Real-Time Fluorimetry Loop-Mediated Isothermal Amplification for Diagnosis of Leishmaniasis and as a Tool for Assessment of Cure for Post-Kala-Azar Dermal Leishmaniasis.

Despite the dwindling number of visceral leishmaniasis (VL) cases in India, there is an urgent need for early and unequivocal diagnostics for controlling and preventing the reemergence of VL. Post-kala-azar dermal leishmaniasis (PKDL), a dermal sequela of VL, serves as a reservoir of the parasite. Diagnosis of PKDL, especially the macular variant, is challenging and poses impediment toward attainment of VL elimination. In this study, a real-time fluorimetry loop-mediated isothermal amplification (RealAmp) assay has been established for the detection of different clinical manifestations of leishmaniasis. The study included 150 leishmaniasis patients (25 VL, 25 cutaneous leishmaniasis [CL], and 100-PKDL) along with 120 controls. The assay demonstrated sensitivity of 100% (95% CI: 86.68-100) for diagnosis of VL and PKDL (95% CI: 79.61-100) and 96% (95% CI: 86.68-100) for CL with 100% specificity. Moreover, considering the cardinal role of PKDL, diagnosis using minimally invasive slit aspirate was explored, which demonstrated remarkable sensitivity of 96% (95% CI: 87.64-98.47). As a test of cure for PKDL, RealAmp successfully detected parasite in two of posttreatment cases who later reported relapse on follow-up. Also, direct sample lysis using slit aspirate was attempted in a small group that yielded sensitivity of 89% (95% CI: 67.20-96.90). RealAmp depicted excellent diagnostic accuracy in the diagnosis of leishmaniasis in concordance with the established SYBR Green I-based (Molecular Probes, Eugene, OR) visual loop-mediated isothermal amplification (LAMP) and the reference comparator real-time PCR. The study endorsed the employment of LAMP either as visual-LAMP or RealAmp for an accurate and expeditious diagnosis of PKDL and as a tool for assessment of cure.

Regulatory T cells antagonize proinflammatory response of IL-17 during cutaneous tuberculosis.

BACKGROUND: The clinical forms of cutaneous tuberculosis (CTB) consist of a spectrum that reflects the host's immune response to Mycobacterium tuberculosis; it provides an ideal model to study the immunological dysregulation in humans. IL-17 plays an important role in initial immune response and is involved in both immune-mediated protection and pathology during M. tuberculosis infection. TGF-ß producing regulatory T-cells (Tregs) are high in leprosy patients and responsible for immune suppression. However, in CTB, the involvement of Tregs and Th17 remains unevaluated. OBJECTIVE: To study the role of proinflammatory Th17 and Treg cells in the human CTB. METHODS: Blood and skin biopsies of CTB patients and healthy controls (HC) were included in the study. Flow cytometric analysis of IL-17, FOXP3, and TGF-ß in blood was done followed by immunohistochemistry on paraffin-embedded skin sections. Expression of IFN-γ, TGF-ß, and IL-17 was evaluated by quantitative real-time PCR. RESULTS: We found significant (P<0.0002) lower expression of proinflammatory IL-17 and IFN-γ (P<0.01) in CTB skins as compared to HC. However, the frequency of TGF-ß producing Treg cells was found to be high in CTB patients (P<0.001) as compared to HC. A similar type of profile was observed by flow cytometric analysis. Treg cells produced suppressive cytokine TGF-ß which showed a positive correlation with FOXP3 gene expression. CONCLUSION: Our study found an increase in lineage-specific CD4+ Tregs in CTB as compared to the HC individuals. Such cells secrete TGF-ß, a suppressive cytokine and may play a role in negatively regulating the T-cell immune responses in CTB. In addition, Tregs with TGF-ß may downregulate Th17 cell responses leading to the antigen-specific anergy associated with CTB patients.

How Reliable Is Microscopy and Culture for the Diagnosis of Gonorrhea? An 11-Year Experience from INDIA.

Positivity of microscopy and culture was greater (P < 0.0001) in men with urethral discharge syndrome (65.8%) than in women with vaginal/cervical discharge (0.5%), indicating that basic diagnostic tests may not be cost-effective for diagnosis of vaginal/cervical discharge syndrome. Microscopy when compared with culture showed sensitivity, specificity, positive predictive value and negative predictive value of 95.4%, 77.6%, 84.6%, and 95.3%, in men, whereas in women, it was 77.8%, 99.9%, 92.1%, and 99.9%, respectively.

