An IL-10 dominant polarization of monocytes is a feature of Indian Visceral Leishmaniasis.

Leishmania donovani, the causative parasite of Visceral Leishmaniasis (VL), deviously manipulates host monocytes/macrophages to ensure its survival. Although monocytes/macrophages from patients with VL have demonstrated an impaired oxidative burst and antigen presentation, an unanswered yet pertinent question remains as to whether they are deactivated or alternatively activated. The significantly raised plasma levels of IL-4/IL-13 and IL-10 in VL patients suggested a microenvironment conducive for alternative activation of monocytes/macrophages. Accordingly, the classical markers for IL-4-driven monocytes/macrophages [M(IL-4)] were studied namely intramonocytic CD206+ , circulating CCL22 and CCL17, and were unchanged. Furthermore, the mRNA expression of Kruppel-like factor 4 (KLF4), peroxisome proliferator-activated receptors (PPAR)-γ and arginase-I (ARG-I) in peripheral blood mononuclear cells was unaltered. However, markers for IL-10-driven monocytes/macrophages [M(IL-10)], namely soluble CD163, intramonocytic IL-10, and circulating CXCL13 were significantly increased. Monocytes/macrophages of patients with VL demonstrated an increased expression of markers for M(IL-10), along with the absence of markers for M(IL-4). Taken together, in human VL, manipulation of these IL-10 polarized monocytes-macrophages may pave the way for improved therapeutic outcomes.

Molecular monitoring of treatment efficacy in human visceral leishmaniasis.

BACKGROUND: Focused efforts of the visceral leishmaniasis elimination program have led to a drastic decline in cases, and the present challenge is disease monitoring, which this study aimed to assess. METHODS: A Leishmania kinetoplastid-targeted qPCR quantified parasite load at disease presentation, and following treatment completion (n=49); an additional 80 cases were monitored after completion of treatment. RESULTS: The parasite load at disease presentation was 13 461.00 (2560.00-37764.00)/µg gDNA, which upon completion of treatment reduced in 47 of 49 cases to 1(1-1)/µg gDNA, p<0.0001. In 80 cases that presented >2 months post-treatment, their parasite burden similarly decreased to 1(1-1)/µg gDNA except in 6 of 80 cases, which were qPCR positive. CONCLUSION: In 129 cases of visceral leishmaniasis, qPCR by quantification of parasite burden proved effective for monitoring treatment.

A foe incognito: an interesting case of pleurisy.

Though pleuritis and pleural effusion are common in lupus patients they are distinctly rare as the initial manifestation of lupus. Diagnosis of lupus pleuritis is also a difficult task and often costly and lengthy immunological panels are employed to diagnose it. We report one case of systemic lupus erythematosus (SLE) presenting with lupus pleuritis as the first manifestation. We propose that demonstration LE cells have a very prominent role in differentiating lupus pleuritis from other causes of pleural effusions in SLE patients. We believe that our case is the first report from India which shows pleuritis may be a first manifestation of lupus.

Early seizures in first-ever acute stroke patients in India: incidence, predictive factors and impact on early outcome.

BACKGROUND AND PURPOSE: Stroke-associated early seizures (ES) often complicate the initial course of acute stroke. This study intended to estimate the rate of and the predictive factors for ES and the impact of ES on the clinical outcome in patients with first-ever acute stroke. MATERIALS AND METHODS: Consecutive patients with first-ever acute stroke admitted in the Department of Medicine from June 2010 to December 2011 were prospectively included. ES were defined as seizures occurring within 7 days from acute stroke. Patients with history of epilepsy, transient ischaemic attack, subarachnoid haemorrhage and cerebral venous thrombosis were excluded. Clinical outcomes were measured under the subheadings of mortality and disability at discharge, according to modified Rankin score. RESULTS: Of the 441 (56.92% male patients, median age 55 years, 49.43% had haemorrhagic stroke) patients, 79 (17.91%, 95% confidence interval (CI): 14.61-21.78%) suffered from ES. At discharge, 37.64% were disabled, and 19.5% were dead. In multivariate analysis, alcoholism, NIHSS at admission, haemorrhagic stroke and cortical location were significant predictors of ES. Thirty-day mortality was predicted by NIHSS at admission [hazard ratio (HR): 1.14, 95% CI: 1.11-1.18, P < 0.001], history of hypertension (HR: 3.79, 95% CI: 2.1-6.85, P < 0.001), history of alcoholism (HR: 2.43, 95% CI: 1.49-3.95, P < 0.001) and early seizure (HR: 2.58, 95% CI: 1.54-4.34, P = 0.001). CONCLUSIONS: Early seizures occurred in about 18% acute stroke patients. Alcoholism, haemorrhagic stroke, cortical and severe strokes predict development of ES. ES are an independent important risk factor for early mortality.

