic institutions had the highest rating with greater availability of consulting services and capacity to perform specific signal functions including breathing interventions and sepsis care. Private institutions had the highest infrastructure availability and diagnostic testing capacity. Across all facilities, common barriers included lack of training of key emergency procedures, written protocols, point-of-care testing, and ancillary patient services. CONCLUSION: This pilot assessment demonstrates that the current emergency care capacity at representative tertiary referral hospitals in Kathmandu, Nepal is variable with some consistent barriers which preclude meeting the ECSA goals. The results can be used to inform emergency care development within Nepal and demonstrate that the WHO HEAT assessment is feasible and may be instructive in systematically advancing emergency care delivery at the national level if implemented more broadly.

Post-kala-azar dermal leishmaniasis in a patient treated with injectable paromomycin for visceral leishmaniasis in India.

Post kala-azar dermal leishmaniasis (PKDL) is a skin manifestation that usually develops after treatment of visceral leishmaniasis (VL), a major public health problem in India. The diagnosis and management of PKDL is complex. This is the first case report from India in which PKDL occurred after paromomycin treatment for VL in an Indian patient.

Fusarium oxysporum as an Opportunistic Fungal Pathogen on Zinnia hybrida Plants Grown on board the International Space Station.

A plant production system called Veggie was launched to the International Space Station (ISS) in 2014. In late 2015, during the growth of Zinnia hybrida cv. 'Profusion' in the Veggie hardware, plants developed chlorosis, leaf curling, fungal growth that damaged leaves and stems, and eventually necrosis. The development of symptoms was correlated to reduced air flow leading to a significant buildup of water enveloping the leaves and stems in microgravity. Symptomatic tissues were returned to Earth on 18 May 2016 and were immediately processed to determine the primary causal agent of the disease. The presumptive pathogen was identified as Fusarium oxysporum by morphological features of microconidia and conidiophores on symptomatic tissues; that is, by epifluorescent microscopy (EFM), scanning electron microscopy (SEM), metabolic microarrays, and ITS sequencing. Both EFM and SEM imaging of infected tissues showed that germinating conidia were capable of stomatal penetration and thus acted as the primary method for infecting host tissues. A series of ground-based pathogenicity assays were conducted with healthy Z. hybrida plants that were exposed to reduced-airflow and high-water stress (i.e., encased in sealed bags) or were kept in an unstressed configuration. Koch's postulates were successfully completed with Z. hybrida plants in the lab, but symptoms only matched ISS-flown symptomatic tissues when the plants were stressed with high-water exposure. Unstressed plants grown under similar lab conditions failed to develop the symptoms observed with plants on board the ISS. The overall results of the pathogenicity tests imply that F. oxysporum acted as an opportunistic pathogen on severely high-water stressed plants. The source of the opportunistic pathogen is not known, but virulent strains of F. oxysporum were not recovered from unused materials in the Veggie plant pillow growth units assayed after the flight.

SARS-CoV-2 and Orientia tsutsugamushi co-infection in a young teen, Nepal: Significant burden in limited-resource countries in Asia?

Scrub typhus is caused by Orientia tsutsugamushi, transmitted through bites of infected chiggers (larval mites). During the coronavirus disease 2019 (COVID-19) pandemic, reports of co-infections with endemic pathogens are increasing around the world. Disease with similar clinical presentation may mask other disease diagnosis and increase the morbidity and mortality of the patients. We report co-infection between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and O. tsutsugamushi in a patient in Nepal presenting with fever, headache, retro-orbital pain, generalized body ache, and knee joints pain with a history of dry cough and dyspnea at night. Since scrub typhus is prevalent and considerate as one of the public health consents in Asian countries and the possible overlapping clinical manifestation with other infections including COVID-19, a further investigation required to determine the burden of SARS-CoV-2 and O. tsutsugamushi co-infection in scrub typhus-endemic countries in Asia.

