Isolation and identification of cutaneous leishmaniasis species by PCR-RFLP in Ilam province, the west of Iran.

Cutaneous leishmaniasis (CL) is one of the most common parasitic diseases and public health problems in Iran. CL is endemic in most parts of Ilam province, in the west of Iran. The distance from the center of country, the great number of divers rural areas, and lack of specialists and laboratory facilities have been the major causes of Leishmania species remaining unknown in this region. Polymerase chain reaction followed by restriction fragment length polymorphism was performed to identify the Leishmania species in 61 patients with cutaneous lesions. Eventually L. major was confirmed as the cause of cutaneous leishmaniasis in Ilam province, the west of Iran.

SYBR green-based detection of Leishmania infantum DNA using peripheral blood samples.

Parasitological methods for the diagnosis of visceral leishmaniasis (VL) require invasive sampling procedures. The aim of this study was to detect Leishmania infantum (L. infantum) DNA by real time-PCR method in peripheral blood of symptomatic VL patient and compared its performance with nested PCR, an established molecular method with very high diagnostic indices. 47 parasitologically confirmed VL patients diagnosed by direct agglutination test (DAT > 3200), bone marrow aspiration and presented characteristic clinical features (fever, hepatosplenomegaly, and anemia) and 40 controls (non-endemic healthy control-30, Malaria-2, Toxoplasma gondii-2, Mycobacterium tuberculosis-2, HBV-1, HCV-1, HSV-1 and CMV-1) were enrolled in this study. SYBR-green based real time-PCR and nested PCR was performed to amplify the Kinetoplast DNA minicircle gene using the DNA extracted from Buffy coat. From among 47 patients, 45 (95.7 %) were positive by both nested-PCR and real time-PCR. These results indicate that real time-PCR was not only as sensitive as a nested-PCR assay for detection of Leishmania kDNA in clinical sample, but also more rapid. The advantage of real time-PCR based methods over nested-PCR is simple to perform, more faster in which nested-PCR requires post-PCR processing and reducing contamination risk.

Bioconjugated fluorescent silica nanoparticles for the rapid detection of Entamoeba histolytica.

Rapid detection of Entamoeba histolytica based on fluorescent silica nanoparticle (FSNP) indirect immunofluorescence microscopy was evaluated. Silica nanoparticles were synthesized using Stöber's method, with their surface activated to covalently bind to, and immobilize, protein A. For biolabeling, FSNP was added to conjugated E. histolytica trophozoites with monoclonal anti-E. histolytica IgG1 for microscopic observation of fluorescence. Fluorescent silica nanoparticle sensitivity was determined with axenically cultured E. histolytica serially diluted to seven concentrations. Specificity was evaluated using other intestinal protozoa. Fluorescent silica nanoparticles detected E. histolytica at the lowest tested concentration with no cross-reaction with Entamoeba dispar, Entamoeba moshkovskii, Blastocystis sp., or Giardia lamblia. Visualization of E. histolytica trophozoites with anti-E. histolytica antibody labeled with fluorescein isothiocyanate (FITC) was compared with that using anti-E. histolytica antibody bioconjugated FSNP. Although FITC and FSNP produced similar results, the amount of specific antibody required for FITC to induce fluorescence of similar intensity was fivefold that for FSNP. Fluorescent silica nanoparticles delivered a rapid, simple, cost-effective, and highly sensitive and specific method of detecting E. histolytica. Further study is needed before introducing FSNP for laboratory diagnosis of amoebiasis.

Mitochondrial PCR-based malaria detection in saliva and urine of symptomatic patients.

BACKGROUND: Malaria can be diagnosed in saliva and urine using mitochondrial PCR detection of Plasmodium DNA. METHODS: Blood, saliva and urine were collected from 99 febrile patients referred to health centers in Sistan and Baluchestan Province, southeastern Iran, from May to November 2011. The mitochondrial cytochrome b gene of Plasmodium falciparum and Plasmodium vivax was targeted in saliva, urine and blood samples using nested PCR. RESULTS: Nested PCR proved to be more sensitive than microscopy for the diagnosis of sub-microscopic and mixed-species infections. The results of nested PCR amplifications of saliva and urine samples showed the same specificity of 97% and sensitivity of 91% and 70%, respectively. Nested PCR amplifications of saliva samples and microscopy showed the greatest area under the receiver operating characteristic (ROC) curve and were more accurate than nested PCR amplifications of urine samples. CONCLUSION: Nested PCR amplification of saliva samples showed good levels of detection of mitochondrial Plasmodium DNA as compared to nested PCR of blood (к=0.84; AUC=0.94), which was used as a reference standard. Based on the results of nested PCR as well as the advantages of saliva sampling, we suggest that saliva could be an alternative to blood, in malaria diagnosis, in cases where repeat sampling is required. Further studies are needed to validate these findings.

