Coinfection of Leptomonas seymouri and Leishmania donovani in Indian leishmaniasis

Leishmania donovani is considered the causative organism of visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). Testing of 4/29 DNA samples from VL and PKDL patients as well as 2/7 field isolates showed an aberrant internal transcribed spacer 1 (ITS1) restriction fragment length polymorphism (RFLP) pattern, which upon sequencing strongly matched Leptomonas seymouri, thus confirming its presence in Indian leishmaniasis.     

IFNG and IFNGR1 gene polymorphisms and susceptibility to post-kala-azar dermal leishmaniasis in Sudan

Post-kala-azar dermal leishmanaisis (PKDL) in Sudan is associated with elevated interferon-gamma (IFN-gamma). To study interferon-gamma pathways in PKDL, we genotyped 80 trios from the Masalit ethnic group for polymorphisms at -470 ins/delTT, -270T/C, -56T/C and +95T/C in IFNGR1 and at -179G/A and +874T/A in IFNG. No associations occurred at IFNG. Global association with haplotypes comprising all four markers at IFNGR1 (chi(2)(10df)=21.97, P=0.015) was observed, associated with a significant (chi(2)(1df)=4.54, P=0.033) bias in transmission of the haplotype insTT T T T and less (chi(2)(1df)=5.59, P=0.018) than expected transmission of insTT C C C. When compared with data on malaria associations from Gambia, the results suggest a complex pattern of haplotypic variation at the IFNGR1 promoter locus associated with different infectious disease in African populations that reflect the complex roles of IFN-gamma in parasite killing versus inflammation and pathogenesis.     

Genes at human chromosome 5q31.1 regulate delayed-type hypersensitivity responses associated with Leishmania chagasi infection

Visceral leishmaniasis (VL) caused by Leishmania chagasi is endemic to northeast Brazil. A positive delayed-type hypersensitivity skin test response (DTH+) is a marker for acquired resistance to disease, clusters in families and may be genetically controlled. Twenty-three single nucleotide polymorphisms (SNPs) were genotyped in the cytokine 5q23.3-q31.1 region IRF1-IL5-IL13-IL4-IL9-LECT2-TGFBI in 102 families (323 DTH+; 190 DTH-; 123 VL individuals) from a VL endemic region in northeast Brazil. Data from 20 SNPs were analyzed for association with DTH+/- status and VL using family-based, stepwise conditional logistic regression analysis. Independent associations were observed between the DTH+ phenotype and markers in separate linkage disequilibrium blocks in LECT2 (OR 2.25; P=0.005; 95% CI=1.28-3.97) and TGFBI (OR 1.94; P=0.003; 95% CI=1.24-3.03). VL child/parent trios gave no evidence of association, but the DTH- phenotype was associated with SNP rs2070874 at IL4 (OR 3.14; P=0.006; 95% CI=1.38-7.14), and SNP rs30740 between LECT2 and TGFBI (OR 3.00; P=0.042; 95% CI=1.04-8.65). These results indicate several genes in the immune response gene cluster at 5q23.3-q31.1 influence outcomes of L. chagasi infection in this region of Brazil.     

Polymorphisms in proinflammatory genes and susceptibility to typhoid fever and paratyphoid fever.

Host genetic factors are thought to contribute to susceptibility and outcome in infectious diseases. A polymorphism in a proinflammatory gene, tumor necrosis factor-alpha (TNFA - 308), was recently found to be associated with susceptibility to typhoid fever. As the observation was made in hospitalized patients, a potential confounder could be that the TNFA polymorphism is associated with the severity of established illness resulting in hospital admission rather than susceptibility to disease. We tested whether the association with TNFA - 308 is present also in typhoid fever patients enrolled in a community-based case-control study in an endemic area in Indonesia. Common polymorphisms in other proinflammatory genes were assayed as well. Samples of patients with blood culture-confirmed typhoid fever (n = 90) and paratyphoid fever (n = 26) and fever controls (n = 337) were compared with those of community controls (n = 322). In these groups, we analyzed polymorphisms in TNFA by PCR and RFLP, polymorphisms of IFNG, IL1A, IL1B, IL1R1, TNFRSF1A, CASP1, and CRP by Sequenom MassArray (San Diego, CA), and polymorphisms in IL12B and IFNGR1 by fragment length analysis. The IL1R1 polymorphisms were nearly absent in the Indonesian population. The TNFA - 308 polymorphism was not associated with typhoid fever (OR 0.35, 95% CI 0.1-1.0) in this population. The polymorphisms at TNFA - 238 or in IFNG, IL1A, IL1B, IL12B, TNFRSF1A, IFNGR1, CASP1, and CRP were also not associated with typhoid or paratyphoid fever. We conclude that polymorphisms in proinflammatory genes do not contribute to susceptibility to typhoid fever and, in view of earlier findings, suggest that the TNFA - 308 polymorphism is likely related to severity of established disease rather than to susceptibility per se.     