Testing urine samples with rK39 strip as the simplest non-invasive field diagnosis for visceral leishmaniasis: an early report from eastern India.

BACKGROUND: Diagnosis of visceral leishmaniasis (VL) is a major obstacle in the control of this disease. The rK39 strip-test using patient's blood is a breakthrough; however, it still requires a blood sample, which is a concern for safety in the field. We tried to simplify the test using the patient's urine instead of blood. AIMS: To observe the sensitivity and specificity of the urine test in comparison with the blood test. MATERIALS AND METHODS: We tested active and post-treatment VL patients, Post Kala azar dermal leishmaniasis (PKDL), VL/HIV and control subjects (healthy, disease suspects and diseased other than VL) with the rK39 strip-test using blood and urine samples. STATISTICAL ANALYSIS: The level of agreement between the urine and blood testing was calculated by inter-rater agreement (kappa) statistics. RESULTS: Forty-two active VL, 40 treated VL, six PKDL, three VL/HIV and 139 controls (54 healthy, 21 disease suspects and 64 diseased other than VL) were tested. All VL-related cases showed positive results with urine as well as blood samples (100%). The urine testing was found to have 100% sensitivity and 86.33% specificity for the diagnosis of VL. Kappa statistic between the two methods was 0.916 (P<0.001). Urine testing had more false-positive results in comparison with blood testing (13.67% vs. 9.45%), but the test subjects were from VL-endemic areas and they might be exposed to Leishmania donovani infection. CONCLUSIONS: The present study has the potentiality of providing a new, yet simplest non-invasive screening tool for VL in remote rural areas.

Monitoring of intracellular nitric oxide in leishmaniasis: its applicability in patients with visceral leishmaniasis.

Nitric oxide (NO) has been demonstrated to be a principal effector molecule responsible for mediating intracellular killing of Leishmania parasites, the causative organism of leishmaniasis. As measurement of intracellular NO remains a challenge to biologists, we have developed a flow cytometric approach to perform real time biological detection of NO within Leishmania parasites and parasitized macrophages using a membrane permeable derivative of diaminofluorescein [4,5-diaminofluorescein diacetate (DAF-2DA)]. Initially, assay optimization was performed in Leishmania donovani promastigotes, assay specificity being confirmed using both a NO donor [S-nitroso-N-acetyl-penicillamine (SNAP)] and a NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, C-PTIO]. Using 40 µM DAF-2DA, basal levels of intracellular NO were measured which varied in different Leishmania species; addition of conventional anti-leishmanial drugs, antimony and miltefosine translated into a dramatic increase in DAF-2T fluorescence. Furthermore, the assay also measured levels of NO in macrophages, but needed a 20 fold lower concentration of DAF-2DA, being 2 µM. Following parasitization, levels of NO decreased which was normalized following treatment with anti-leishmanial drugs. Similarly monocytes of patients with visceral leishmaniasis at disease presentation showed decreased levels of NO which too reverted on completion of treatment. Taken together, this study opens new perspectives of research regarding monocyte function and provides a real time approach for monitoring the effect of anti-leishmanial compounds.

Cardiac tamponade in systemic lupus erythematosus.

BACKGROUND: Cardiac tamponade is a rare but life-threatening complication of systemic lupus erythematosus (SLE). AIMS/OBJECTIVES: To describe incidence, risk factors and treatment of cardiac tamponade in a large cohort of Indian patients with SLE. METHODS: This retrospective study was conducted at the Department of Rheumatology, IPGMER, Kolkata, India from May 2014 to December 2016 on admitted patients with SLE. Lupus-related serositis was diagnosed after excluding other causes, such as infection, malignancy or heart failure. RESULTS: Of 409 patients with SLE, pericarditis was diagnosed in 25.4% (104/409) and cardiac tamponade in 5.9% (24/409). Tamponade was the presenting feature of SLE in 50% (12/24). Tamponade occurred in 77.8% (14/18) of large effusions and in 11.63% (10/86) of small-to-moderate effusions. The commonest autoantibody in serum and pericardial fluid was anti-nucleosme antibody. Large pericardial effusion (>20 mm) (Odd's ratio (OR): 93.2, 95% confidence interval (CI): 11.1-782.5, P < 0.001) predicted tamponade. In the subset of patients with small-to-moderate sized pericardial effusion, tamponade was associated with pleuritis (OR: 44.5, 95% CI: 1.6-1243, P = 0.025), anti-nucleosome antibody (OR: 42.9, 95% CI: 1.6-1176, P = 0.026) and size of pericardial effusion (OR: 1.36, 95% CI: 1.04-1.76, P = 0.025). Repeated pericardiocentesis was required in 3 patients and one needed surgical intervention. Immunosuppressives used were: prednisolone with monthly intravenous cyclophosphamide (in 33.33%) and intravenous methylprednisolone with monthly cyclophosphamide (in 50%). CONCLUSIONS: Pleuritis, anti-nucleosome antibody and size of pericardial effusion predicted development of tamponade. High dose immunosuppression (methylprednisolone and IV cyclophosphamide) alleviated need for surgery in majority.

