Bidirectional cytokine-microRNA control: A novel immunoregulatory framework in leishmaniasis.

As effector innate immune cells and as a host to the protozoan parasite Leishmania, macrophages play a dual role in antileishmanial immunoregulation. The 2 key players in this immunoregulation are the macrophage-expressed microRNAs (miRNAs) and the macrophage-secreted cytokines. miRNAs, as small noncoding RNAs, play vital roles in macrophage functions including cytokines and chemokines production. In the reverse direction, Leishmania-regulated cytokines alter miRNAs expression to regulate the antileishmanial functions of macrophages. The miRNA patterns vary with the time and stage of infection. The cytokine-regulated macrophage miRNAs not only help parasite elimination or persistence but also regulate cytokine production from macrophages. Based on these observations, we propose a novel immunoregulatory framework as a scientific rationale for antileishmanial therapy.

Aberrant expression of SOCS impairs the anti-leishmanial immune response.

Establishing a balance between Th1 and Th2 subsets and M1- and M2-type macrophages is essential for the control of Leishmania infection. The suppressors of cytokine secretion (SOCS) proteins, particularly SOCS1 and SOCS3, play a significant role in regulating cytokine-triggered signaling pathways, thereby impacting the macrophage-and effector T-cell mediated antileishmanial immune response. In addition to the pro-inflammatory cytokines, Leishmania-derived lipophosphoglycan (LPG) and CpG-DNA interact with TLR2 and TLR9 to trigger SOCS expression. The aberrant levels of SOCS1 and SOCS3 expression in Leishmania-infected macrophages impair macrophage-T-cell interaction perturbing the balance in macrophage subsets polarization. This hinders macrophage apoptosis and macrophage-mediated leishmanicidal activity, both support the establishment of infection and parasite replication. Furthermore, aberrant SOCS3 levels in T-cells disrupt Th1 differentiation and aid in parasite replication, lesion development, and pathological immune responses. Strategically, selective modulation of SOCS expression and function in immune effector cells may reduce parasite survival and prevent disease progression.

Monitoring of airborne fungi during the second wave of COVID-19 in selected wards of the referral university hospital in southeastern Iran.

Microbiological monitoring of the air hospital is essential for prevention and control, due to the possible airborne route of infection transmission, especially in high-risk wards. This study aimed to monitor the airborne fungi during the second wave of the COVID-19 pandemic in selected wards of the biggest university educational hospital in Kerman, southeastern Iran. This study was conducted in 11 different wards, separated into the patient room and nursing station, of the Afzalipour hospital from May to August 2021. Fungal isolates were characterized to the species level by conventional and sequencing methods. Out of 93 obtained fungal colonies, 70 (75.3%) isolates were filamentous and 23 (24.7%) isolates were yeast. Aspergillus species were the predominant fungal isolates among the filamentous colonies (n=19; 27.1%), and Naganishia albida (formerly Cryptococcus albidus) was identified as the most common yeast isolate (n=13/23; 56.8%). The infectious ward was the most contaminated unit (n=19/93), while the least contaminated units were the neonatal intensive care unit (n=3/93), and oncology (n=3/93). The statistical findings displayed that the number of fungal isolates in patients' rooms is significantly higher than in nurses' stations (p-value=0.013). Our study demonstrated the presence of diverse fungal species in all wards of the hospital. Considering the presence of airborne fungi in hospitals and related public health problems is one of the critical issues for health systems management. In this regard, efficient monitoring of airborne fungi might play an influential role in hospital infection control and surveillance, particularly in high-risk hospitalization patients in critical wards.

The expression of PD-1 and its ligands increases in Leishmania infection and its blockade reduces the parasite burden.

The expression of programmed cell death protein-1 (PD-1) and its ligands- PD-L1 and PD-L2- on T cells and macrophages', respectively, increases in Leishmania infection. The PD-1/PD-L1 interaction induces T cell anergy, T cell apoptosis and exhaustion, diversion of T cells toward TH2 and T-reg cells but inhibits M1 macrophage activities by suppression of nitric oxide (NO) and reactive oxygen species (ROS) production. These changes exacerbate Leishmania infection. As PD-L1-deficient, but not PD-L2-deficient, mice were protected againstL. mexicanainfection, differential roles have been proposed for PD-L1 and PD-L2 in mouse models of leishmaniasis. Blockade of PD-1/PD-L1 interaction in various in vitro and Leishmania-infected mouse, hamster and dog models enhanced IFN-γ and NO production, reduced IL-10 and TGF-ß generation, promoted T cell proliferation and reduced parasite burden. Therefore, PD-1/PD-L1 blockade is being considered as a potential therapeutic strategy to restore protective immunity during leishmaniasis, particularly, in drug-resistant cases.

