Evaluation of Multiplex/Nested Polymerase Chain Reaction and Loop-Mediated Isothermal Amplification for Malaria Diagnosis in Southeastern Iran.

Malaria is one of the most serious health problems in many countries, including Iran. Accurate diagnosis is important regardless of the elimination status of a country. A cross-sectional study was performed on 105 people who were suspected to be positive for malaria infection in Sistan and Baluchistan, Iran. Blood smears (thin and thick films) were stained with 10% Giemsa. DNA was extracted from the prepared thin and thick films for molecular methods. Multiplex/nested polymerase chain reaction (mn-PCR), loop-mediated isothermal amplification (LAMP), and light microscopy (LM) were compared with nested PCR (nPCR) as a gold standard. Of 105 subjects, 52 (49.5%), 58 (55.2%), 58 (55.2%), and 63 (60%) were positive for malaria by LM, nPCR, mn-PCR, and LAMP, respectively. The sensitivity, specificity, and kappa were 92.1%, 100%, and 0.9 for LAMP and 100%, 100%, and 1 for mn-PCR, respectively. Eight cases of coinfection (Plasmodium vivax and Plasmodium falciparum) that were not detected by LM method were diagnosed by mn-PCR and LAMP. In the present study, the high sensitivity and specificity of LAMP and mn-PCR indicate that these two tests are good alternatives to nPCR for malaria diagnosis.

Prevalence of camel babesiosis in southeast of Iran.

Babesiosis is a globally distributed zoonotic parasitic disease in a broad range of vertebrates with great importance in the veterinary field. The standard diagnostic test for Babesiosis in animals is microscopic identification of the parasite in a venous blood smear stained with Giemsa combined with assessment of clinical manifestations throughout the acute phase of the disease. The present study was planned to determine the presence of Babesia species in camels from the southeastern regions of Iran. A total of 140 blood samples of camels were randomly collected in four selected cities including Qaen, Nehbandan, Iranshahr, and Zahedan from March to August 2019. Blood smears of each case were also examined by the Giemsa staining method and extracted DNA samples were subjected to internal transcribed spacers (ITS1) polymerase chain reaction (PCR) amplification. The prevalence rates using microscopically and molecular examinations were 10% and 19.28%, respectively. The prevalence rates significantly vary between the selected regions (p = 0.003). PCR technique showed higher sensitivity than microscopy. We found that all infected camels were positive for Babesia caballi. The rate of infection with Babesia among the camel in Zahedan is remarkable. Early diagnosis and early treatment can prevent further spread of the disease in this area.

Investigation of LAMP Technique in Diagnosis Type of Plasmodium Species in Anopheles Mosquitoes :A Fast and Practical Technique to Detect Malaria Pathogens in the Field.

BACKGROUND: Malaria is one of the main parasitic diseases and a major health issue in some countries. This study aims to determine the rate and type of infections of Anopheles mosquitoes with malaria parasites using the molecular LAMP method in the Southeastern Iran. METHODS: In this study, 400 Anopheles mosquitoes were collected by the Zahedan Medical Insecticide Center in Nikshahr City, a high-risk area of malaria transmission in Sistan-Baluchestan Province. The mosquitoes were caught manually (by hand) in domestic (humans and animals), natural, and artificial outdoor places (Shelter pits). After DNA extraction, the LAMP method was used, which was compared with Multiplex Nested-PCR as a standard method. RESULTS: Out of 400 samples collected from Nikshahr City, 6 samples (1.5%) were infected with Plasmodium vivax. No Plasmodium falciparum or a mix (Plasmodium vivax and Plasmodium falciparum) was detected in this study. CONCLUSIONS: The results of this study indicate that in places with transmission of both species, i.e. Plasmodium vivax and Plasmodium falciparum, detection of malaria parasites by the LAMP method could be very useful in spotting infections in the field. Thus, molecular epidemiological studies could be conducted annually to monitor malaria in endemic regions. The results of this research show that contamination with mosquito malaria vectors is increasing in Nikshahr City, and it seems that more studies will be required to eliminate malaria until 2026.

Nanoliposome-loaded antifungal drugs for dermal administration: A review.