Histoplasmosis in eastern India: the tip of the iceberg?

Systemic histoplasmosis has various clinical presentations and is of especially concern in immunocompromised patients. A high index of suspicion is required for its diagnosis. A total of 38 cases had been reported from India up to 1996. The most frequent occurrence of cases was around Calcutta in eastern India where the previous case was detected 20 years earlier. However, we have diagnosed 5 cases in the past 2 years from eastern India which are reported here. These cases may indicate under-diagnosis and under-reporting of histoplasmosis in India. All 5 patients had disseminated disease with multisystem involvement including 2 with bilateral adrenal enlargement. Two were diabetic and only 1 patient was infected with HIV.

K39 strip test--easy, reliable and cost-effective field diagnosis for visceral leishmaniasis in India.

OBJECTIVE: A firm diagnosis of visceral leishmaniasis (VL) requires demonstration of the parasite in splenic or bone marrow aspirate. The aim of this prospective study was to assess the usefulness of K39 strip test as a noninvasive method of diagnosing visceral leishmaniasis under field conditions by testing serum antibody to the leishmanial antigen K39. MATERIAL AND METHODS: One drop of serum/blood was applied to the sample application pad on the test strip, which was diluted with 2 drops of chase buffer solution. The development of two visible red lines indicates the presence of IgG anti-K39. In the first phase of the study (2001), a total of 200 patients (Active VL-70, ex-VL-30, healthy endemic control-20 and patients with other tropical diseases-80) were tested with the K39 strip test at the School of Tropical Medicine, Kolkata. In the second phase of the study (2002), the test was applied in a remote tribal area of West Bengal where an epidemic of VL had occurred. Thirty-two patients were identified in 207 villagers of the affected area; all of them were tested with the K39 strip test. RESULTS: In the first phase, all VL and ex-VL cases gave positive results (100%). Ten percent of the healthy endemic controls were positive. The test results were negative in all other prevalent tropical diseases (100%). The estimated sensitivity of the test was 100% and the specificity was 98.18%. In the second phase of the study, all 32 patients of the epidemic were shown to be positive. All patients were treated with sodium stibogluconate injections and they recovered uneventfully. CONCLUSIONS: K39 strip test is ideal for rapid reliable field diagnosis of visceral leishmaniasis. The test has high sensitivity and specificity but it remains positive long after treatment (up to 3 years).

Cholelithiasis in a child--an unusual presentation of Wilson's disease.

A nine year old mentally retarded girl with moderate splenomegaly and ascites presented with chronic cholelithiasis. The presence of Kayser-Fleischer rings and low serum ceruloplasmin level confirmed the diagnosis of Wilson's disease. Cirrhosis of liver and recurrent episodes of hemolysis--these two common complications of Wilson's disease make an ideal setting for gall stone formation. Only three such cases have been reported worldwide and ours is the first case report from India. We suggest that cholelithiasis and splenomegaly in a child without evidence of congenital hemolytic disease should be taken as a suspect of Wilson's disease.

Type 3 renal tubular acidosis.

Renal tubular acidosis (RTA) is a group of transport defects in the reabsorption of bicarbonate, the excretion of hydrogen ion (H(+)), or both, resulting in systemic acidosis and hypokalemia with a normal glomerular filtration rate. Although isolated proximal (type 2) or distal (type 1) tubular pathologies are well characterized, a combined pathology leading to type 3 RTA is very rare. Here, we report a case of type 3 RTA, using an algorithmic approach to classify a scenario of hypokalemic metabolic acidosis in the setting of episodic flaccid paralysis.

Diuretic loading test and use of Bartter's Normogram in diagnosing a case of Gitelman's syndrome: Relook into pathophysiology.

Gitelman's syndrome is a rare autosomal recessive, renal tubular disorder, characterized by chronic hypokalemia, hypomagnesemia, metabolic alkalosis, hypocalciuria, and normal blood pressure. Patients usually present at a later age with episodic mild muscle weakness. Unexplained hypokalemia arouses suspicion. The diuretic loading test with furosemide and thiazide and the use of Bartter's normogram provides a practical and simple tool in comparison to the complex and costly genetic analysis, to confirm the diagnosis. Here we report a case of Gitelman's syndrome to show the utility of these simple techniques to explain the pathophysiology of the disease, as well as to localize the site of the renal tubular defect, to confirm the diagnosis.