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.     

Identification of a novel G245R polymorphism in the IL-2 receptor beta membrane proximal domain associated with human visceral leishmaniasis

Binding of the interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) triggers a series of intracellular events culminating in lymphocyte proliferation and differentiation. We report here the identification of a novel G245R polymorphism in the membrane proximal domain of the IL-2 receptor beta chain (IL-2Rbeta). Present at a frequency of 7.2%, the IL-2-Rbeta G245R was identified in a population of Eastern Sudan exposed to a severe outbreak of visceral leishmaniasis (VL), a disease associated with a marked depression of T-cell antigen-specific responses. The location of the G245R polymorphism next to the box1/box2 proximal cytokine receptor homology segment and suggestive genetic association with the development of disease (P=0.043), suggest that it may affect Janus kinase (JAK) association and impair growth signal transduction. However, additional genetic association with a synonymous single nucleotide polymorphism (IL2RB+8777T) suggests that other variations of IL2RB or nearby genes participate in the highly significant linkage with VL at 22q12 previously reported for this population.     

Visceral and post-Kala-Azar dermal leishmaniasis isolates show significant difference in their in vitro drug susceptibility pattern

Visceral leishmaniasis (VL) remains a major health problem in old world, and India accounts for half of the world burden. The widespread emergence of resistance to standard drug in India poses a major obstacle in the control of leishmaniasis. Post-Kala-Azar dermal leishmaniasis (PKDL) is considered as main source of drug resistance. Experimental data indicate that resistance against newer drugs is also imminent. Therefore, in vitro studies were carried out to test minimum parasiticidal concentration of five conventional and newly introduced anti-leishmanial drugs against 20 field isolates of Leishmania donovani obtained from visceral and post-Kala-Azar dermal leishmaniasis patients of India. Study revealed wide range of variation in minimum inhibitory concentration of sodium antimony gluconate (SAG). PKDL isolates displayed significantly lower susceptibility to SAG and miltefosine than VL isolates with P value of 0.0006 and 0.0243, respectively. All clinical isolates had higher IC₅₀ value for paromomycin and miltefosine as compared to reference strain indicating their vulnerability to develop unresponsiveness. However, isolates were uniformly susceptible to pentamidine and amphotericin B. The results of gene expression analysis of AQP1 were largely in agreement with phenotypic drug sensitivity results. Interestingly, significant down-regulation of AQP1 was observed in PKDL isolates as compared to VL isolates indicating their increased propensity for drug unresponsiveness. However, no significant difference in mRNA expression of LdMT and LdRos3 gene was found for two groups. The present study unravels valuable baseline scientific data showing variation in the drug susceptibility pattern in the L. donovani isolates. The information might have impact on the management and control of Indian visceral leishmaniasis.     

Validation of a simple resazurin-based promastigote assay for the routine monitoring of miltefosine susceptibility in clinical isolates of Leishmania donovani

Simple, cost-effective approach for routine surveillance of parasite susceptibility to antileishmanial drug miltefosine (MIL) is highly desirable for controlling emergence of drug resistance in visceral leishmaniasis (VL). We validated a simple resazurin-based fluorimetric assay using promastigotes to track natural MIL tolerance in Leishmania donovani parasites from VL cases (n?=?17) against standard amastigote assay, in two different labs in India. The inter-stage MIL susceptibility correlated strongly (r?=?0.70, p?=?0.0018) using J774.A.1 macrophage cell line-based amastigote assay and fluorescence-based resazurin assay for promastigotes. Investigation of inter-stage MIL susceptibility for the same set of clinical isolates in another lab also showed a strong correlation (r?=?0.72, p?=?0.0012) using mouse peritoneal macrophages for amastigote assay and resazurin-based alamar blue assay for promastigotes. Additionally, parasites from post-kala-azar dermal leishmaniasis (PKDL) lesions (n?=?7, r?=?0.78, p?=?0.046) and MIL-induced parasites (r?=?0.92, p?=?0.0001; n?=?3) also exhibited a strongly correlated inter-stage miltefosine susceptibility. Thus, our results support the utility of resazurin assay as a simplified biological tool for MIL susceptibility monitoring in clinical isolates from MIL-treated VL/PKDL patients.     