Cutaneous fungal infections are the fourth most common health problem, which involves approximately one billion people worldwide. Drug delivery to the skin seems to be the best choice for superficial fungal infections. Topical formulations can release a sufficient amount of drug in therapeutical concentrations and permeate higher layers of the skin like the stratum corneum. As the outermost layer of the epidermis, the stratum corneum prevents the drug from penetrating the skin. Liposomes, especially nanosized as topical drug delivery systems to the skin, can show various functions depending on their size, lipids and cholesterol components, the percent of ingredients, lamellarity, and surface charge. Nanoliposomes can increase permeation through the stratum corneum, decrease systemic effects with their localizing actions, and overcome many dermal drug delivery obstacles. Antifungal drugs, such as croconazole, econazole, fluconazole, ketoconazole, terbinafine hydrochloride, tolnaftate, and miconazole entrapped in liposomes have indicated improved skin penetration and localizing effects. According to the literature review summarized in this paper, many studies have identified liposomes as a powerful carrier for topical antifungal drug delivery to the skin. However, a few studies introduced new generations of liposomes like ethosomes and transfersomes. This paper was conducted on almost all liposomal studies of antifungal drugs with dermal application.

Sequence Analysis of Pvama-1 among Plasmodium Vivax Isolates in Sistan-Baluchistan.

BACKGROUND: Apical Membrane antigen 1 (AMA-1) is an important membrane protein that presents in all Plasmodium species and participates in critical phases in the attraction of cells. In human, it is one of the most immunodominant antigens with a protective immune response simulation role Apical Membrane antigen 1 (AMA-1) is an important membrane protein which presents in all Plasmodium species and is located on the surface of merozoite and sporozoites that participates in critical phases in attraction of human red blood cells by merozoites and hepatocytes by sporozoites, so in human, it is one of the most immunodominant antigens with a protective immune response simulation role. Since extra information is necessary to lighten of AMA-1 scope, we equaled genetic variation in P.vivax AMA-1 from 40 Iranian isolates with those reported from the other malarious countries. METHODS: Blood samples were collected from 40 patients' positive of P.vivax, and genomic DNA was extracted from the blood. The nucleotide sequence for 446 amino acid (AA) residues (42-488 of PvAMA-1) of AMA-1 gene was amplified via PCR and then sequenced. RESULT: A total of 24 different haplotypes were recognized between samples. No new haplotype was determined in this research that was reported previously in other regions of Iran and the world. We detected 37-point mutations at the nucleotide level in their sequences and showed 43 amino acid variations, at 37 positions in which 6 sites demonstrate trimorphic polymorphism, and the others were dimorphic. CONCLUSION: Sequence analysis of the major haplotype showed 95% similarity with P.vivax Sal-1 AMA-1 gene and high level of allelic diversity at the domain I of PvAMA-1 among P. vivax isolates of Iran. Because PvAMA-1 is noticeable as vaccine candidate antigen, these documents provide valuable information for the development of malaria vaccine.

Immunogenicity and protection effects of cationic liposome containing imiquimod adjuvant on leishmaniasis in BALB/c mice.

OBJECTIVES: Protection against leishmaniasis, in the murine model, is dependent on developing a potent CD4+ mediated Th1 type response. Liposomes can be applied as immunoadjuvants to stimulate immune responses to different antigens. In the present study, it was investigated whether DOTAP liposomes having SLA and imiquimod adjuvant, can induce a Th1 response and protect against Leishmania major challenge in BALB/c mice. MATERIALS AND METHODS: Liposomes were provided applying the lipid film procedure. BALB/C mice were subcutaneously immunized, three times with 2-week intervals, with various formulations. Assessment of lesion development and parasite burden in the foot and spleen after challenge with L. major, assessment of Th1 cytokine (IFN-γ), and titration of IgG isotypes assessed the type of generated immune reaction and the protection extent. RESULTS: The mice immunized with Liposome DOTAP+imiquimod+SLA showed smaller footpad swelling which was meaningfully different (P<0.05) compared with other groups. The highest level of IgG2a was observed with Lip DOTAP+imiquimod+SLA more than the control (P<0.001). Mice immunized with Lip DOTAP+SLA+imiquimod demonstrated the least number of live parasites in the footpad and spleen. Cytokine assay showed that the greatest IFN- γ secretion was seen in the splenocytes of mice immunized with all formulations as compared to the control group (P<0.0001). In contrast, the lowest IL-4 production was detectable in Lip+imiquimod+SLA spleen, which was not significantly different compared with other groups. CONCLUSION: The results of this study show that liposome DOTAP+SLA+imiquimod formulation generates a cellular immune response that is protective against challenge against L. major.

Genotyping determination of Acanthamoeba strains: an original study and a systematic review in Iran.