A novel rapid LAMP test for identification of cutaneous leishmaniasis: An evaluation and comparative analysis of three molecular methods.

Leishmaniasis is a neglected and widespread parasitic disease that can lead to serious health problems. The conventional method in diagnostic health clinics is direct smear preparation of the lesion and staining with standard Giemsa to visualize the amastigote stage and by culturing the organism in an NNN (Novy-MacNeal-Nicolle) to observe the promastigote form of the parasite. In the case of urban-type leishmaniasis, microscopic diagnosis is sometimes not possible due to the reduction of amastigotes in patients' wounds. Because most endemic areas are located in regions that do not have access to laboratories equipped with molecular tools, access to a rapid test to diagnose the disease is essential. In this study, for the first time for DNA extraction, the scalpel used for sampling was washed and extracted by boiling method. Also, the LAMP technique in this study was modified so that the test can be performed in 10 min and the results can be recognized by color. We used four microscopic methods, conventional PCR, real-time PCR, and LAMP, to diagnose urban-type leishmaniasis and compared the results of these methods with each other. The sensitivity and specificity of LAMP were higher than other techniques used. Therefore, it allows rapid diagnosis for timely treatment of the disease to control the primary reservoir host more quickly in ACL as humans are the principal source of infection. This test is performed at a high-speed and is cost-effective. For its convenience, this test is highly recommended to be used in endemic areas.

Global distribution of treatment resistance gene markers for leishmaniasis.

BACKGROUND: Pentavalent antimonials (Sb(V)) such as meglumine antimoniate (Glucantime®) and sodium stibogluconate (Pentostam®) are used as first-line treatments for leishmaniasis, either alone or in combination with second-line drugs such as amphotericin B (Amp B), miltefosine (MIL), methotrexate (MTX), or cryotherapy. Therapeutic aspects of these drugs are now challenged because of clinical resistance worldwide. METHODS: We reviewedthe recent original studies were assessed by searching in electronic databases such as Scopus, Pubmed, Embase, and Web of Science. RESULTS: Studies on molecular biomarkers involved in drug resistance are essential for monitoring the disease. We reviewed genes and mechanisms of resistance to leishmaniasis, and the geographical distribution of these biomarkers in each country has also been thoroughly investigated. CONCLUSION: Due to the emergence of resistant genes mainly in anthroponotic Leishmania species such as L. donovani and L. tropica, as the causative agents of ACL and AVL, respectively, selection of an appropriate treatment modality is essential. Physicians should be aware of the presence of such resistance for the selection of proper treatment modalities in endemic countries.

Vulvovaginal candidiasis in Iranian women: Molecular identification and antifungal susceptibility pattern.

AIM: Vulvovaginal candidiasis (VVC), is a common fungal infection that remains a global concern. The objectives of this study were molecular identification and assessment of the antifungal susceptibility profile of Candida species, causing VVC in southeast Iran. METHODS: A cross-sectional investigation was carried out on 119 nonpregnant females suspected of VVC between February 2019 and May 2021. Yeast samples were characterized to the species level by conventional and molecular methods. All Candida isolates were examined for in vitro susceptibility profile to six conventional antifungal drugs using Clinical and Laboratory Standards Institute guidelines. RESULTS: Out of 119 subjects, 52 (43.7%) cases were affected by VVC, out of whom 11 (21.15%) cases had recurrent vulvovaginal candidiasis (RVVC). The species distribution was as follows; Candida albicans (n = 21; 40.4%), C. glabrata (n = 11; 21.2%), C. tropicalis (n = 9; 17.3%), C. parapsilosis (n = 5; 9.7%), C. africana (n = 3; 5.7%), C. famata (n = 1; 1.9%), C. lusitaniae (n = 1; 1.9%), and C. dubliniensis (n = 1; 1.9%). The resistance rate of Candida isolates to fluconazole, itraconazole, and voriconazole were 15.38%, 11.5%, and 3.8%, respectively. Resistance to fluconazole was obtained in 46% (5/11) of RVVC cases but only in 7% (3/41) of VVC cases. CONCLUSION: This study demonstrated that the majority of VVC cases were caused by non-albicans Candida species which also were resistant to some antifungal agents. Hence, our findings revealed the importance of conducting periodical epidemiological studies to determine changes in species distribution. Moreover, for effective management of treatment and infection, it is imperative to evaluate the susceptibility profiles of Candida species isolated from VVC patients.

