Revealing a Novel Antigen Repressor of Differentiation Kinase 2 for Diagnosis of Human Visceral Leishmaniasis in India.

Visceral leishmaniasis (VL) is one of the major global health concerns due to its association with morbidity and mortality. All available diagnostic tools have been, until now, unable to provide a very specific and cost-effective mode of detection for VL globally. Therefore, the design of robust, specific, and commercially translatable diagnostic tests is urgently required. Currently, we are attempting to identify and explore the diagnostic potential of a novel parasite antigen. Repressor of differentiation kinase 2 (RDK2), a serine/threonine kinase, has a versatile role in parasite life cycle progression. However, its role as a diagnostic candidate for VL has not been investigated. Herein, we cloned and over-expressed LdRDK2 and studied the recombinant RDK2 for the diagnosis of human VL using serum and urine samples. In silico analysis predicted that RDK2 is conserved among Leishmania species with the least conservation in humans. RDK2 developed immune-reactive bands with antibodies present in VL patients' sera, and it demonstrated no cross-reactivity with sera from healthy controls and other diseases. Additionally, RDK2 antigen demonstrated a significant reactivity with IgG antibodies of VL patients' sera, with 78% sensitivity and 86.67% specificity as compared to healthy controls and other diseases. Furthermore, we evaluated its utility for non-invasive diagnosis of VL using patients' urine samples and found 93.8% sensitivity and 85.7% specificity. RDK2 was found to have better sensitivity and treatment response in patients' urine compared to serum samples, indicating its role as a promising point of care (POC) antigen. In a nutshell, we explored the role of RDK2 as a potential diagnostic marker for VL in both invasive and non-invasive modes as well as its utility as a promising POC antigen for treatment response cases.

Development and Clinical Evaluation of Serum and Urine-Based Lateral Flow Tests for Diagnosis of Human Visceral Leishmaniasis.

Visceral leishmaniasis (VL), a fatal parasitic infection, is categorized as being neglected among tropical diseases. The use of conventional tissue aspiration for diagnosis is not possible in every setting. The immunochromatography-based lateral flow assay (LFA) has attracted attention for a long time due to its ability to give results within a few minutes, mainly in resource-poor settings. In the present study, we optimized and developed the LFA to detect anti-Leishmania antibodies for VL diagnosis. The performance of the developed test was evaluated with serum and urine samples of Indian VL patients and Brazilian sera. The new test exploits well-studied and highly-sensitive purified antigens, LAg isolated from Leishmania donovani promastigotes and protein G conjugated colloidal-gold as a signal reporter. The intensity of the bands depicting the antigen-antibody complex was optimized under different experimental conditions and quantitatively analyzed by the ImageJ software. For the diagnosis of human VL in India, LFA was found to be 96.49% sensitive and 95% specific with serum, and 95.12% sensitive and 96.36% specific with urine samples, respectively. The sensitivity and specificity of LFA were 88.57% and 94.73%, respectively, for the diagnosis of Brazilian VL using patients' sera infected with Leishmania infantum. LFA is rapid and simple to apply, suitable for field usage where results can be interpreted visually and particularly sensitive and specific in the diagnosis of human VL. Serum and urine LFA may improve diagnostic outcomes and could be an alternative for VL diagnosis in settings where tissue aspiration is difficult to perform.

A Novel Antigen, Otubain Cysteine Peptidase of Leishmania donovani, for the Serodiagnosis of Visceral Leishmaniasis and for Monitoring Treatment Response.

Tests for visceral leishmaniasis (VL) are not uniformly effective for all endemic regions. In a serological assay, a novel antigen, otubain cysteine peptidase, compared with rK39, showed comparable sensitivity with Indian VL serum samples and prominently increased sensitivity with Brazilian samples, as well as improved monitoring of the treatment response.

Evaluation of Cysteine Protease C of Leishmania donovani in Comparison with Glycoprotein 63 and Elongation Factor 1α for Diagnosis of Human Visceral Leishmaniasis and for Posttreatment Follow-Up Response.

