Menberework Chanyalew

Background: Conventional polymerase chain reaction (PCR) assays are well‐established molecular techniques that can be integrated as standard diagnostic tools, especially in referral settings. This study aimed to assess the diagnostic potential of a multiplex PCR (mPCR) assay for the diagnosis of cutaneous leishmaniasis (CL), skin tuberculosis, and leprosy. Method: A cross‐sectional study was carried out involving 62 patients in the study group, comprising 45 with CL, 9 with leprosy, 4 with skin tuberculosis, and 4 with coinfections. Additionally, 112 positive control DNA samples were analyzed, including 37 of M. tuberculosis , 46 of M. leprae , and 29 of L. aethiopica . The study assessed sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and detection limits. Results: Sensitivity and specificity of the mPCR on positive and negative control samples were 100% (95% CI: 96.8%–100%) and 100% (95% CI: 94.9%–100%), respectively. Its sensitivity and specificity among the study group were 75.8% (95% CI: 63.3%–85.8%) and 100% (95% CI: 94.9%–100.0%), respectively. Conclusions: With further validation on more clinical suspects, mPCR has the potential to facilitate diagnosis in settings with coendemic CL, leprosy, and skin tuberculosis.

Epidemiological, histological, and immunogenetic studies suggest that podoconiosis (a non-infectious tropical lymphoedema affecting approximately 4 million people globally) is an HLA class II-associated inflammatory condition that develops in response to an unknown trigger found in volcanic red clay soils. Silicate particles of the kaolinite and aluminum types have been identified in femoral lymph node biopsy samples from endemic area residents, suggesting a possible role in the pathogenesis of podoconiosis. We measured in vitro peripheral blood mononuclear cell cytokine responses (TNF-α, IL-1β, and IFN-γ) to stimulation with the minerals kaolinite, chlorite, and beryllium sulfate (all at 100 µM) using ELISA. Real time PCR was used to measure gene expression of signature cytokines in fresh whole blood, comparing podoconiosis patients and endemic healthy controls. Our results showed that the levels of TNF-α and IL-1β from in vitro cell cultures were significantly higher in unstimulated samples from patients compared to controls (p = 0.04 and p = 0.005, respectively). The minerals kaolinite and chlorite induced two and three-fold higher levels of IL-1β following 24 h of stimulation in healthy controls compared to patients, respectively. We did not find significant differences in mRNA expression of the cytokine genes assayed, though a slight fold increment in IL-1β and TGF-β was observed. In conclusion, our data suggest that the immune system is in a state of persistent activation in vivo in podoconiosis patients, and additional studies of immune regulation and exhaustion are needed to further characterize immune dysfunction in the pathogenesis of the disease. A better understanding of the underlying processes could lead to the development of a ‘biosignature’ detectable in the early reversible stages that could ultimately contribute to the elimination of this preventable, disabling, neglected tropical disease.

Background: Cutaneous leishmaniasis (CL) is an endemic disease in Ethiopia, mainly caused by L. aethiopica. Limited reports are available related to histopathological features of the skin lesion caused by L. aethiopica. This study aimed to analyze the histopathological features of CL due to L. aethiopica.Materials and Methods: A similar cohort polymerase chain reaction (PCR) confirmed CL patients from a previous own study, who were prospectively enrolled from All Africa Leprosy, Tuberculosis and Rehabilitation Training (ALERT) Hospital Addis Ababa, Kela Health Center in Gurage Zone, Siliti Health Center in Silit zone of southern nations and nationalities, as well as Ankober Health Center in Amhara region was used for data analysis. The histopathology was analyzed by performing hematoxylin and eosin (H&E) staining to look for the presence of general and specific histopathology patterns of the disease. Descriptive statistics was utilized using SPSS version 26.0 (SPSS, Inc., Chicago, United States of America).Results: Amastigotes were observed in skin biopsies of 29% (n = 2) mucocutaneous leishmaniasis (MCL) and 58% (n = 6) localized cutaneous leishmaniasis (LCL) patients. Diffused inflammatory cell infiltrate was observed in the dermal compartment of 77% (n = 20) samples while the remaining 23% (n = 6) had patchy or nodular inflammatory cell infiltrate. The dominant type of inflammatory cell infiltrate in the dermal compartments is macrophages and lymphocytes with a similar proportion, 23/26 (88.5%), followed by plasma cells, 21/26 (80.8%). Among all cases, 38.5% (n = 10) of them were categorized under the Type I pattern while Types IV and V patterns were reported in 26.9% (n = 7) and 34.6% (n = 9) of the remaining samples, respectively. The study found statistically significant correlations between necrosis and MCL (p = 0.01), unorganized granulomas and LCL (p = 0.04), and the presence of eosinophils and giant cell Langerhans with MCL (p = 0.002 and p < 0.001, respectively).Conclusion: In our study, the histopathological patterns of the CL caused by L. aethiopica were shown to have a dermal change that was characterized by a domination of diffused inflammatory cell infiltrate. Most of the cell types in the infiltrate were macrophages and lymphocytes. In addition, amastigote resided in the histiocyte with a varying degree of intensity, and both the organized and unorganized granulomas were shown with a considerable proportion.