T-cell response in human leishmaniasis

In the present communication we provide evidence for the existence of a Th1/Th2 dichotomy in the T-cell response to Leishmania antigens in human leishmaniasis. Our data suggest that the pattern of IL-4 and IFN-gamma response is polarised in these patients. Lymphocytes from individuals recovered from cutaneous leishmaniasis (CL) responded by IFN-gamma production following stimulation with Leishmania antigens whereas cells from patients recovered from visceral leishmaniasis (VL) showed a mixed pattern of IFN-gamma and IL-4 responses. The cells producing these cytokines were predominantly CD4+. Furthermore, IL-10 plays an important role in the development of post kala azar dermal leishmaniasis (PKDL) from VL. The balance between the parasitic-specific T-cell response plays an important regulatory role in determining the outcome of Leishmania infections in humans.     

Fine‐needle sampling provides appreciable diagnostic yield in lesions of post kala azar dermal leishmaniasis: Analysis of four cases from North Eastern India

Post‐kala‐azar dermal leishmaniasis (PKDL) is a sequel of visceral leishmaniasis (VL), usually occurring 6 months to 3 years after VL. Spectrum of cutaneous lesions in PKDL can be hypopigmented macules, nodules, plaques, or erythema. It is usually diagnosed clinically, supplemented by ancillary techniques like skin smear examination, histopathology, polymerase chain reaction, and monoclonal antibody test. Literature on the role of cytology in the diagnosis of PKDL is extremely limited. Here we highlight the appreciable yield of fine‐needle sampling in four cases of PKDL, which may be considered as a useful diagnostic aid. Diagn. Cytopathol. 2014;42:525–529. © 2013 Wiley Periodicals, Inc.     

Elevated levels of interferon-gamma, interleukin-10, and interleukin-6 during active disease in Indian kala azar

We have evaluated levels of 6 cytokines in sera of 35 patients of kala azar (KA), 29 post kala azar dermal leishmaniasis (PKDL), and 18 healthy controls using cytometric bead array technology. Results indicated significantly high levels of interferon gamma (IFN-gamma), interleukin (IL)-10, and IL-6 during active KA, while tumor necrosis factor alpha (TNF-alpha), IL-2, and IL-4 were minimal. Serum level of cytokines in PKDL was comparable to the controls while TNF-alpha was significantly elevated compared to KA or control. At post-treatment stage, KA patients showed a significant decrement in the levels of IFN-gamma, IL-10, and IL-6; however, IL-6 remained significantly elevated above control levels. Further, comparison of cytokine levels in children and adults revealed elevated level of IL-10 in pediatric cases. SAG unresponsive cases showed significantly elevated levels of IFN-gamma in comparison with the responsive cases. The results depict that type1 response is not depressed during active KA and suggest the possibility that unresponsiveness to type1 stimuli may prevail.     

Polymorphisms in the interleukin-4 and interleukin-4 receptor α chain genes confer susceptibility to asthma and atopy in a Caucasian population

BACKGROUND : IL-4 by binding to its receptor (IL-4R) is essential for the development of airway inflammation present in asthma, through the induction of IgE synthesis in B cells and differentiation of T cells to a Th2 phenotype. OBJECTIVE : To investigate the role of four common polymorphisms in the IL-4 (IL4-34CT and IL4-589CT) and IL-4Ralpha chain (IL4RAI50V and IL4RAQ576R) genes in conferring susceptibility to the development of atopy and/or asthma. METHODS : Two polymorphisms in the IL-4 gene promoter, IL4-34CT and IL4-589CT, and two polymorphisms in the IL-4Ralpha chain gene, IL4RAI50V and IL4RAQ576R, have been genotyped using PCR-based methods in 341 asthmatic families and in 184 non-asthmatic adults recruited from the south of England. RESULTS : Case-control analysis did not reveal differences in the distribution of the four polymorphisms between asthmatics and controls. However, the transmission disequilibrium test showed that the IL4-589 T allele was preferentially transmitted to asthmatic children (P=0.036) and that the IL4RAQ576 was preferentially transmitted to children with atopic asthma (P=0.018). Haplotype analysis showed a strong association between the IL4-34T/-589T haplotype and asthma per se (P=0.041), and a strong association between the IL4RA I50/Q576 haplotype and atopic asthma (P=0.006). CONCLUSION : Our data suggest that polymorphisms in the IL-4 and IL-4Ralpha chain genes might play a role both conferring susceptibility to and modulating severity of atopy and asthma.     