Visceral leishmaniasis (VL) is a threat in many developing countries. Much effort has been put to eliminating this disease, for which serodiagnosis remains the mainstay for VL control programs. New and improved antigens as diagnostic candidates are required, though, as the available antigens fail to demonstrate equal optimum performance in all areas of endemicity. Moreover, these diagnoses are dependent on invasive serum sampling. In the current study, we cloned and expressed Leishmania donovani cysteine protease C (CPC) and evaluated its diagnostic and test-of-cure possibilities by detecting the antibody levels in human serum and urine through ELISA and immunoblot assays. Two immunodominant antigens, recombinant glycoprotein 63 (GP63) and elongation factor 1α (EF1α), identified earlier by our group, were also assessed by employing human serum and urine samples. Of these three antigens in ELISAs, CPC demonstrated the highest sensitivities of 98.15% and 96% positive testing in serum and urine of VL patients, respectively. Moreover, CPC yielded 100% specificity with serum and urine of nonendemic healthy controls compared to GP63 and EF1α. Urine samples were found to be more specific than serum for distinguishing endemic healthy controls and other diseases by means of all three antigens. In all cases, CPC gave the most promising results. Unlike serum, urine tests demonstrated a significant decrease in antibody levels for CPC, GP63, and EF1α after 6 months of treatment. The diagnostic and test-of-cure performances of CPC in the immunoblot assay were found to be better than those of GP63 and EF1α. In conclusion, CPC, followed by GP63 and EF1α, may be utilized as candidates for diagnosis of VL and to assess treatment response.

Investigation of the antigenicity and protective efficacy of Leishmania promastigote membrane antigens in search of potential diagnostic and vaccine candidates against visceral leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL), is a parasitic disease that causes serious medical consequences if treatment is delayed. Despite a decline in the number of VL cases in the Indian subcontinent, the commencement of the disease in newer areas continues to be a major concern. Although serological diagnosis mainly by immunochromatographic tests has been found to be effective, a test of cure in different phases of treatment is still desired. Even though a good prophylactic response has been obtained in murine models by a number of vaccine candidates, few have been proposed for human use. METHODS: In this study, nine antigenic components (31, 34, 36, 45, 51, 63, 72, 91 and 97 kDa) of Leishmania promastigote membrane antigens (LAg), were electroeluted and evaluated through ELISA to diagnose and distinguish active VL from one month cured and six months post-treatment patients. Further, to investigate the immunogenicity of electroeluted proteins, human PBMCs of cured VL patients were stimulated with 31, 34, 51, 63, 72 and 91 kDa proteins. RESULTS: We found that 34 and 51 kDa proteins show 100% sensitivity and specificity with healthy controls and other diseases. After six months post-treatment, antibodies to 72 and 91 kDa antigens show a significant decline to almost normal levels. This suggests that 34 and 51 kDa proteins are efficient in diagnosis, whereas 72 and 91 kDa proteins may be used to monitor treatment outcome. In another assay, 51 and 63 kDa proteins demonstrated maximum ability to upregulate IFN-γ and IL-12 with minimum induction of IL-10 and TGF-ß. The results indicating that 51 and 63 kDa proteins could be strong candidates for human immunization against VL. In contrast, 34 and 91 kDa proteins demonstrated a reverse profile and may not be a good vaccine candidate. CONCLUSIONS: The preliminary data obtained in this study proposes the potential of some of the antigens in Leishmania diagnosis and for test of cure. Additionally, some antigens demonstrated good immunoprophylactic cytokine production through T cell-mediated immune response, suggesting future vaccine candidates for VL. However, further studies are necessary to explore these antigens in diagnosis and to access the long-term immune response.

Combination Therapy Against Indian Visceral Leishmaniasis with Liposomal Amphotericin B (FungisomeTM) and Short-Course Miltefosine in Comparison to Miltefosine Monotherapy.