Post-kala-azar dermal leishmaniasis (PKDL), HIV and pulmonary tuberculosis.

Post-kala-azar dermal leishmaniasis is usually a sequel to visceral leishmaniasis. A 25-year-old woman presented with hypopigmented maculopapular lesions all over the body for the past 4 years without any previous history of visceral leishmaniasis. She was on treatment for leprosy and pulmonary tuberculosis for the past 2 months, but did not show any improvement. Investigations confirmed that she had post-kala-azar dermal leishmaniasis associated with pulmonary tuberculosis and HIV-1 infection. She was started on treatment for the triad of diseases, and showed improvement.

Leishmania donovani vs immunity: T-cells sensitized from Leishmania of one donor may modulate their cytokines pattern on re-stimulation with Leishmania from different donor in visceral leishmaniasis.

Antimony resistance is frequently encountered during treatment of visceral leishmaniasis (VL) and the differences are well characterized by inadequate IFN-gamma dominant type-1 protection mechanisms. The part played by Leishmania parasites derived from antimony treated patients in the outcome of an immune response largely remains to be investigated. In the present study we observed that macrophages of BALB/c mice infected with antimony non-responder (SAG-NR) isolates had a greater amastigote burden than antimony responder (SAG-R) isolates. Later it was observed that antigen from SAG-NR and R L. donovani isolates elicit different cytokine responses in peritoneal blood mononuclear cells (PBMCs) from patients with VL. The production of IFN-gamma by T-cells in VL patients increased in response to Leishmania derived from responder patients but this response within same T-cells was lower when sensitized from Leishmania from a non-responder VL patient. On the other hand, IL-4 and IL-10 expression was increased when primed with parasites from non-responder VL source. Such a differential pattern of cytokine expression by the same T-cell population produced to Leishmania from different donors, needs further exploration.

Wilson disease with visceral leishmaniasis: an extremely uncommon presentation.

Visceral leishmaniasis (VL), which is caused by the protozoa Leishmania donovani and transmitted by the bite of the female sand fly Phlebotomus argentipes, is common in Bihar, India. Wilson disease is an autosomal recessive disorder of copper metabolism in which copper is deposited in the brain and liver. We report a case of an extremely uncommon combination of these diseases in a patient. Treatment options for such a combination of diseases are limited and difficult.

Visceral leishmaniasis and tuberculosis in patients with HIV co-infection.

We describe here two cases, one male and one female, both age 40 years, with visceral leishmaniasis and HIV-1 co-infection. The female patient had features of Koch's abdomen. The male patient had features of tuberculous lymphadenitis and bilateral pleural effusion more marked on the right side. Both were treated with highly active antiretroviral therapy, antituberculous drugs, antibiotics, antifungal medicine (fluconazole) and miltefosine. Both patients showed marked improvement with therapy.

Transcriptome Profiling of Buffalograss Challenged with the Leaf Spot Pathogen Curvularia inaequalis.

Buffalograss (Bouteloua dactyloides) is a low maintenance U. S. native turfgrass species with exceptional drought, heat, and cold tolerance. Leaf spot caused by Curvularia inaequalis negatively impacts buffalograss visual quality. Two leaf spot susceptible and two resistant buffalograss lines were challenged with C. inaequalis. Samples were collected from treated and untreated leaves when susceptible lines showed symptoms. Transcriptome sequencing was done and differentially expressed genes were identified. Approximately 27 million raw sequencing reads were produced per sample. More than 86% of the sequencing reads mapped to an existing buffalograss reference transcriptome. De novo assembly of unmapped reads was merged with the existing reference to produce a more complete transcriptome. There were 461 differentially expressed transcripts between the resistant and susceptible lines when challenged with the pathogen and 1552 in its absence. Previously characterized defense-related genes were identified among the differentially expressed transcripts. Twenty one resistant line transcripts were similar to genes regulating pattern triggered immunity and 20 transcripts were similar to genes regulating effector triggered immunity. There were also nine up-regulated transcripts in resistance lines which showed potential to initiate systemic acquired resistance (SAR) and three transcripts encoding pathogenesis-related proteins which are downstream products of SAR. This is the first study characterizing changes in the buffalograss transcriptome when challenged with C. inaequalis.