Indian erythrodermic postkala-azar dermal leishmaniasis.

Postkala-azar dermal leishmaniasis (PKDL) is a complication of kala-azar or visceral leishmaniasis and is caused by Leishmania donovani. We describe an Indian male patient with the rarer erythrodermic form of PKDL and multiple unusual skin lesions viz. verrucous, annular and mucosal ulceration.

Leprosy Reactions Show Increased Th17 Cell Activity and Reduced FOXP3+ Tregs with Concomitant Decrease in TGF-ß and Increase in IL-6.

BACKGROUND: 50% of leprosy patients suffer from episodes of Type 1/ reversal reactions (RR) and Type 2/ Erythema Nodosum Leprosum (ENL) reactions which lead to morbidity and nerve damage. CD4+ subsets of Th17 cells and CD25+FOXP3+ regulatory T cells (Tregs) have been shown to play a major role in disease associated immunopathology and in stable leprosy as reported by us and others. The aim of our study was to analyze their role in leprosy reactions. METHODOLOGY AND PRINCIPLE FINDINGS: Quantitative reverse transcribed PCR (qPCR), flowcytometry and ELISA were used to respectively investigate gene expression, cell phenotypes and supernatant levels of cytokines in antigen stimulated PBMC cultures in patients with stable disease and those undergoing leprosy reactions. Both types of reactions are associated with significant increase of Th17 cells and associated cytokines IL-17A, IL-17F, IL-21, IL-23 and chemokines CCL20, CCL22 as compared to matching stable forms of leprosy. Concurrently patients in reactions show reduction in FOXP3+ Treg cells as well as reduction in TGF-ß and increase in IL-6. Moreover, expression of many T cell markers, cytokines, chemokines and signaling factors were observed to be increased in RR as compared to ENL reaction patients. CONCLUSIONS: Patients with leprosy reactions show an imbalance in Th17 and Treg populations. The reduction in Treg suppressor activity is associated withhigherTh17cell activity. The combined effect of reduced TGF-ß and enhanced IL-6, IL-21 cytokines influence the balance between Th17 or Treg cells in leprosy reactions as reported in the murine models and autoimmune diseases. The increase in Th17 cell associated cytokines may contribute to lesional inflammation.

Increase in TGF-ß secreting CD4⁺CD25⁺ FOXP3⁺ T regulatory cells in anergic lepromatous leprosy patients.

BACKGROUND: Lepromatous leprosy caused by Mycobacterium leprae is associated with antigen specific T cell unresponsiveness/anergy whose underlying mechanisms are not fully defined. We investigated the role of CD25(+)FOXP3(+) regulatory T cells in both skin lesions and M.leprae stimulated PBMC cultures of 28 each of freshly diagnosed patients with borderline tuberculoid (BT) and lepromatous leprosy (LL) as well as 7 healthy household contacts of leprosy patients and 4 normal skin samples. METHODOLOGY/PRINCIPLE FINDINGS: Quantitative reverse transcribed PCR (qPCR), immuno-histochemistry/flowcytometry and ELISA were used respectively for gene expression, phenotype characterization and cytokine levels in PBMC culture supernatants. Both skin lesions as well as in vitro antigen stimulated PBMC showed increased percentage/mean fluorescence intensity of cells and higher gene expression for FOXP3(+), TGF-ß in lepromatous (p<0.01) as compared to tuberculoid leprosy patients. CD4(+)CD25(+)FOXP3(+) T cells (Tregs) were increased in unstimulated basal cultures (p<0.0003) and showed further increase in in vitro antigen but not mitogen (phytohemaglutinin) stimulated PBMC (iTreg) in lepromatous as compared to tuberculoid leprosy patients (p<0.002). iTregs of lepromatous patients showed intracellular TGF-ß which was further confirmed by increase in TGF-ß in culture supernatants (p<0.003). Furthermore, TGF-ß in iTreg cells was associated with phosphorylation of STAT5A. TGF-ß was seen in CD25(+) cells of the CD4(+) but not that of CD8(+) T cell lineage in leprosy patients. iTregs did not show intracellular IFN-γ or IL-17 in lepromatous leprosy patients. CONCLUSIONS/SIGNIFICANCE: Our results indicate that FOXP3(+) iTregs with TGF-ß may down regulate T cell responses leading to the antigen specific anergy associated with lepromatous leprosy.