Visceral leishmaniasis (VL) is endemic in Asia, East and North Africa, South America, and Southern Europe, and is a major public health problem in the Indian subcontinent. Miltefosine received approval in 2002 to treat VL in India, and the Indian National Vector Borne Disease Control Programme later adopted a single dose (10 mg/kg) of liposomal amphotericin B. We report results of a randomized trial comparing the efficacy of combination therapy with an Indian preparation of liposomal amphotericin B (single dose of 7.5 mg/kg) and short-course miltefosine (2.5 mg/kg/day for 14 days; n = 66) in comparison to miltefosine monotherapy (2.5 mg/kg/day for 28 days; n = 78). Nine patients in the miltefosine group and three in the combination therapy group had to discontinue therapy because of serious adverse events. At the end of the therapy, the clinical and parasitological cure rate was 100% in both groups. By per-protocol analysis, by 6 months after completion of treatment, 12 of 69 patients in the miltefosine monotherapy arm (17.4%, 95% CI: 10.24-28%) and none in the combination therapy arm had relapse. Over 5 years of follow-up, 10 patients in the miltefosine monotherapy arm (all within 0.5-2 years after completing therapy) and none in the combination therapy arm experienced post-kala-azar dermal leishmaniasis. Combination therapy offered benefits over miltefosine monotherapy for VL in India.

Pediatric case of Graham Little Piccardi Lassueur syndrome - A rare entity.

Graham Little Piccardi Lassueur syndrome (GLPLS) is a rare dermatosis characterized by patchy cicatricial alopecia of scalp, rapidly developing keratosis pilaris like follicular papules over trunk and extremities, and noncicatricial loss of axillary and pubic hair. This syndrome which is mostly seen in middle aged post-menopausal females (between ages 30-70)has rarely ever been described in the pediatric age group. We report a case of a 15 year old girl presenting to us with this rare syndrome.

A multicentric evaluation of dipstick test for serodiagnosis of visceral leishmaniasis in India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain.

Visceral leishmaniasis (VL) is one of the leading infectious diseases affecting developing countries. Colloidal gold-based diagnostic tests are rapid tools to detect blood/serum antibodies for VL diagnosis. Lack of uniformity in the performance of these tests in different endemic regions is a hurdle in early disease diagnosis. This study is designed to validate a serum-based dipstick test in eight centres of six countries, India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain with archived and fresh sera from 1003 subjects. The dipstick detects antibodies against Leishmania donovani membrane antigens (LAg). The overall sensitivity and specificity of the test with 95% confidence intervals were found to be 97.10% and 93.44%, respectively. The test showed good sensitivity and specificity in the Indian subcontinent (>95%). In Brazil, Ethiopia, and Spain the sensitivity and specificity of the dipstick test (83.78-100% and 79.06-100%) were better as compared to the earlier reports of the performance of rK39 rapid test in these regions. Interestingly, less cross-reactivity was found with the cutaneous form of the disease in Spain, Brazil, and Sri Lanka demonstrating 91.58% specificity. This dipstick test can therefore be a useful tool for diagnosing VL from other symptomatically similar diseases and against cutaneous form of leishmaniasis.

SLE in a Male Patient Presented Initially as Rowell's Syndrome.

A 22 year old male Indian patient presented with high grade fever, multiple joint pain, low back pain, generalized body ache since 6 months and erythematous pruritic rashes and atypical annular target like lesions over face, arm, leg and back and ulcers on hard palate and buccal mucosa for 2 months. Laboratory investigations showed a speckled pattern anti-nuclear antibody with a titer >1:160 and positive SS-A, dsDNA auto-antibodies and Rheumatoid factor. Diagnosis of Rowell's syndrome was made based on clinical and laboratory finding and the patient was treated with oral prednisolone (50 mg/day), hydroxychloroquine (200 mg q12h) and pulse cyclophosphamide (700 mg) chemotherapy. Majority of skin lesions and oral ulcerations subsided after 4 weeks of therapy. Till date only 11 male patients out of the total 71 cases of Rowell's syndrome were reported in the world's literature.

Immunoproteomic Identification and Characterization of Leishmania Membrane Proteins as Non-Invasive Diagnostic Candidates for Clinical Visceral Leishmaniasis.