Immunoglobulin subclass distribution and diagnostic value of Leishmania donovani antigen-specific immunoglobulin G3 in Indian kala-azar patients.

Visceral leishmaniasis, or kala-azar, a fatal tropical disease, remains problematic, as early diagnosis is difficult and treatment often results in drug resistance and relapse. We have developed a sensitive enzyme-linked immunosorbent assay (ELISA), using leishmanial membrane antigenic extracts (LAg) to detect specific antibody responses in 25 untreated Indian visceral leishmaniasis patients. To investigate the pathogenetic significance of isotype markers in kala-azar, relative levels of specific immunoglobulin G (IgG), IgM, IgA, IgE, and IgG subclasses were analyzed under clinically established diseased conditions. Since LAg showed higher sensitivity for specific IgG than lysate, the immunoglobulin isotype responses were evaluated, with LAg as antigen. Compared to 60 controls, which included patients with malaria, tuberculosis, leprosy, and typhoid and healthy subjects, visceral leishmaniasis patients showed significantly higher IgG (100% sensitivity, 85% specificity), IgM (48% sensitivity, 100% specificity), and IgE (44% sensitivity, 98.3% specificity) responses. Low levels of IgA in visceral leishmaniasis patients contrasted with a 13-fold-higher reactivity in sera from patients with leprosy. Among IgG subclasses, IgG1, -3, and -4 responses were significantly higher in visceral leishmaniasis patients than in the controls. IgG2 response, however, was significantly higher (twofold) in leprosy than even visceral leishmaniasis patients. The rank orders for sensitivity (IgG = IgG1 = IgG3 = IgG4 > IgG2 > IgM > IgE > IgA) and specificity (IgM = IgG3 > IgE > IgG4 > IgG2 > IgG > IgG1 > IgA) for LAg-specific antibody responses suggest the potentiality of IgG3 as a diagnostic marker for visceral leishmaniasis.

Distribution of IgG subclasses in antimonial unresponsive Indian kala-azar patients.

Sodium antimony gluconate (SAG) is the mainstay of treatment for visceral leishmaniasis (VL) or kala-azar. In view of the increasing incidence of refractoriness to SAG in India, we compared the levels of parasite-specific IgG and IgG subclasses in 20 longitudinally followed up kala-azar patients. In both SAG-responsive (n = 10) and unresponsive patients (n = 10), the levels of total IgG, IgG1, IgG2, IgG3 and IgG4 were increased, the rank order being IgG1 > IgG2 > IgG3 = IgG4. Following treatment, a significant decrease in total IgG and the four subclasses occurred in the SAG-responsive group, whereas in the SAG-unresponsive group these levels were unchanged or slightly increased. Therefore, monitoring of IgG1 and IgG2 levels in Indian kala-azar patients is a good serologic alternative to monitoring the disease status.

Differential decline in Leishmania membrane antigen-specific immunoglobulin G (IgG), IgM, IgE, and IgG subclass antibodies in Indian kala-azar patients after chemotherapy.

Pathogenesis in kala-azar is associated with depressed cellular immunity and significant elevation of antileishmanial antibodies. Since these antibodies are present even after cure, analysis of the parasite-specific isotypes and immunoglobulin G (IgG) subclasses in kala-azar patients may shed new light on the immune responses during progression and resolution of infection. Using leishmanial membrane antigenic extracts, we investigated the relative levels of specific IgG, IgM, IgA, IgE, and IgG subclasses in Indian kala-azar patient sera during disease, drug resistance, and cure. Acute-phase sera showed strong stimulation of IgG, followed by IgE and IgM and lastly by IgA antibodies. IgG subclass analysis revealed expression of all of the subclasses, with a predominance of IgG1 during disease. Following sodium stibogluconate (SAG) resistance, the levels of IgG, IgM, IgE, and IgG4 remained constant, while there was a decrease in the titers of IgG2 and IgG3. In contrast, a significant (2.2-fold) increase in IgG1 was observed in these individuals. Cure, in both SAG-responsive and unresponsive patients, correlated with a decline in the levels of IgG, IgM, IgE, and all of the IgG subclasses. The stimulation of IgG1 and the persistence, most importantly, of IgE and IgG4 following drug resistance, along with a decline in IgE, IgG4, and IgG1 with cure, demonstrate the potential of these isotypes as possible markers for monitoring effective treatment in kala-azar.