Application of loop-mediated isothermal amplification assay for the sensitive and rapid diagnosis of visceral leishmaniasis and post-kala-azar dermal leishmaniasis.

Loop-mediated isothermal amplification (LAMP) is at the forefront in the search for innovative diagnostics for rapid and specific amplification of target DNA under isothermal conditions. We have applied LAMP assay using SYBR Green for clear-cut naked eye detection of Leishmania (Leishmania) donovani in 200 clinical samples of visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). The assay was positive in 53/55 VL blood samples (sensitivity, 96.4%; 95% confidence interval [CI], 87.7-99%), 15/15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI, 79.6-100%), 60/62 PKDL tissue biopsy samples (sensitivity, 96.8%; 95% CI, 88.9-99.1%), and 1/68 control samples (specificity, 98.5%; 95% CI, 92.1-99.7%). The assay was specific for L. (L.) donovani, the causative species for VL and negative for L. (L.) infantum, L. (L.) tropica, and L. (L.) major. This is the first comprehensive clinical study demonstrating the applicability of the LAMP assay for a rapid and reliable molecular diagnosis of VL and PKDL.

Reliable diagnosis of post-kala-azar dermal leishmaniasis (PKDL) using slit aspirate specimen to avoid invasive sampling procedures.

OBJECTIVE: Confirmatory diagnosis of post-kala-azar dermal leishmaniasis (PKDL) is primarily based on invasive skin biopsy procedure. We evaluated the utility of minimally invasive slit aspirate specimen for serological and molecular diagnosis of PKDL. We compared the PKDL diagnosis using slit aspirate and skin biopsy specimens from the same patients. METHODS: Serological diagnosis using rK39 strip test was performed with serum and slit aspirate sample; molecular diagnosis for parasite detection and quantification was carried out by quantitative real-time PCR (Q-PCR) with skin biopsy and slit aspirate sample. RESULTS: The rK39 serological strip test was positive in all PKDL cases with both slit aspirate and serum samples (n = 50) and negative in all control cases (n = 24), giving a sensitivity of 100% (95% CI: 92.9-100%) and a specificity of 100% (95% CI: 86.2-100%). Quantitative-PCR detected parasite in all PKDL slit aspirates (n = 50, sensitivity = 100%, 95% CI: 92.9-100%) and tissue biopsies (n = 46, sensitivity = 100%, 95% CI: 92.3-100; it was negative in all controls including dermal tissues (n = 24) and slit aspirates (n = 24), giving specificity of 100% (95% CI: 86.2-100%). The parasite load in tissue and slit aspirate samples was significantly (P < 0.0001) correlated (r = 0.82). CONCLUSIONS: Slit aspirates are a simpler and minimally invasive sampling technique for initial screening by serology followed by confirmatory diagnosis of PKDL with microscopy and/or Q-PCR. The simplified procedure has the potential for epidemiological studies and assessment of cure in PKDL.

Cutaneous tuberculosis in children.

Cutaneous tuberculosis is a rare form of extrapulmonary tuberculosis that accounts for 1% to 2% of cases. Childhood skin tuberculosis represents 18% to 82% of all cutaneous tuberculosis cases. Scrofuloderma and lupus vulgaris are the two most common clinical forms in children. An increase in the number of tuberculids, especially lichen scrofulosorum, has been observed in the last several years. Cutaneous tuberculosis in children can be severe and have a protracted course. Multiplicity of lesions and multifocal disseminated involvement in scrofuloderma and lupus vulgaris is common. Scrofuloderma progressing to gummatous lesions (scrofulous gumma) is mostly described in children. Morbidities and deformities are more severe in children.

Novel and recurrent FERMT1 gene mutations in Kindler syndrome.