Visceral leishmaniasis (VL), a potentially fatal disease is an outcome of infection caused by the parasite Leishmania donovani. The clinical diagnostic tests for this disease are still related to invasive tissue aspiration or serological immunochromatography. Advancements in immunoproteomics such as two-dimensional gel electrophoresis, mass spectrometry, B cell epitope prediction, and peptide synthesis have enabled researchers to discover newer biomarkers for disease diagnosis. In this study, we have screened several urine-reactive leishmanial membrane proteins as potential biomarker candidates. In the immunoblot assay, three proteins 51, 55 and 63 kDa showed 100% reactivity to the urine of 47 VL patients and nonreactive to 18 healthy and other diseases. Mass spectrometry revealed the identity of 51, 55 and 63 kDa proteins as elongation factor 1α (EF1-α), α-tubulin, and glycoprotein 63, respectively. B cell reactive epitopes of these proteins were mapped through bioinformatic tools and one epitope from each protein that had the highest score were synthesized. All the three native electroeluted proteins and their corresponding synthetic peptides were tested through ELISA for reactivity with VL and control urine samples. While all three demonstrated good reactivity, the diagnostic performance of EF1-α was the best. Our findings illustrate the use of urine-based proteomic approach for biomarker discovery in non-invasive clinical diagnosis of VL.

Scleroderma-like Initial Presentation of Multiple Myeloma.

Systemic sclerosis (SSc) is a multisystem connective tissue disease affecting skin and internal organs. Certain drugs, environmental toxins and some viruses have been implicated in SSc-like illnesses. Scleroderma may be associated with some connective tissue disorders or autoimmune diseases but coexistence of scleroderma with multiple myeloma (MM) is an unusual finding. We here report a case of a 59 years old female patient with 5 months history of progressive thickening of skin all over the body. Multiple myeloma was diagnosed by osteolytic lesion in skull X-ray, increase in clonal plasma cells by bone marrow biopsy, very high Kappa light chain in serum light chain assay and detection of M band by serum protein electrophoresis.

Noninvasive Diagnosis of Visceral Leishmaniasis: Development and Evaluation of Two Urine-Based Immunoassays for Detection of Leishmania donovani Infection in India.

BACKGROUND: Visceral Leishmaniasis (VL), a severe parasitic disease, could be fatal if diagnosis and treatment is delayed. Post kala-azar dermal leishmaniasis (PKDL), a skin related outcome, is a potential reservoir for the spread of VL. Diagnostic tests available for VL such as tissue aspiration are invasive and painful although they are capable of evaluating the treatment response. Serological tests although less invasive than tissue aspiration are incompetent to assess cure. Parasitological examination of slit-skin smear along with the clinical symptoms is routinely used for diagnosis of PKDL. Therefore, a noninvasive test with acceptable sensitivity and competency, additionally, to decide cure would be an asset in disease management and control. METHODOLOGY/PRINCIPAL FINDINGS: We describe here, the development of antibody-capture ELISA and field adaptable dipstick test as noninvasive diagnostic tools for VL and PKDL and as a test of cure in VL treatment. Sensitivity and specificity of urine-ELISA were 97.94% (95/97) and 100% (75/75) respectively, for VL. Importantly, dipstick test demonstrated 100% sensitivity (97/97) and specificity (75/75) in VL diagnosis. Degree of agreement of the two methods with tissue aspiration was 98.83% (κ = 0.97) and 100% (κ = 1), for ELISA and dipstick test, respectively. Both the tests had 100% positivity for PKDL (14/14) cases. ELISA and dipstick test illustrated treatment efficacy in about 90% (16/18) VL cases when eventually turned negative after six months of treatment. CONCLUSIONS/SIGNIFICANCE: ELISA and dipstick test found immensely effective for diagnosis of VL and PKDL through urine samples thus, may substitute the existing invasive diagnostics. Utility of these tests as indirect methods of monitoring parasite clearance can define infected versus cured. Urine-based dipstick test is simple, sensitive and above all noninvasive method that may help not only in active VL case detection but also to ascertain treatment response. It can therefore, be deployed widely for interventions in disease management of VL particularly in poor resource outskirts.