This study aimed to detect the presence of Acanthamoeba spp. in different water resources of Zahedan, southeast of Iran, and also systematically reviewed all publications regarding Acanthamoeba in Iran (2005-2018). Fifty water samples were collected from different water resources in Zahedan. The positive samples were identified morphologically and subjected to polymerase chain reaction (PCR) using fragments of 18S rRNA. In the systematic review, data collection using particular terms was carried out using the following electronic databases including Science Direct, ISI Web of Science, MEDLINE, EBSCO, Scopus, and Google Scholar. A total of 17 (34%) samples were positive for Acanthamoeba spp., and nucleotide sequencing indicated that 15 samples (88.23%) belonged to the T4 genotype and the rest belonged to the T5 genotype. A total of 39 studies reported genotyping of Acanthamoeba spp. from various geographical areas of Iran and revealed that T4 (35 studies), T5 (19 studies), T3 (11 studies), T11 (8 studies), and T2 (6 studies) genotypes were the most prevalent in Iran. The T4 genotype of Acanthamoeba is a prevalent free-living amoeba and widely distributed not only in Zahedan but also in other provinces of Iran. Phylogenetic analysis reveals that A. castellanii and A. griffini predominantly colocalize with the T4 genotype.

Protection induced by Leishmania Major antigens and the imiquimod adjuvant encapsulated on liposomes in experimental cutaneous leishmaniasis.

There is a need for new, effective, and less expensive and toxic treatment for Leishmaniasis. It seems that the use of a suitable adjuvant and a delivery system is effective in inducing immune reactions for protection. Liposomes can be applied as immunoadjuvants to trigger immune reactions to different antigens. The adjuvant effects of imiquimod using DSPC liposomes containing SLA (soluble Leishmania antigens) were studied on the type and intensity of the produced immune reaction to the challenge of Leishmania major in BALB/c mice. Liposomes were produced by the lipid film procedure. BALB/C mice were immunized subcutaneously, three times at 2-week intervals and with various formulations. Lesion development and the parasite burden in the spleens and feet after the challenge with Leishmania major, Th1 cytokine (IFN-γ), and the IgG isotype titration were assessed to evaluate the induced immune reaction and the protection level. The group of mice immunized with Liposome DSPC +Imiquimod +SLA revealed less severe footpad swelling, being significantly different (P < .05) from other groups. A higher level of IgG2a and IFN-γ secretion was observed in the mice immunized with Liposome DSPC +Imiquimod +SLA than the control group. These observations imply that the DSPC liposome containing imiquimod induces the Th1 immune response that is protective against the challenge of Leishmania major.

Evaluation of malaria multiplex/nested PCR performance at low parasite densities and mixed infection in Iran: A country close to malaria elimination.

BACKGROUND: Malaria is still a major public health problem in subtropical and tropical regions. The rapid and accurate diagnosis of malaria remains a challenge in most of the endemic areas. The primary objective of the present study was to evaluate the performance of multiplex/nested PCR in detecting Plasmodium falciparum at low parasite densities and mixed infection. METHODS: The study was performed in the Sistan-Baluchestan province of the southeastern Iran, from May 2015 to July 2016. A total of 105 patients suspected to malaria infection were enrolled in the study. The obtained DNA products, extracted from the thick/thin films, were analyzed by multiplex/nested PCR using genus-specific primers and compared with light microscopy. RESULTS: 43 samples were confirmed to be infected by microscopic examination. Among 43 microscopically diagnosed P. falciparum cases, 11.4% (12/105) were confirmed by multiplex/nested PCR, 36.2% (38/105) were confirmed as P. vivax, 1.9% (2/105) had mixed infections with P. falciparum and P. vivax. Among microscopy-negative samples, 10 samples turned malaria-positive in nPCR. In multiplex/nested PCR, the rate of mixed infections was 8.6% (9/105). When compared to LM, the sensitivity, specificity, positive predictive value and negative predictive value of multiplex/nested PCR were calculated to be 82.8, 91.5, 92.3 and 81.1%, respectively. CONCLUSION: In this study, we showed that microscopic examination of blood smears does not reliably distinguish Plasmodium species in the case of mixed infections. Therefore, it seems that multiplex/nested PCR is a good candidate for examining the presence of malaria parasites in clinically suspected but microscopically negative cases.

Detection of species and molecular typing of Leishmania in suspected patients by targeting cytochrome b gene in Zahedan, southeast of Iran.