Kindler syndrome (OMIM 173650) is an autosomal recessive condition characterized by skin blistering, skin atrophy, photosensitivity, colonic inflammation and mucosal stenosis. Fewer than 100 cases have been described in the literature. First reported in 1954, the molecular basis of Kindler syndrome was elucidated in 2003 with the discovery of FERMT1 (KIND1) loss-of-function mutations in affected individuals. The FERMT1 gene encodes kindlin-1 (also known as fermitin family homologue 1), a 77 kDa protein that localizes at focal adhesions, where it plays an important role in integrin signalling. In the current study, we describe five novel and three recurrent loss-of-function FERMT1 mutations in eight individuals with Kindler syndrome, and provide an overview of genotype-phenotype correlation in this disorder.

Interferon (IFN)-gamma , tumor necrosis factor-alpha , interleukin-6, and IFN-gamma receptor 1 are the major immunological determinants associated with post-kala azar dermal leishmaniasis.

Semiquantitative reverse-transcription polymerase chain reaction was used to analyze intralesional cytokine gene expression in 28 patients with post-kala azar dermal leishmaniasis (PKDL) and 14 patients with kala azar (KA). The data revealed mixed T helper cell type 1 (Th1) and T helper cell type 2 (Th2) responses, as reflected by elevated expression of interferon (IFN)-gamma , tumor necrosis factor (TNF)-alpha , transforming growth factor (TGF)-beta , interleukin (IL)-10, IL-6, and IL-4 mRNA, with minimal expression of IFN-gamma receptor 1 (IFN-gamma R1) mRNA in PKDL lesions, compared with that in normal skin tissue. In comparison with those in KA lesions, mRNA levels for IFN-gamma , TNF-alpha , and IL-6 were found to be significantly elevated in PKDL lesions, implying that these cytokines play an important role in PKDL pathogenesis. In the presence of elevated levels of IFN-gamma and TNF-alpha , interference with type 1 effector activity in PKDL may be due to minimal expression of the IFN-gamma R1 gene or the simultaneous presence of elevated levels of IL-10, IL-6, and TGF-beta , which have counteracting effects. After treatment, the restoration of IFN-gamma R1 at both mRNA and protein levels, coupled with down-regulation of counteracting cytokines, may facilitate the action of signals associated with IFN-gamma , yielding parasite clearance. Therefore, unfavorable clinical evolution in PKDL may not be due to the absence of an intralesional Th1 response but rather may be due to the presence of counteracting cytokines along with the down-modulation of IFN-gamma R1.

Visceral leishmaniasis, or kala azar (KA): high incidence of refractoriness to antimony is contributed by anthroponotic transmission via post-KA dermal leishmaniasis.

Individuals with visceral leishmaniasis, or kala azar (KA) and individuals with post-KA dermal leishmaniasis (PKDL) are considered to be reservoirs of transmission of Leishmania donovani in India. When intracellular amastigotes were used to assess the natural susceptibility that PKDL isolates and KA isolates have to sodium antimony gluconate (SAG), the mean ED(50) was found to be 12.0+/-2.49 and 11.0+/-1.38 microg/mL, respectively; and there was a significant correlation with the clinical response (r rank=0.99). All KA isolates, as well as a significant proportion (55%) of PKDL isolates from high-endemicity zones, were resistant to SAG. The median ED(50) for SAG-resistant PKDL isolates (20.0 microg/mL) was significantly higher (P<.05) than that for SAG-resistant KA isolates (15.7 microg/mL). SAG-resistant PKDL isolates may contribute to KA's increased refractoriness to SAG, via anthroponotic transmission of SAG-resistant strains.

Clinical Utility of LSR/A 15 Gene for Micobacterium leprae Detection in Leprosy Tissues Using the Polymerase Chain Reaction

Resumo: Skin biopsy and slit-skin smears from 46 leprosy patients and 4 nonleprosy patients were tested for the presence of Mycobacterium leprae by the polymerase chain reaction (PCR) using primers based on the sequence of the LSR/15 kD gene. The PCR was found to be specific and sensitive, with a detection level of 10 and 100 bacilli. PCR using skin biopsies gave a higher detection rate than did slit-skin smears, probably due to the higher density of bacilli in a 4-mm punch biopsy. Dot blot hybridization with radioactive probes was 10-fold more sensitive than the ethidium bromide staining. Eight patients who did not show acid-fast bacilli in tissues by the conventional methods were shown to have PCR-amplified M. leprae DNA. False-negative results were obtained in 3 cases even though formal evidence for tissue inhibitors was absent