Role of HLA-DR antigen on T-cell activation in visceral leishmaniasis.

Ability of peripheral blood monocytes in association with HLA-DR molecules to support T-cell activation in response to soluble Leishmania donovani antigen was investigated. Adherent cells were stained with monoclonal antibodies. The increased number of cells with DR expression was more efficient in presenting L. donovani antigen to sensitized T-cells. The results suggest that quantitative variation in monocytes with expression of DR molecules, correlates with their ability to support T-cell response to L. donovani antigen, in vitro, as assessed by migration inhibition factor (MIF). However, it is not clear whether this is due to only HLA-DR antigen on the surface or whether other factors are involved.

Reversal of T-cell unresponsiveness through serine-esterase inhibitors mediated enhanced lymphokine induced microbicidal activities in kala-azar.

After presenting processed glycoprotein of Leishmania donovani to T-cell, macrophage seeks the help of a panel of T-cells lymphokines to transform from a state that sustains intra cellular replication of parasite to an effector state for destructing parasites. But esterase and trypsin of macrophage membrane prevent T-cells to release MIF. Role of soya-bean trypsin inhibitor (STI) has been exposed in the present study with a view to alter esterase functional behaviour of macrophage for control of T-cell activation and also, if T-cells once made responsive to antigen by STI do alter macrophage response to T-cells or not. Results establish STI as potent effector molecule, which can serve as an adjuvant to candidate T-cell epitope and synthetic peptide for development of anti-Kala-azar vaccine protocol in future.

Effect of immunization with lipid associated polysaccharide antigen and anti CD-2 antibodies on class II MHC expression and cellular immune response in BALB/C mice infected with Leishmania donovani.

In a bid to characterize the antigens and immunization mechanisms which may be used to produce a protective response against L. donovani, role of lipid associated polysaccharide (LPS) antigen and whole antigen was evaluated. BALB/C mice were immunized with whole or LPS antigen in combination with one of three putative adjuvents (anti CD-2 antibody/FIA/0.85% Saline). LPS antigen emulsified in anti CD-2 antibody was found to induce significant antibodies in mice on day 28 against challenge with lethal dose of L. donovani. Immunoprophylactic properties of LPS and whole antigen was investigated on day 40 through cytokine elicitation (IL-2), MIF) in culture supernatants of spleen cells, but before that MHC-II expressed on macrophage was studied. The LPS antigen in combination with anti CD-2 antibody was found to be most immuno-reactive inducing higher MHC-II expression on macrophages which was associated with substantial rise in the level of MIF and IL-2. It coincided with decline in antibody titre in 100% mice immunized with LPS antigen while Leishmania injected as whole antigen failed to induce specific macrophage and T-cell response with all the above formulations. We surmise from our data that lipid associated polysaccharide antigen linked to anti CD-2 antibody has potential for eliciting protective immunity against Leishmania.

Relevance of direct agglutination test in seroepidemiological survey of Kala-azar in epidemic foci of Bihar.

The performance of direct agglutination test (DAT) was evaluated under field conditions in three endemic districts of Bihar. In three villages of these districts, 197 households were surveyed with a population of 1167 individuals. Out of 1167 individuals, 603 were clinically examined for VL and blood samples of each examined individual were collected for DAT. The proportion of DAT positivity in both males (32.33%) and females (32.94%) was statistically not significant (p > 0.05). The proportion of DAT positivity in all age groups was observed to be similar (p > 0.05). The GMRT was observed highest in active VL cases. Out of 20 DAT positive individuals having fever, 5 were examined parasitologically (bone marrow aspiration) and in three cases positivity was observed. This showed the relevance of DAT in early diagnosis of visceral leishmaniasis and usefulness of DAT in field conditions.