AIM: Cutaneous leishmaniasis (CL) is one of the most important health problems that are capable of involving both tropical and subtropical areas, especially in Iran. This cross-sectional study aimed to differentiate the species that are able to cause CL in Zahedan city by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. MATERIALS AND METHODS: It was conducted on 145 suspected CL patients in Zahedan city between 2014 and 2016. The smears were initially prepared, air-dried, fixed with absolute methanol, and stained with 10% Giemsa. Then, we examined the stained samples by a light microscope under 1000× magnifications. PCR assay targeted cytochrome b(cyt b) gene using LCBF1 and LCBR2 primers and the products digested by Ssp1 enzymes. RESULTS: From 145 suspected CL patients, 76 (52.4%) were positive in microscopic examination. In addition, we detected gene of interest (cyt b) in 98 (67.5%). The results of PCR-RFLP indicated that 53/98 (54%) cases were Leishmania major and 45/98 (46%) were Leishmania tropica, and the main species in these areas was L. major. CONCLUSION: We concluded that the microscopic examination is not sensitive enough and is not able to distinguish between different Leishmania species. Instead, molecular methods like PCR-RFLP can be appropriately used with promising results.

Niosome-loaded antifungal drugs as an effective nanocarrier system: A mini review.

Skin is an important organ of the body due to offering an accessible and convenient site for drug administration. One of the disadvantages of transdermal drug delivery is the low penetration rate of drugs through the skin. Over the past decades, nanoparticles have been used as drug delivery systems to increase therapeutic effects or reduce toxicity. Encapsulation of drugs in nanoparticulate vesicles simplifies the transports of drugs into and across the skin. Niosome nanoparticles are among these drug delivery systems, which have numerous applications in drug delivery and targeting. Niosomes are frequently used for loading drugs serving different purposes (e.g., anticancer, antiviral, and antibacterial agents). In recent years, there has been much research on the use of niosomal systems for the delivery of fungal drugs. A review of the literature investigating the advantages of niosomes in antifungal drug delivery can elucidate the efficiency and superiority of this nanocarrier over other nanocarriers.

Cytochrome b and Molecular Typing of Leishmania spp. in a Passive Sampling of Suspected Patients with Cutaneous Leishmaniasis in Sistan and Baluchestan Province, Eastern Iran.

BACKGROUND: Despite the high prevalence and drug resistance of disease in Sistan and Baluchestan Province of Iran, the species of cutaneous leishmaniasis (CL) has not been identified. In the present study, cytochrome b (Cyt b) was used in Sistan and Baluchestan to find species of Leishmania in suspected patients of CL using PCR-RFLP and DNA sequencing. METHODS: This study was conducted from Oct 2015 to Oct 2016. The samples were collected from the individuals clinically suspected to CL and referred to Iran Shahr, Chabahar, Khash, Zabol, Zahedan, Mirjaveh, and Nikshahr health centers. Overall, 700 Giemsa-Stained slides from the wound of patients suspected of CL were passive collected and examined under a light microscope at ×1000. After DNA extraction, positive samples were used for Cyt b detection by PCR-RFLP to determine the parasite species. One hundred positive slides were selected for molecular studies. Among positive samples, 20% were sequenced. To compare the results of sequences, molecular evolutionary genetic analysis (MEGA6) was used. RESULTS: Overall, 53 samples were identified as L. major and 47 samples (47%) L. tropica. Cyt b in L. major and L. tropica is converted to 400 and 480 bp and 130, 215 and 535 bp pieces respectively. In the isolated L. tropica and L. major, nucleotide changes were 3-5 (mainly in wobble site). CONCLUSION: Infection was more related to L. major. PCR-RFLP method has a high sensitivity for diagnosis of Leishmania species.

The role of ISCOMATRIX bilayer composition to induce a cell mediated immunity and protection against leishmaniasis in BALB/c mice.

OBJECTIVES: Development of new generation of vaccines against leishmaniasis is possible because long-term protection is usually seen after recovery from cutaneous leishmaniasis. ISCOMATRIX is particulate antigen delivery system composed of antigen, cholesterol, phospholipid and saponin. In this study, the role of ISCOMATRIX bilayer composition made by different phase transition temperature (Tc) to induce a type of immune response and protection against leishmaniasis was assessed. MATERIALS AND METHODS: ISCOMATRIX formulations with different bilayer compositions consisting of EPC (Tc <0 °C), DMPC (Tc 23 °C) and DSPC (Tc 54 °C) were prepared. Different ISCOMATRIX formulations were mixed with soluble Leishmania antigens (SLA). BALB/c mice were immunized subcutaneously, three times with 2-week intervals. As criteria for protection, footpads swelling, parasite burden, determination of IgG isotypes and the level of IFN-γ and IL-4 were assessed. RESULTS: Although the groups of mice immunized with ISCOMATRIX DMPC or ISCOMATRIX DSPC showed the smallest footpad swelling and least parasite burden compared with the buffer group, the difference was not significant. Moreover, the highest level of IFN- γ and IL-4 was observed in the splenocytes of mice immunized with ISCOMATRIX DMPC or ISCOMATRIX DSPC, respectively. After challenge, the mice immunized with ISCOMATRIX DSPC showed the highest elevation of IgG, IgG1 and IgG2a antibodies (P<0.01) compared with control group. However, our results indicated that ISCOMATRIX EPC, DMPC or DSPC generated a mixed Th1/Th2 response that was not protective. CONCLUSION: Our results showed that the adjuvanticity of prepared ISCOMATRIX doesn't influence with different phospholipids at least in our mice model.