Leishmania promastigote membrane antigen-based enzyme-linked immunosorbent assay and immunoblotting for differential diagnosis of Indian post-kala-azar dermal leishmaniasis

Diagnosis of post-kala-azar dermal leishmaniasis (PKDL), caused by Leishmania donovani, is difficult, as the dermal lesions are of several types and resemble those caused by other skin diseases, especially leprosy. Since the disease generally appears very late after the clinical cure of kala-azar in India, it is also difficult to correlate PKDL with a previous exposure to L. donovani. Very few attempts have been made so far to diagnose PKDL serologically, and the diagnostic methods vary in their sensitivities and specificities. Diagnosis of PKDL through sophisticated PCR methods, although highly sensitive, has limited practical use. We have developed a serodiagnostic method using an enzyme-linked immunosorbent assay to detect specific immunoglobulin (Ig) isotypes and IgG subclass antibodies in the sera of Indian PKDL patients. Our assay, which uses L. donovani promastigote membrane antigens, was 100% sensitive for the detection of IgG and 96.7% specific for the detection of IgG and IgG1. Optical density values for individual patients, however, demonstrated wide variations. Western blot analysis based on IgG reactivity could differentiate patients with PKDL from control subjects, which included patients with leprosy, patients from areas where kala-azar is endemic, and healthy subjects, by the detection of polypeptides of 67, 72, and 120 kDa. The recognition patterns of the majority of serum samples from patients with PKDL were also distinct from those of the serum samples from patients with visceral leishmaniasis (VL), at least for a 31-kDa polypeptide. To further differentiate patients with PKDL from those with active and cured VL, we analyzed the specific titers of the Ig isotypes and IgG subclasses. High levels of IgG, IgG1, IgG2, and IgG3 antibodies significantly differentiated patients with PKDL from patients cured of VL. The absence of antileishmanial IgE and IgG4 in patients with PKDL differentiated these patients from those with active VL. These results imply intrinsic differences in the antibodies generated in the sera from patients with PKDL and VL.     

Immunopathology of post kala-azar dermal leishmaniasis (PKDL): T-cell phenotypes and cytokine profile

In Sudan, post kala-azar dermal leishmaniasis (PKDL) caused by Leishmania donovani develops in half of the patients treated for visceral leishmaniasis (kala-azar). In most patients lesions heal spontaneously, but in others symptoms are severe and persist for years. This study examined the immunological response in lesions of PKDL patients by immunohistochemistry and compared the findings with results obtained using peripheral blood mononuclear cells (PBMCs). In all lesions, parasites or parasite antigen were present and provoked the formation of an inflammatory infiltrate consisting of a mixture of macrophages, lymphocytes, and plasma cells. In patients who had high interferon-gamma (IFNgamma) responses to Leishmania antigen in vitro, compact epithelioid granulomas were formed. The inflammatory cells were mainly CD3(+) and interleukin-10 (IL10) was the most prominent cytokine in the lesions. However, IFNgamma was found in all and IL4 in most lesions, in varying amounts. PBMCs from all patients responded to Leishmania antigen by IFNgamma production or proliferation. The results indicate that PKDL develops as a result of an influx of immunocompetent cells into skin, which harbours parasites. The inflammatory response to the parasites is complex. It involves several cell types and cytokines, of which some are antagonistic. It is conceivable that the balance between these cytokines determines the outcome of the disease.     

Polymorphisms in the genes encoding interferon-gamma and interferon-gamma receptors in multiple sclerosis.

Genome screens suggest that several genes, each contributing to a small extent, are involved in multiple sclerosis (MS) susceptibility. Simultaneous analysis of related genes may improve the power to detect such small effects. Interferon-gamma (IFN-gamma), mediating its effects through the IFN-gamma receptor, is a pleiotropic, pro-inflammatory cytokine for which a detrimental effect on the course of MS has been reported. The role of IFN-gamma receptor 1 (IFNGR1) and IFN-gamma receptor 2 (IFNGR2) gene polymorphisms has not been studied in MS, and, for the IFNG gene polymorphism there is only one previous study, which incorporates clinical, but not imaging, data. The aim of this study was to investigate whether polymorphisms in the IFNG and IFNGR1 and IFNGR2 genes are associated with susceptibility to MS, or disease characteristics, as defined by clinical and imaging criteria. Genotypes for IFNG, IFNGR1 and IFNGR2 were determined in 509 patients with MS and in 193 healthy controls. Patient files were reviewed for disease course, age at onset of disease, and rate of progression. Serial magnetic resonance imaging (MRI) data were available for 107 patients. No significant differences in the distribution of IFNG, IFNGR1 and IFNGR2 genotype and allele frequencies were found between patients and controls. A progressive, as opposed to a relapsing, onset was significantly more frequent in carriers of the IFNGR2 allele Arg64 (P = 0.028). Moreover, IFNGR2 allele Arg64 carriers had a lower black hole ratio than non-carriers (P = 0.016). No other associations with clinical parameters, such as age at onset or rate of progression, or with imaging parameters, were observed. The IFNG intron 1 gene polymorphism studied is unlikely to play a major role in MS susceptibility or disease course. The IFNGR1 and IFNGR2 gene polymorphisms studied do not exert an important influence on MS susceptibility, but allele IFNGR2*Arg64 may be associated with a progressive disease onset.     