The importance of T cell-derived cytokines in post-kala-azar dermal leishmaniasis.

Infection with the same species of Leishmania (L)donovani causes different manifestations including visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL), indicating that the host-related immunological parameters perform a decisive role in the pathogenesis of diseases. As PKDL is a reservoir of the parasite, a better understanding of the host immune responses is necessary to restrict the L. donovani transmission. The proper local production of Th1 cell-related cytokines (including IFN-γ, TNF-α and IL-12), Th17 cell-derived cytokines (such as IL-17A, IL-17F and IL-22), and CD8+ cytotoxic T lymphocyte (CTL)-derived IFN-γ are protective against PKDL. However, dominant production of regulatory CD4+ T cell-derived cytokines (such as IL-10 and TGF-ß), Th2 cell-derived cytokines (such as IL-4/IL-13), M2 macrophage-derived cytokines (such as IL-4 and IL-10), keratinocyte-derived IL-10, regulatory CD8+ T cell-derived IL-10, and dendritic cell-derived IL-10, IL-27 and IL-21 can contribute to the parasite persistence and PKDL development. Understanding of the T cell-related cytokine network within PKDL lesions gives rise to novel insights concerning the role of each cytokine in the protection or susceptibility to disease. Manipulation of the cytokine network can be considered as an interesting immunotherapeutic strategy for the treatment of L. donovani-mediated PKDL.

The immunity of the recombinant prokaryotic and eukaryotic subunit vaccines against cutaneous leishmaniasis.

Leishmaniasis counts as one of the most neglected tropical diseases. Despite the amount of research perceived in this field, no fully effective and approved vaccine against this disease is yet available in humans. In this study, LACK and KMP11 antigens were constructed simultaneously by recombinant methods in prokaryotic and eukaryotic expression systems and were compared and assessed along with the CpG adjuvant in BALB/c mice. In the prokaryotic method, LACK and KMP11 protein gene sequences were synthesized in pET28a-TEV vector. In order to extract these two proteins after expression, His-tag and S-tag sequences were added to the constructs, respectively for LACK and KMP11. The pET28a-TEV-LACK/KMP11 construct was transformed into Escherichia coli, and the inserts were verified by Colony PCR. Pure proteins were verified by western blot, and groups of BALB/c mice were injected with the created prokaryotic recombinant proteins along with an ODN CpG adjuvant. In the eukaryotic method, antigen sequences were constructed in the pLEXSY-neo 2.1 vector, E.coli Top10 strain was cloned in the bacteria, and after being linearized were transfected into Leishmania tarentolae genome. After recombinant strains were selected, they were verified by molecular methods. After the extraction and purification of the protein using the method above, groups of mice were injected with the recombinant antigens and ODN CpG adjuvant. Eukaryotic subunit vaccines showed more effective immunization compared with prokaryotic vaccines and caused an immune system shift towards Th1 and protection. Protein expression in L. tarentolae by the constructs created in this host contains Post-Translational Modifications. The constructed protein will be significantly similar to eukaryotic proteins, considering that they are identical epitopes. More comprehensive studies aiming to improve the effectiveness of this vaccine are being conducted to improve immune profiles and immunological memory stimulation in future designs.

Seroepidemiology of visceral leishmaniasis among free-roaming dogs and children in Zahedan city, southeast of Iran, 2018-2020.