Induction of IL-10 and TGFß from CD4+CD25+FoxP3+ T Cells Correlates with Parasite Load in Indian Kala-azar Patients Infected with Leishmania donovani.

BACKGROUND: Visceral leishmaniasis (VL) is distinguished by a complex interplay of immune response and parasite multiplication inside host cells. However, the direct association between different immunological correlates and parasite numbers remains largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We examined the plasma levels of different disease promoting/protective as well as Th17 cytokines and found IL-10, TGFß and IL-17 to be significantly correlated with parasite load in VL patients (r = 0.52, 0.53 and 0.51 for IL-10, TGFß and IL-17, respectively). We then extended our investigation to a more antigen-specific response and found leishmanial antigen stimulated levels of both IL-10 and TGFß to be significantly associated with parasite load (r = 0.71 and 0.72 for IL-10 and TGFß respectively). In addition to cytokines we also looked for different cellular subtypes that could contribute to cytokine secretion and parasite persistence. Our observations manifested an association between different Treg cell markers and disease progression as absolute numbers of CD4+CD25+ (r = 0.55), CD4+CD25hi (r = 0.61) as well as percentages of CD4+CD25+FoxP3+ T cells (r = 0.68) all correlated with parasite load. Encouraged by these results, we investigated a link between these immunological components and interestingly found both CD4+CD25+ and CD4+CD25+FoxP3+ Treg cells to secrete significantly (p<0.05) higher amounts of not only IL-10 but also TGFß in comparison to corresponding CD25- T cells. CONCLUSIONS/SIGNIFICANCE: Our findings shed some light on source(s) of TGFß and suggest an association between these disease promoting cytokines and Treg cells with parasite load during active disease. Moreover, the direct evidence of CD4+CD25+FoxP3+ Treg cells as a source of IL-10 and TGFß during active VL could open new avenues for immunotherapy towards cure of this potentially fatal disease.

Alternative treatment approach to cerebral toxoplasmosis in HIV/AIDS: experience from a resource-poor setting.

The current standard treatment for cerebral toxoplasmosis (pyrimethamine/sulfadiazine) often encounters problems of poor tolerability, adverse effects, frequent dropouts and non-availability of pyrimethamine/sulfadiazine in some parts of India. We have had to use the combination of two effective alternative agents for toxoplasmosis, cotrimoxazole and clindamycin, on compassionate grounds. This retrospective observational study reports superior efficacy and better tolerability of cotrimoxazole/clindamycin compared to the recommended regimen. Primary end-point (complete response) was defined as more than 50% improvement of clinical status or more than 50% decrease in the size of brain lesions after two weeks of treatment initiation. Complete response occurred more commonly with cotrimoxazole/clindamycin than with pyrimethamine/sulfadiazine group (80% vs. 31.25%, respectively, relative risk 2.56, 95% confidence interval: 1.21-5.43). There was a trend towards higher on-treatment mortality in the pyrimethamine/sulfadiazine group in comparison to the cotrimoxazole/clindamycin (mortality rate 37.5% in pyrimethamine/sulfadiazine vs 12% in cotrimoxazole/clindamycin, p = 0.07, relative risk = 3.125, 95% confidence interval: 0.91-10.75). Overall, 62.5% (10/16) of patients on pyrimethamine/sulfadiazine suffered drug-related adverse reactions compared to 24% (6/25) on cotrimoxazole/clindamycin (p = 0.02, relative risk = 2.60, 95% confidence interval: 1.17-5.76). The commonest complication of pyrimethamine/sulfadiazine was severe thrombocytopenia with major bleeding (4/16, 25%). We propose that the new combination chemotherapy, which is widely available, effective and safe, can be used in developing countries.

A Defective Oxidative Burst and Impaired Antigen Presentation are Hallmarks of Human Visceral Leishmaniasis.