In cutaneous Leishmaniasis the parasitic control in human host macrophages is still poorly understood. We found an increased expression of the human cathelicidin CAMP in skin lesions of Ethiopian patients with cutaneous leishmaniasis. Vitamin D driven, Cathelicidin-type antimicrobial peptides (CAMP) play an important role in the elimination of invading microorganisms. Recombinant cathelicidin was able to induce cell-death characteristics in Leishmania in a dose dependent manner. Using human primary macrophages, we demonstrated pro-inflammatory macrophages (hMDM1) to express a higher level of human cathelicidin, both on gene and protein level, compared to anti-inflammatory macrophages (hMDM2). Activating the CAMP pathway using Vitamin D in hMDM1 resulted in a cathelicidin-mediated-Leishmania restriction. Finally, a reduction of cathelicidin in hMDM1, using a RNA interference (RNAi) approach, increased Leishmania parasite survival. In all, these data show the human cathelicidin to contribute to the innate immune response against Leishmaniasis in a human primary cell model.

Following successful malaria control during the last decade, Ethiopia instituted a stepwise malaria elimination strategy in selected low-transmission areas. Cross-sectional surveys were conducted in Babile district, Oromia, Ethiopia from July to November 2017 to evaluate malaria infection status using microscopy and nested polymerase chain reaction (nPCR) and serological markers of exposure targeting Plasmodium falciparum and Plasmodium vivax apical membrane antigen-1 (AMA-1). Parasite prevalence was 1.2% (14/1135) and 5.1% (58/1143) for P. falciparum and 0.4% (5/1135) and 3.6% (41/1143) for P. vivax by microscopy and nPCR, respectively. Antibody prevalence was associated with current infection by nPCR for both P. falciparum (pP. vivax (p=0.014) and showed an age-dependent increase (pP. falciparum and 11.5 y prior to sampling for P. vivax, broadly following malaria incidence data from district health offices, with higher antibody titres in adults than children for both species. Malaria transmission declined substantially in the region with continuing heterogeneous but measurable local transmission, arguing in favour of continued and tailored control efforts to accelerate the progress towards elimination efforts.

Low vitamin D (vitD₃) is one of the most common nutritional deficiencies in the world known to be associated with numerous medical conditions including infections such as tuberculosis (TB). In this study, vitD₃ status and its association with the antimicrobial peptide, human cathelicidin (LL-37), was investigated in Ethiopian patients with different clinical forms of TB. Patients with active TB (n = 77) and non-TB controls (n = 78) were enrolled in Ethiopia, while another group of non-TB controls (n = 62) was from Sweden. Active TB included pulmonary TB (n = 32), pleural TB (n = 20), and lymph node TB (n = 25). Concentrations of 25-hydroxyvitamin D₃ (25(OH)D₃) were assessed in plasma, while LL-37 mRNA was measured in peripheral blood and in samples obtained from the site of infection. Median 25(OH)D₃ plasma levels in active TB patients were similar to Ethiopian non-TB controls (38.5 versus 35.0 nmol/L) and vitD₃ deficiency (3 levels compared with Swedish non-TB controls (51.0 nmol/L, high latitude), but vitD₃ status was not affected by tuberculin-positivity or HIV infection. Patients with local lymph node TB had significantly higher 25(OH)D₃ levels compared with pulmonary TB patients (48.0 versus 29.0 nmol/L). Moreover, plasma 25(OH)D₃ levels correlated with local LL-37 expression in granulomatous lesions in TB infected lymph nodes. Instead, systemic LL-37 mRNA expression in blood cells was elevated compared with the site of infection in pulmonary and pleural TB. Low vitD₃ status may be associated with an enhanced peripheral expression of LL-37 in patients with intrathoracic TB that could result from chronic inflammation.

Resistant mouse strains mount a protective T cell-mediated immune response upon infection with Leishmania (L.) parasites. Healing correlates with a T helper (Th) cell-type 1 response characterized by a pronounced IFN-γ production, while susceptibility is associated with an IL-4-dependent Th2-type response. It has been shown that dermal dendritic cells are crucial for inducing protective Th1-mediated immunity. Additionally, there is growing evidence that C-type lectin receptor (CLR)-mediated signaling is involved in directing adaptive immunity against pathogens. However, little is known about the function of the CLR Dectin-1 in modulating Th1- or Th2-type immune responses by DC subsets in leishmaniasis. We characterized the expression of Dectin-1 on CD11c⁺ DCs in peripheral blood, at the site of infection, and skin-draining lymph nodes of L. major-infected C57BL/6 and BALB/c mice and in peripheral blood of patients suffering from cutaneous leishmaniasis (CL). Both mouse strains responded with an expansion of Dectin-1⁺ DCs within the analyzed tissues. In accordance with the experimental model, Dectin-1⁺ DCs expanded as well in the peripheral blood of CL patients. To study the role of Dectin-1⁺ DCs in adaptive immunity against L. major, we analyzed the T cell stimulating potential of bone marrow-derived dendritic cells (BMDCs) in the presence of the Dectin-1 agonist Curdlan. These experiments revealed that Curdlan induces the maturation of BMDCs and the expansion of Leishmania-specific CD4⁺ T cells. Based on these findings, we evaluated the impact of Curdlan/Dectin-1 interactions in experimental leishmaniasis and were able to demonstrate that the presence of Curdlan at the site of infection modulates the course of disease in BALB/c mice: wild-type BALB/c mice treated intradermally with Curdlan developed a protective immune response against L. major whereas Dectin-1^−/− BALB/c mice still developed the fatal course of disease after Curdlan treatment. Furthermore, the vaccination of BALB/c mice with a combination of soluble L. major antigens and Curdlan was able to provide a partial protection from severe leishmaniasis. These findings indicate that the ligation of Dectin-1 on DCs acts as an important checkpoint in adaptive immunity against L. major and should therefore be considered in future whole-organism vaccination strategies.