BACKGROUND: Mediterranean visceral leishmaniasis (VL) or kala-azar is an endemic zoonotic disease in Iran. Domestic dogs are the primary reservoir host and source of VL infection. The high-risk populations are children and immune-deficient adults. OBJECTIVE: Based on the lack of published reports about the VL in Sistan and Baluchestan province in the southeast of Iran, this study aimed to assess the seroprevalence of diseae in free-roaming dogs and children under 12 years old using indirect fluorescent antibody (IFA) test. METHODS: This cross-sectional study was performed between 2018 and 2020 in Zahedan city, Sistan, and Baluchestan province. Blood samples were taken from 400 children under 12 years old with a fever history accompanied by at least another specific clinical presentation. In the same period, blood samples were collected from 150 stray dogs. Demographic characteristics and clinical manifestations in both humans and dogs were recorded. The IFA test examined all blood samples for the detection of anti-Leishmania infantum antibodies. RESULTS: Overall, the IFA test results were positive in 8 dogs (5.33%). Only two seropositive dogs (25%) showed obvious clinical symptoms. There was a significant correlation between the positive cases, clinical signs (P = 0.046), and age (P = 0.037) in infected dogs. None of the collected sera from 400 febrile children were positive. CONCLUSION: According to the present finding, it seems that VL is not endemic in Zahedan city, Sistan, and Baluchestan province, but the domestic cycle of L. infantum has been established in this area. Further investigations would be needed to estimate the status of VL infection in wild canines as a secondary potential reservoir host. Furthermore, periodic monitoring of disease must not be neglected.

Dirofilariosis caused by Dirofilaria immitis in the south of Kerman province, Iran.

BACKGROUND: Dirofilariosis due to Dirofilaria immitis is endemic in various areas of Iran. Domestic dogs are the main reservoirs and represent a major potential infection source for the vector and humans. OBJECTIVE: This study aimed to explore the prevalence of dirofilariosis due to D.immitis and its public health importance in domestic dogs in the Jiroft district, south of Kerman province, Iran, by serological and parasitological methods. METHODS: This descriptive study was carried out as a cross-sectional investigation. A questionnaire was completed for 100 domestic dogs from May 2017 to February 2018 and recorded their age, sex, and clinical features. Also, we used enzyme-linked immunosorbent assay (ELISA) to identify antigens of heartworms in the bloodstream, with 98% sensitivity and 100% specificity, and parasitological techniques (Knott's test) to detect microfilariae in canine blood in Jiroft district, south of Kerman province, Iran. RESULTS: Overall, 10 (10%) and 4 (4%) domestic dogs were infected as confirmed by ELISA and modified Knott's tests, respectively. The rate of occult infections in the ELISA test than Knott's test was 60%. No significant difference was found between dirofilariosis and gender. In contrast, there was a significant difference between dirofilariosis infection and age (P < 0.05). CONCLUSION: The present findings could help understand the epidemiological aspects of D. immitis for future control programs and take appropriate preventive and therapeutic strategies against the disease.

Linguatulosis in small ruminants in southeastern Iran: Epidemiological, histopathological and phylogenetic findings and its public health importance.

Linguatulosis, as a zoonotic disease, can infect most ruminants and cause accidental infections in humans. The objective of this study was to explore the epidemiological, histopathological and phylogenetic profiles of Linguatula serrata infection in sheep and goats and its public health importance during 2015-2018. Mesenteric lymph nodes (MLNs) and liver tissue of goats and sheep were selected randomly in Kerman slaughterhouse. Nymphal samples were used for DNA extraction, amplification and subsequently phylogenetic analysis using 18s rRNA and cytochrome C oxidase subunit 1 (cox1). Overall, of 828 examined livestock, 179 (42.4%) goats and 71 (17.5%) sheep were found to be infected with the nymphal stage of L. serrata. A significant difference was observed between linguatulosis and age. In the histopathological assessment, longitudinal and transverse sections of L. serrata nymphs were observed within the cyst-like spaces surrounded by a wall of fine fibrosis and compact lymphocytes. Moreover, comparing with the L. serrata reference sequences, we found only a single nucleotide change in our goat haplotype in 18s genetic region; while much nucleotide variations were observed in cox1 gene sequences. The results of the present study showed a high infection rate among goats and sheep in southeastern Iran. A better understanding of the disease could be achieved when the parasite species, their molecular characterization and the extent of infection in the area are determined. It is fundamental to select a comprehensive control program in order to take proper preventive and therapeutic measures against the infection.

Immunological role of keratinocytes in leishmaniasis.