Detection of Plasmodium vivax and Plasmodium falciparum DNA in human saliva and urine: loop-mediated isothermal amplification for malaria diagnosis.

This study investigated loop-mediated isothermal amplification (LAMP) detection of Plasmodium falciparum and Plasmodium vivax in urine and saliva of malaria patients. From May to November 2011, 108 febrile patients referred to health centers in Sistan and Baluchestan Province of south-eastern Iran participated in the study. Saliva, urine, and blood samples were analyzed with nested PCR and LAMP targeting the species-specific nucleotide sequence of small subunit ribosomal RNA gene (18S rRNA) of P. falciparum and P. vivax and evaluated for diagnostic accuracy by comparison to blood nested PCR assay. When nested PCR of blood is used as standard, microscopy and nested PCR of saliva and urine samples showed sensitivity of 97.2%, 89.4% and 71% and specificity of 100%, 97.3% and 100%, respectively. LAMP sensitivity of blood, saliva, and urine was 95.8%, 47% and 29%, respectively, whereas LAMP specificity of these samples was 100%. Microscopy and nested PCR of saliva and LAMP of blood were comparable to nested PCR of blood (к=0.95, 0.83, and 0.94, respectively), but agreement for nested PCR of urine was moderate (к=0.64) and poor to fair for saliva LAMP and urine LAMP (к=0.38 and 0.23, respectively). LAMP assay showed low sensitivity for detection of Plasmodium DNA in human saliva and urine compared to results with blood and to nested PCR of blood, saliva, and urine. However, considering the advantages of LAMP technology and of saliva and urine sampling, further research into the method is worthwhile. LAMP protocol and precise preparation protocols need to be defined and optimized for template DNA of saliva and urine.

Development and Assessment of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran.

BACKGROUND: Parasitological methods for the diagnosis of Visceral leishmaniasis (VL) require invasive procedures, so serological and molecular approaches have been developed but are not generally applicable in the field. We evaluated a loop mediated isothermal amplification (LAMP) assay using blood from VL patients and compared it to nested PCR. METHODS: Forty-seven subjects with clinical features (fever, hepatosplenomegaly and anemia) were confirmed positive for VL by the direct agglutination test (DAT) at titers >3200. Forty DAT negative individuals from non-endemic areas with no clinical signs or symptoms of VL served as controls. A LAMP assay was performed using a set of six primers targeting Leishmania infantum kinetoplast DNA (kDNA) minicircle gene under isothermal (64 °C) conditions. For nested PCR we used primers targeting the kDNA minicircle gene. RESULTS: The LAMP assay provided a detection limit of 1 parasite in 1 ml of peripheral blood and detected L. infantum DNA in 44 of 47 DAT-confirmed VL cases, with diagnostic sensitivity of 93.6% (95% CI). No L. infantum DNA was amplified in controls, indicating a specificity of 100%. The nested PCR yielded sensitivity of 96% (95% CI) and a specificity of 100% (95% CI). CONCLUSION: The LAMP assay gave results similar to those of nested PCR but in a shorter time. The LAMP method is simple; requires no sophisticated equipment; has a short reaction time; and results, indicated by turbidity of the reaction mixture, are observable with the naked eye.

Prevalence of zoonotic intestinal parasites in domestic and stray dogs in a rural area of Iran.