Variants in the 5q31 cytokine gene cluster are associated with psoriasis

A multitiered genetic association study of 25 215 single-nucleotide polymorphisms (SNPs) in three case-control sample sets (1446 patients and 1432 controls) identified three IL13-linked SNPs (rs1800925, rs20541 and rs848) associated with psoriasis. Although the susceptibility effects at these SNPs were modest (joint allelic odds ratios (ORs): 0.76 to 0.78; P(comb): 1.3E-03 to 2.50E-04), the association patterns were consistent across the sample sets, with the minor alleles being protective. Haplotype analyses identified one common, susceptible haplotype CCG (joint allelic OR=1.27; P(comb)=1.88E-04) and a less common, protective haplotype TTT (joint allelic OR=0.74; P(comb)=7.05E-04). In combination with the other known genetic risk factors, HLA-C, IL12B and IL23R, the variants reported here generate an 11-fold psoriasis-risk differential. Residing in the 5q31 cytokine gene cluster, IL13 encodes an important T-cell-derived cytokine that regulates cell-mediated immunity. These results provide the foundation for additional studies required to fully dissect the associations within this cytokine-rich genomic region, as polymorphisms in closely linked candidate genes, such as IRF1, IL5 or IL4, may be driving these results through linkage disequilibrium.     

Associations between cytokine receptor polymorphisms and variability in laboratory immune parameters in normal humans

In every study involving human immune parameters, large inter-subject variability occurs which can make interpretation of results difficult. The aim of this study was to evaluate whether genetic variants in cytokine receptors could associate with variability in laboratory immune measures. A total of 207 normal volunteers were recruited in this study. Immunoregulatory profiles were measured by flow cytometry and genotyping assays were performed by allelic discrimination real-time PCR. Immunoregulatory profiles were categorized according to various single nucleotide polymorphisms (SNPs) of cytokine receptors including T-56C and G-611A of IFN-γ receptor 1 (IFNGR1); Q64R of IFNGR2; and Ile50Val, Q576R and S503P of IL4R. Results reveal that Th1 levels were significantly higher in the heterozygous of the IFNGR1 T-56C polymorphism (minor allele) compared to wild-type (WT, major allele) (p = 0.006). For the Q576R of IL4R, Th1/Th2 ratio was significantly lower for the homozygous SNP (Arg/Arg) compared to the WT (Gln/Gln) (p = 0.035). In addition, the significant interaction effects of demographic characteristics on SNP-immune parameter associations were reported as well. We conclude that cytokine receptor polymorphisms might associate with variability in laboratory immune measures. Approach of SNP analysis of cytokine receptors can be useful in categorizing baseline immune responses to more accurately evaluate clinical immune data.     

The development of post-kala-azar dermal leishmaniasis (PKDL) is associated with acquisition of Leishmania reactivity by peripheral blood mononuclear cells (PBMC)

PKDL develops in about 50% of Sudanese patients treated for visceral leishmaniasis (kala-azar). Patients with kala-azar were entered into this study and followed for a period of up to 2 years. During follow up 12 patients developed PKDL and eight did not. Proliferative responses and cytokine production to Leishmania donovani and control antigens were measured in vitro using PBMC isolated at the time of diagnosis of kala-azar, after treatment of visceral leishmaniasis, during follow up, and at the time of diagnosis of PKDL. Proliferative responses and interferon-gamma (IFN-gamma) production were low at diagnosis and increased after treatment of kala-azar in both patients who developed (group 1) and those who did not develop PKDL later (group 2). In group 1, development of PKDL was always associated by an increased PBMC response to Leishmania antigen in proliferation and IFN-gamma production assays. There were no differences in Leishmania antigen-induced production of IL-4, IL-5 and IL-10 between or within the two groups. We have previously shown that Leishmania parasites spread to the skin during visceral leishmaniasis and proposed that PKDL was the result of an immunological attack on parasites, which have survived in the skin despite the drug treatment. The finding that PKDL develops after treatment of kala-azar as Leishmania-reactive T cells start to circulate in peripheral blood in sufficient numbers to be detected in in vitro assays supports this hypothesis.