Following inoculation of Leishmania, a protozoan parasite, into the skin of a mammal, the epidermal keratinocytes recognize the parasite and influence the local immune response that can give rise to different outcomes of leishmaniasis. The early keratinocyte-derived cytokines and keratinocytes-T cells interactions shape the anti-leishmanial immune responses that contribute to the resistance or susceptibility to leishmaniasis. The keratinocyte-derived cytokines can directly potentiate the leishmanicidal activity of monocytes and macrophages. As keratinocytes express MHC-II and enhance the expression of costimulatory molecules, these cells act as antigen-presenting cells (APCs) in cutaneous leishmaniasis (CL). Depending on the epidermal microenvironment, the keratinocytes induce various types of effector CD4+ T cells. Keratinocyte apoptosis and necrosis have been also implicated in ulceration in CL. Further, keratinocytes contribute to the healing of Leishmania-related cutaneous wounds. However, keratinocyte-derived IL-10 may play a key role in the development of post-kala-azar dermal leishmaniasis (PKDL). In this review, a comprehensive discussion regarding the multiple roles played by keratinocytes during leishmaniasis was provided, while highlighting novel insights concerning the immunological and pathological roles of these cells.

The potential therapeutic role of PTR1 gene in non-healing anthroponotic cutaneous leishmaniasis due to Leishmania tropica.

BACKGROUND: Drug resistance is a common phenomenon frequently observed in countries where leishmaniasis is endemic. Due to the production of the pteridine reductase enzyme (PTR1), drugs lose their efficacy, and consequently, the patient becomes unresponsive to treatment. This study aimed to compare the in vitro effect of meglumine antimoniate (MA) on non- healing Leishmania tropica isolates and on MA transfected non-healing one to PTR1. METHODS: Two non-healing and one healing isolates of L. tropica were collected from patients who received two courses or one cycle of intralesional MA along with biweekly liquid nitrogen cryotherapy or systemic treatment alone, respectively. After confirmation of L. tropica isolates by polymerase chain reaction (PCR), the recombinant plasmid pcDNA-rPTR (antisense) was transfected via electroporation and cultured on M199. Isolates in form of promastigotes were treated with different concentrations of MA and read using an enzyme-linked immunosorbent assay (ELISA) reader and the half inhibitory concentration (IC50 ) value was calculated. The amastigotes were grown in mouse macrophages and were similarly treated with various concentrations of MA. The culture glass slides were stained, and the mean number of intramacrophage amastigotes and infected macrophages were assessed in triplicate for both stages. RESULTS: All three transfected isolates displayed a reduction in optical density compared with the promastigotes in respective isolates, although there was no significant difference between non-healing and healing isolates. In contrast, in the clinical form (amastigotes), there was a significant difference between non-healing and healing isolates (p < 0.05). CONCLUSION: The results indicated that the PTR1 gene reduced the efficacy of the drug, and its inhibition by antisense and could improve the treatment of non-healing cases. These findings have future implications in the prophylactic and therapeutic modality of non- healing Leishmania isolates to drug.

The impact of diabetes on cutaneous leishmaniasis: a case-control field assessment.

This study aimed to assess the associated-risk determinants for cutaneous leishmaniasis (CL) in patients with diabetes mellitus (DM) compared to patients without DM. This case-control study was performed between 2017 and 2019 in southeastern Iran. Overall, 206 participants were selected from patients with DM without CL (11.2%), patients with CL without DM (6.2%), and DM patients concomitance with CL (27.6%) as case groups and healthy individuals as a control group 64 (76%). These cases were compared for parasitological, immunological, biochemical, and hematological parameters. The findings demonstrated that parasitological factors regarding the number, duration, and size of the lesion in CL patients showed a significant difference among patients with and without DM (p < 0.05). Data analysis showed that six major risk factors, including female (odds ratio (OR) = 3.47, confidence interval (CI) = 1.84-6.53, p < 0.001), total protein in CL group (OR = 4.9, CI = 2.3-10.44, p < 0.001), alanine aminotransferase (ALT) concentration in CL group (OR = 0.87, CI = 0.81-0.93, p < 0.001) and DM co-infected with CL group (OR = 0.8, CI = 0.72-0.88, p < 0.001) than healthy group, aspartate aminotransferase (AST) concentration in DM group (OR = 0.86, CI = 0.76-0.98, p = 0.02), transforming growth factor beta)TGF-ß( level in the CL group (OR = 1.03, CI = 1.003-1.05, p = 0.02), and presence of diabetes disease (OR = 2.07, CI = 1.16-3.7, p < 0.05), were significantly linked with the induction of CL lesion. The findings demonstrated a significant relationship between DM and CL in distinct risk determinants. Also, the study revealed that DM enhanced the severity of active CL.