Leishmania donovani: influence of anti-leishmanial therapy on expression of lymphocyte function-associated antigen-3 and its relevance to pathogenisis in visceral leishmaniasis.

Lymphocyte function associated antigen 3 (LFA-3) is known as adhesion molecule with its role in T-cell activation signaling as well as in Foxp3 expression. Its influences on IL-10 production is also available, whose role in pathogenesis is well documented. However, this molecule is not yet directly addressed for its association with visceral leishmaniasis (VL). We investigated the relationship between Leishmania donovani infection and expression of LFA-3 in VL patients in their pre and post treatment stage through this case control study. Present study reports L. donovani mediated expression of LFA-3 on CD14(+) monocytes in human VL. Active cases of VL was observed with 2.91-fold increased mean florescence intensity (MFI) of LFA-3 expression on CD14(+) cells compared to healthy control (p = 0.0001). This increased MFI of untreated VL cases was reduced 1.92-fold in successfully treated cases (p = 0.0001). This observation was also accorded by mRNA expression for LFA-3 in monocytes of corresponding samples. The expression of LFA-3 was also observed influenced by L. donovani load in splenic aspirates, as it was 1.71-fold elevated in patients with Ld grade ≥3+ compared to patients with ≤2+ Ld grade (p = 0.0121). To evaluate the possibility that L. donovani utilize LFA-3 mediated evasion pathway in human visceral leishmaniasis; in vitro experiments were performed for measurement of pathogenic cytokine IL-10 and Foxp3 mRNA expression. The IL-10 production and Foxp3 expression in peripheral blood mononuclear cells from VL subjects were observed regulated significantly (p = 0.0131 and 0.0436 when compared with untreated samples) in presence of an antagonist to LFA-3. This study recommends further investigations to strengthen the pathogenic and prognostic significance of LFA-3 in visceral leishmaniasis.

Vertical migration of Haemonchus contortus infective larvae on Cynodon dactylon and Paspalum notatum pastures in response to climatic conditions.

Observations were made on vertical migration patterns of Haemonchus contortus infective larvae on Cynodon dactylon (bermudagrass) and Paspalum notatum (bahiagrass) pastures under summer climatic conditions typical of East Texas. Ten thousand H. contortus infective larvae (L3) were introduced to 100 cm(2) subplots of each pasture species within a plot area of 1m(2). Subplots were inoculated with larvae by applying them in an aqueous medium to the soil or mat beneath the vegetation. Herbage from the inoculated areas was harvested on 5 sampling days over a span of 21 days. L3 recoveries were observed and recorded each day on four herbage strata viz. 0-5, 5-10, 10-20 and >20 cm from ground level. The log transformed larval recovery data were analyzed for effect of day, stratum, and day x stratum interaction for each grass species during two separate experimental periods. Precipitation, relative humidity and temperature during the study were subjected to correlation and multiple regression analyses with the larval counts. Significant (Por=0.93) between rainfall and total average daily larval counts was apparent. The multiple regression analysis did not show significant results for any of the climatic factors tested. This study showed that the H. contortus infective larvae can survive beyond 21 days in the soil and infest pasture grasses when the climatic conditions are favorable. Avoiding use of H. contortus contaminated pasturelands in summer at the onset of rainfall following a dry spell may effectively reduce nematode loads in susceptible farm animals. Additional studies should focus on factors affecting long term L3 survivability, migrational pattern on these and other plant species and the relationship between climatic factors and larval migration patterns throughout the year. Total larval recovery of H. contortus in this study was greater in bahiagrass than bermudagrass. While the design of this study did not allow for testing one pasture species against another, studies with potted plants would allow for some valid comparisons. Soil characteristics may also play a role in L3 survival and subsequent migration.