Certain zoonotic parasites are enteropathogens in dogs that cause serious human disease such as cystic echinococcosis, human alveolar echinococcosis, visceral larva migrans, and ocular larva migrans. This study investigated the prevalence of intestinal parasites in dogs in the Chenaran County, Razavi Khorasan Province, Iran. Sampling was carried out randomly in 17 villages from November 2009 to January 2010. Seventy-seven fecal samples from 28 domestic and 49 stray dogs were examined using sieving/flotation and modified Ziehl-Neelsen staining. Intestinal parasites were found in 51 of the 77 (66%) dogs most common being Toxascaris leonina (29%, 22/77), Toxocara spp. (25%, 19/77), Eimeria spp. (19%, 15/77), Taenia/Echinococcus spp. (18%, 14/77), Sarcocystis spp. (17%, 13/77), and Dicrocoelium dendriticum (14%, 11/77). Lower infection rates of parasites were observed for Trichuris vulpis (6%, 5/77), Cryptosporidium spp. (5%, 4/77), and Physaloptera spp. (3%, 2/77). Prevalence of infection by Dipylidium caninum, Capillaria spp., Cystoisospora spp., and hookworms was similar (1%, 1/77). This study is the first report of the prevalence of intestinal parasites of domestic and stray dogs in Chenaran County, Northeast Iran. The higher prevalence of zoonotic intestinal parasites such as Toxascaris leonina, Toxocara spp. and Taenia/Echinococcus spp. compared to other parasites indicates the need for control programs to minimize the risk of transmission of zoonotic disease, particularly cystic echinococcosis, alveolar echinococcosis, visceral larva migrans, and ocular larva migrans to people living in these areas.

Detection of Echinococcus multilocularis in carnivores in Razavi Khorasan province, Iran using mitochondrial DNA.

BACKGROUND: Echinococcus multilocularis is the source of alveolar echinococcosis, a potentially fatal zoonotic disease. This investigation assessed the presence of E. multilocularis infection in definitive hosts in the Chenaran region of Razavi Khorasan Province, northeastern Iran. METHODOLOGY/PRINCIPAL FINDINGS: Fecal samples from 77 domestic and stray dogs and 14 wild carnivores were examined using the flotation/sieving method followed by multiplex PCR of mitochondrial genes. The intestinal scraping technique (IST) and the sedimentation and counting technique (SCT) revealed adult Echinococcus in the intestines of five of 10 jackals and of the single wolf examined. Three jackals were infected only with E. multilocularis but two, and the wolf, were infected with both E. multilocularis and E. granulosus. Multiplex PCR revealed E. multilocularis, E. granulosus, and Taenia spp. in 19, 24, and 28 fecal samples, respectively. Echinococcus multilocularis infection was detected in the feces of all wild carnivores sampled including nine jackals, three foxes, one wolf, one hyena, and five dogs (6.5%). Echinococcus granulosus was found in the fecal samples of 16.9% of dogs, 66.7% of jackals, and all of the foxes, the wolf, and the hyena. The feces of 16 (21.8%) dogs, 7 of 9 (77.8%) jackals, and all three foxes, one wolf and one hyena were infected with Taenia spp. CONCLUSIONS/SIGNIFICANCE: The prevalence of E. multilocularis in wild carnivores of rural areas of the Chenaran region is high, indicating that the life cycle is being maintained in northeastern Iran with the red fox, jackal, wolf, hyena, and dog as definitive hosts.

Giardia intestinalis: DNA extraction approaches to improve PCR results.

Difficulty in disrupting cysts of Giardia intestinalis, a cosmopolitan protozoan parasite, decreases the yield of DNA extracted and reduces the effectiveness of the polymerase chain reaction (PCR). To improve the detection of the Giardia Glutamate Dehydrogenase (gdh) gene, we re-evaluated the effects of deoxyribonucleic acid (DNA) extraction methods. Purified and concentrated cysts from 33 fecal samples were disrupted using conventional methods, and DNA extraction was conducted using two protocols: the QIAamp Stool Mini Kit and phenol/chloroform/isoamyl alcohol (PCI). PCR amplification was successful for 12 extracted DNA samples (36%) using PCI following a glass bead and freeze/thaw pretreatment and for all 33 samples (100%) using the QIAamp Stool Mini Kit following the aforementioned pretreatment. Consequently, the pretreatment of cysts with glass beads and freeze/thaw cycles followed by extraction of DNA with the QIAamp Stool Mini kit was the more effective protocol.