Molecular characterization of ocular dirofilariasis: a case report of Dirofilaria immitis in south-eastern Iran.

BACKGROUND: Dirofilariasis is a zoonotic parasitic infection transmitted from animals to humans by culicid mosquitoes. Although the disease can be caused by Dirofilaria spp. including Dirofilaria immitis and Dirofilaria repens, human ocular dirofilariasis due to D. immitis is relatively rare in the world. This study was aimed to present a case of ocular dirofilariasis caused by D. immitis in southeastern Iran. CASE PRESENTATION: A nematode extracted from the right eye of a 69-year-old man referred with clinical symptoms including itching and redness was examined. After the morphometric analysis, Dirofilaria parasite was detected. Afterwards, a piece of worm body was cut and DNA was extracted and a 680-bp gene fragment amplification and nucleotide sequencing were performed. Phylogenetic analysis revealed a D. immitis roundworm as the causative agent of infection. The patient was treated with antibiotics and corticosteroid and followed up for 1 month. CONCLUSION: The present study provides the second report on ocular dirofilariasis caused by D. immitis isolated from a human in southeast Iran. Based on the available evidence, dirofilariasis in dogs has significantly increased in endemic areas such as Iran. Therefore, physicians should be aware of such zoonotic nematodes so as to take proper and timely action and treatment against the disease.

Leishmania species-dependent functional duality of toll-like receptor 2.

Toll-like receptors (TLRs) are a subset of pattern recognition receptors (PRR) in innate immunity and act as a connecting link between innate and adaptive immune systems. During Leishmania infection, the activation of TLRs influences the pathogen-specific immune responses, which may play a decisive role in determining the outcome of infection, toward elimination or survival of the pathogen. Antigen-presenting cells (APCs) of the innate immune system such as macrophages, dendritic cells (DCs), neutrophils, natural killer (NK) cells, and NKT cells express TLR2, which plays a crucial role in the parasite recognition and elicitation of immune responses in Leishmania infection. Depending on the infecting Leishmania species, the TLR2 pathways may result in a host-protective or a disease-exacerbating response. While Leishmania major and Leishmania donovani infections trigger TLR2-related host-protective and non-protective immune responses, Leishmania mexicana and Leishmania infantum infections are reported to elicit TLR2-mediated host-protective responses and Leishmania amazonensis and Leishmania braziliensis infections are reported to evoke a disease-exacerbating response. These findings illustrate that TLR2-related effector functions are diverse and may be exerted in a species- or strain-dependent manner. TLR2 agonists or antagonists may have therapeutic potentials to trigger the desired immune response during leishmaniasis. In this review, we discuss the TLR2-related immune responses during leishmaniasis and highlight the novel insights into the possible role of TLR2-driven resistance or susceptibility to Leishmania.

Detection of malaria with light microscopy and Nested polymerase chain reaction (Nested PCR) methods in peripheral blood expansions and investigation of the genetic diversity of Plasmodium species by 18S rRNA gene in Southeast of Iran.