Cationic Immune Stimulating Complexes Containing Soluble Leishmania Antigens: Preparation, Characterization and in Vivo Immune Response Evaluation.

BACKGROUND: Cationic immune stimulating complexes (PLUSCOMs) are particulate antigen delivery systems. PLUSCOMs consist of cationic immunostimulatory complexes (ISCOMs) derivatives and are able to elicit in vivo T cell responses against an antigen. OBJECTIVE: To evaluate the effects of PLUSCOMs containing Leishmania major antigens (SLA) on the type of immune response generated in the murine model of leishmaniasis. METHODS: PLUSCOMs consisting of 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used as antigen delivery system/immunoadjuvants for soluble SLA. BALB/c mice were immunized subcutaneously, three times in 2-week intervals. Footpads swellings at the site of challenge and parasite loads were assessed as a measure of protection. The immune responses were also evaluated by determination of IgG subclasses and the level of IFN-γ and IL-4 in cultured splenocytes. RESULTS: There was no significant difference (p<0.05) between the sizes of lesions in mice immunized with different formulations. Also, there was no significant difference in the number of parasites in the footpad or spleen of all groups compared with the control group. The highest level of IFN-γ secretion was observed in the splenocytes of mice immunized with PLUSCOM/SLA (p<0.001) and lower amounts of IL-4 was observed in PLUSCOM group (p<0.001) as compared to negative control. CONCLUSION: Our results indicated that SLA in different formulations generated an immune response with mixed Th1/Th2 response that was not protective enough despite the activation of CD4+ T cells with secreting IFN-γ in groups which received PLUSCOM with antigen.

Molecular detection of Crimean-Congo haemorrhagic fever (CCHF) virus in ticks from southeastern Iran.

Crimean-Congo haemorrhagic fever (CCHF) virus is a tick-borne member of the genus Nairovirus, family Bunyaviridae. CCHF virus has been isolated from at least 31 different species of ticks. The virus is transmitted through the bite of an infected tick or by direct contact with CCHF virus-infected patients or the products of infected livestock. This study was conducted to determine the rate of CCHF virus infection in ticks in the district of Zahedan, in the province of Sistan and Baluchistan, southeastern Iran. A total of 140 ticks were collected from Sistan and Baluchistan. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used for the detection of the CCHF virus genome in the tick population. This genome was detected in 4.3% of ticks collected from livestock of different regions of Zahedan. The infected tick genera belonged to Hyalomma and Haemaphysalis. Although in the epidemiology of CCHF virus Hyalomma ticks are considered to be the most important vectors and reservoirs, the virus has also been reported to occur in other genera of ticks, which conforms to the current data in our study from Sistan and Baluchistan. Given that animals are common hosts for Hyalomma and Haemaphysalis, regular monitoring programmes for livestock should be applied for CCHF virus control.

Ecology of Anopheles stephensi in a malarious area, southeast of Iran.

District of Jiroft is situated in south-east of Iran which is one of the malarious regions. Anopheles stephensi is considered as one of the main malaria vector in this region. Ecology of this species was studied in the area to understand its vector behavior for implementation of effective vector control measures. Different methods like total catch, pit shelter, night bite collection on human and animal, larval dipping methods were used for species identification, seasonal activity, anthropophilic index and egg morphological characteristics. Anthropophilicity index was assessed by ELISA test. Activity of Anopheles species started at the beginning of April, and its peak occurs in late spring. The larvae were found in the river bed with pools, stagnant streams, slow foothill streams, temporary pools, and slowly moving water with and without vegetation, drainage containers of air conditioner and palm irrigation canals. From different methods of adult collection, it was found that spray sheet collection is the appropriate method. ELISA testing of 144 blood meals of females revealed the anthropophilicity of 11.8% indicating host preference on animal, mainly cow. Ridge length and their number on the egg floats confirmed Anopheles stephensi mysorensis form. This study showed that Anopheles stephensi is the main vector of malaria in the region, although some other species may play a role. Our findings could provide a valuable clue for epidemiology and control of malaria in the southeast of Iran.