PURPOSE: Survival of the Leishmania parasite within monocytes hinges on its ability to effectively nullify their microbicidal effector mechanisms. Accordingly, this study aimed to delineate this biological niche in patients with visceral leishmaniasis (VL). METHODS: In monocytes, the redox status, antigen presenting capacity, expression of Toll-like receptors (TLRs), co-stimulatory molecules (CD80/86) and generation of intracellular cytokines (IL-8, IL-1ß, IL-10 and LAP-TGF-ß1) was measured by flow cytometry, levels of circulating cytokines (IL-1ß, IL-6, TNF-α, IL-8, IL-4, IL-13, IL-10 and GM-CSF) by ELISA and arginase activity by spectrophotometry. RESULTS: Within monocytes, generation of an oxidative burst was markedly attenuated as evident by decreased generation of nitric oxide and reactive oxygen species, concomitant with raised levels of thiols. This was accompanied by lowered frequency of TLR4(+) monocytes, but the arginase activity remained unaltered. Pathogen persistence was enhanced by the predominance of anti-inflammatory cytokines within monocytes, notably IL-10. Alongside, development of adaptive immunity was severely attenuated as manifested by a pronounced impairment of antigen presentation and co-stimulation evident by down regulation of CD54, HLA-DR and CD86. Treatment corrected the redox imbalance and reversed the impaired antigen presentation. CONCLUSIONS: In VL, monocyte functions were severely impaired facilitating parasite persistence; anti-leishmanial chemotherapy mediated parasite elimination through modulation of the macrophage microenvironment by restoring its redox status and antigen presenting capacity.

Rare association of coeliac disease with aplastic anaemia: report of a case from India.

Coeliac disease is a systemic autoimmune disorder with major intestinal manifestations. It has multiple hematologic associations including anaemia (mostly due to iron, folate and/or vitamin B12 malabsorption), other cytopenias, coagulation abnormalities, hyposplenism, IgA deficiency and lymphomas. Aplastic anaemia has however, only rarely been described with celiac disease in published literature. We here present a case of atypical coeliac disease in a 40 year male Indian patient, with insignificant gastrointestinal symptoms, presenting with aplastic anaemia manifested by pancytopenia with hypocellular bone marrow. On gluten free diet, his symptoms like weakness, fatigue and malaise were relieved-blood and platelet transfusion requirement also diminished.

A curative immune profile one week after treatment of Indian kala-azar patients predicts success with a short-course liposomal amphotericin B therapy.

BACKGROUND: The present pilot study investigating the minimum dose for short-course single and double-dose treatment of kala-azar with an apparently new liposomal formulation of amphotericin B, Fungisome, led to identification of immunological components for early detection of success and/or failure to cure. METHODS: Patients were treated with 5, 7.5 (single-dose) and 10 mg/kg body weight (5 mg/kg double-dose) of Fungisome. Immunological investigations involving plasma cytokines and antigen-specific lymphoproliferation and cytokine responses from PBMCs were carried out before, 1 week after Fungisome treatment, at the time of relapse, and again after conventional amphotericin B treatment. RESULTS: At 1-month follow-up all the patients showed 100% initial cure. However, total doses of 5, 7.5 and 10 mg/kg Fungisome showed 60%, 50% and 90% cure, respectively, at 6-months posttreatment. Patients successfully cured demonstrated downregulation of IL-12 and IL-10 in plasma, and two-fold or more elevation of IFN-gamma, IL-12 and TNF, and significant down-regulation of IL-10 and TGF-beta in culture supernatants 1-week posttreatment irrespective of drug-dose. A differential immune profile, involving insignificant decline in IL-10 and IL-12 in plasma and negligible elevation of IFN-gamma, IL-12 and TNF, and persistence of IL-10, despite decline in TGF-beta in culture supernatants, in apparently cured individuals, corresponded with relapse within 6-months of treatment. CONCLUSION: Immunological investigations revealed significant curative and non-curative immunomodulation 1-week posttreatment, correlating with successful cure and relapse, respectively. Although immune-correlation was dose-independent, almost consistent curative response in patients treated with the highest dose 10 mg/kg reflected a definitive impact of the higher-dose on the immune response. TRIAL REGISTRATION NAME AND NUMBER: Clinical Trials Registry--India (CTRI) CTRI/2009/091/000764.