BACKGROUND: Malaria is a public health concern that leads to about a million deaths worldwide every year. Malaria is caused by the genus Plasmodium, which includes P. falciparum, P. vivax, P. malariae, and P. ovale. Molecular phylogeny is essential to better recognition the evolution of the genus Plasmodium genus and detection of the relative degree of Plasmodium species in humans. The aim of this study was to detect malaria with Light Microscopy (LM) and Nested polymerase chain reaction (Nested PCR) methods in peripheral blood expansions and to investigate the genetic diversity of Plasmodium species by 18S rRNA gene in the southeast of Iran. METHODS: A total of 97 blood smears were collected from patients suspected to malaria in a 6-year period in the southeast of Iran including Hormozgan, Kerman, and Sistan and Baluchestan provinces. Diagnosis of Plasmodium species on blood smears was performed using LM and Nested PCR methods. In addition, 16 Plasmodium-positive samples were chosen for the determination of genetic diversity. RESULTS: Overall, 97 of 97 (100%) studied cases were positive by LM while 94 of 97 (96.8%) of them were detected as malaria by Nested PCR. Except for seven cases, Nested PCR confirmed the LM results. These samples involved two P. vivax and five P. falciparum in the LM method. Meanwhile, the Nested PCR was detected in all of the cases as a mixed infection with P. vivax and P. falciparum. The results of the phylogenetic analysis revealed two main clades and five different subclades. About 87.5% of the isolates were located in clade I and contained P. vivax. In addition, 12.5% of the studied isolates involved P. falciparum that was in clade II. CONCLUSION: According to our results, Nested PCR method had higher sensitivity than LM and is suggested as a good approach for malaria detection. Consideration the wide diversity of tested isolates and the importance of vaccine development, which is affected by this diversity, further studies are needed in this regard.

Differential expression of TLRs 2, 4, 9, iNOS and TNF-α and arginase activity in peripheral blood monocytes from glucantime unresponsive and responsive patients with anthroponotic cutaneous leishmaniasis caused by Leishmania tropica.

BACKGROUND: Detection of the mechanism of host/parasite interactions in unresponsive forms of anthroponotic cutaneous leishmaniasis (ACL) caused by Leishmania tropica is helpful for immunotherapy and vaccine development. In the present study, the gene expression of toll-like receptors (TLRs), TNF-α, iNOS and also arginase (ARG) activity in monocytes from Glucantime unresponsive in comparison to responsive patients infected with L. tropica was investigated. METHODS: In this case-control study, patients with unresponsive (n = 10) and responsive (n = 10) ACL were recruited. Gene expression of TLR2, TLR4, TLR9, TNF-α and iNOS was analyzed in L. tropica-exposed monocytes. The level of ARG activity in both isolated promastigotes and the lysates of monocytes was also determined. RESULTS: L. tropica-exposed monocytes represented higher expression of all three TLRs and TNF-α and lower expression of iNOS compared to unexposed ones in both groups of patients. Results revealed a significant down-regulation of TLR2 and TNF-α and up-regulation of TLR9 expression in unresponsive isolates in comparison to responsive ones. Besides, ARG level showed a significant increase in L. tropica-stimulated monocytes and cultured promastigotes from unresponsive isolates versus responsive ones. CONCLUSIONS: The decreased TLR2, TLR4, TNF-α and iNOS and the increased level of TLR9 expression in L. tropica-exposed monocytes from unresponsive isolates and also the increment in ARG activity in their promastigotes and monocytes, might possibly be involved in the severity of the disease and leading to Glucantime unresponsiveness.

A single-group trial of end-stage patients with anthroponotic cutaneous leishmaniasis: Levamisole in combination with Glucantime in field and laboratory models.

Currently, there is no satisfactory treatment modality available for cutaneous leishmaniasis (CL). The major objective of the present study was to explore the effect of immunomodulator-levamisole in combination with Glucantime in end-stage unresponsive patients with anthroponotic CL (ACL). Twenty end-stage unresponsive patients with ACL were identified for participation in this single-group trial study. Simultaneously, each patient was received a combination of levamisole pills along with Glucantime during the remedy course. Several in vitro complementary experiments were performed to evaluate the mode of action of levamisole and Glucantime alone and in combination using a macrophage model, in vitro MTT assay, flow cytometry and quantitative real time PCR (qPCR). Overall, 75% of the patients showed complete clinical cure, 10% partially improved and the remaining (15%) had underlying chronic diseases demonstrated no response to the treatment regimen. In in vitro studies, there was no cytotoxic effect associated with these drugs in the range of our experiments. The findings by the flow cytometric analysis represented that the highest apoptotic values corresponded to the drugs combination (32.23%) at 200 µg/ml concentration. Finally, the gene expression level of IL-12 p40, iNOS and TNF-α promoted while the level of IL-10 and TGF-ß genes reduced as anticipated. The findings clearly indicated that the combination of levamisole and Glucantime should be considered in end-stage unresponsive patients with ACL who have not responded to basic treatments. The immunomodulatory role of levamisole in mounting immune system as documented by the in vitro experiments and further substantiated by this single-group trail study was highlighted.