Wild rodents and their ectoparasites in Baluchistan area, southeast of Iran.

During 2008-2009 a total of 67 individuals of rodents, Tetera indica, Meriones hurrianae, Meriones libycus and Gerbillus nanus were trapped in three areas, Bampor, Daman and Qasre Qand from Iranshahr and Nikshahr districts. There is a significant difference between comparative abundance of four species (P<0.05). A total of 1422 ectoparasites collected including 299 mites (21%), 127 fleas (8.9%), 972 lice (68.4%) and 24 ticks (1.7%). Significant findings amongst the ectoparasites is the lice group with three species identified, Laelaps accuninata, Andralaelaps hermophrodita and Paracheylaellaps pyriformis being the first record in the study areas. All four captured genera of rodents are known as main/ potential reservoir hosts of zoonotic cutaneous leishmaniasis. The migration habit of rodents may affect the spatial distribution of parasitic ticks and their transmitted diseases like CCHF, which has been reported in recent years from Sistan and Baluchestan province. Monitoring of rodent populations and their ectoparasites will help to predict the potential of zoonotic arthropod-borne diseases.

Sequence Analysis of Different Domains of Plasmodium vivax Apical Membrane Antigen (PvAMA-1 gene) Locus in Iran.

BACKGROUND: Plasmodium vivax is responsible for approximately 80 million malaria cases in the world. Apical membrane antigen1 (AMA-1) is a type I integral membrane protein present in all Plasmodium species. AMA-1 interferes in critical steps of invasion of human hepatocytes by sporozoites and red blood cells by merozoites and is one of the most immunodominant antigens for eliciting a protective immune response in human. It is considered as a promising antigen for inclusion in a vaccine against P. vivax. Since more knowledge is needed to lighten the scope of such antigen we compared genetic variation in P. vivax AMA-1from an Iranian isolate with those reported from some of the other malarious countries so far. METHODS: P. vivax genomic DNA was extracted from the whole blood of an Iranian patient with patent P. vivax infection. The nucleotide sequence for 446 amino acid (AA) residues (42-488 of PvAMA-1) was amplified by PCR and cloned in pUC19 vector for sequencing. RESULTS: Sequence analysis of the antigen showed a high degree of identity (99%) with strong homology to the PvAMA-1 gene of P. vivax S3 and SKO814 isolates from India and Korea (Asian isolates) respectively, and 96% similarity with P. vivax Sal-1 AMA-1 gene from El Salvador. CONCLUSIONS: We cloned and characterized three domains of PvAMA-1 gene from an Iranian patient. Predicted protein sequence of this gene showed some discrepancies in corresponding protein in comparing with similar genes reported from other malarious countries.

The Role of Malaria Microscopy Training and Refresher Training Courses in Malaria Control Program in Iran during 2001 - 2011.

BACKGROUND: Malaria is still one of the most important infectious diseases in the world. The disease also is a public health problem in south and southeast of Iran. This study programmed to show the correlation between regular malaria microscopy training and refresher training courses and control of malaria in Iran. METHODS: Three types of training courses were conducted in this programme including; five - day, ten - day and bimonthly training courses. Each of the training courses contained theoretical and practical sections and training impact was evaluated by practical examination and multiple-choice quizzes through pre and post tests. RESULTS: Distribution pattern of the participants in the training and refresher training courses showed that the most participants were from Sistan & Baluchistan and Hormozgan provinces where malaria is endemic and most cases of the infection come out from these malarious areas. A total of 695 identified individuals were participated in the training courses. A significant conversely correlation was found between conducting malaria microscopy training courses and annual malaria cases in Iran. CONCLUSION: Conducting a suitable programme for malaria microscopy training and refresher training plays an important role in the control of malaria in endemic areas. Obviously, the decrease of malaria cases in Iran has been achieved due to some activities that malaria diagnosis training was one of them.

High specificity of semi-nested multiplex PCR using dried blood spots on DNA Banking Card in comparison with frozen liquid blood for detection of Plasmodium falciparum and Plasmodium vivax.

BACKGROUND: Venipuncture sampling in test tubes for detecting malaria parasites using PCR assays possesses a number of limitations such as reluctance of patients, some difficulties in transportation of blood samples and freezing them for long time. To overcome the mentioned limitations, some approaches have been employed by a number of authors. This study was proposed to compare between DNA Banking Card (DBC) filter papers containing dried finger-prick blood and venipunctured frozen liquid blood. METHODS: A total of 75 specimens was prepared from the equal enrolled individuals using three blood storage approaches; making Geimsa-stained thin and thick smears from each individual to determine the malaria-positive or -negative specimens, spotting two to three drops of finger-prick blood onto the DBC filter paper, and collecting a 2-ml venous blood sample into EDTA-contained test tube from each individual. A semi-nested Multiplex PCR technique with DNA extracted from the two latter sets of specimens was used for plasmodia diagnosis. RESULTS: DNA samples isolated from dried blood spotted on the DBC filter papers resulted in 32 (42.7%) positive and 43 (57.3%) negative cases comparable with the results outcome of frozen liquid blood with 35 (46.7%) positive and 40 (53.3%) negative cases. Statistical analysis revealed higher sensitivity for SnM-PCR using DNA from liquid blood with 100% vs. dried blood spotted on DBC with 97% but higher specificity for the DBC with 100% vs. liquid blood with 95.2%. CONCLUSIONS: Based on the results obtained from this study to overcome the problems of venipuncture frozen liquid blood sampling, replacement of a reliable filter paper for preserving finger-prick blood samples is a trustable and useful facilitator particularly in remote malaria-endemic areas.

Detection of asymptomatic malaria infection among the Afghani immigrant population in Iranshahr district of southeastern Iran.

Asymptomatic malaria infection is often associated with subpatent level of parasitaemia and normal clinical examination. Such infection becomes a greater cause for concern when involved in blood transfusion and vector transmission. This study was performed to monitor the situation of asymptomatic malaria among the Afghani immigrants and native residents in Iranshahr district, a malaria endemic area in southeastern Iran, by performing conventional light microscopy. Out of 446 samples collected from Afghani immigrant participants, seven (1.6%) thick blood smears were diagnosed as Plasmodium vivax. None of the individuals who tested positive had malaria symptoms and they did not remember having had any malaria signs during the past two years. Out of 496 samples collected from native resident participants, three (0.6%) thick blood smears were detected as P. vivax and Plasmodium falciparum with mild malaria symptoms. An asymptomatic Plasmodium-infected individual can be a source of malaria parasites for transmission of the agents.

Comparison of microscopical examination and semi-nested multiplex polymerase chain reaction in diagnosis of Plasmodiumfaldparum and P. vivax

We compared light microscopy examination and a semi-nested multiplex PCR [SnM-PCR] assay in endemic areas of the Islamic Republic of Iran. A total of 68 individuals with malaria-positive and suspected malaria symptoms were included in the study. Giemsa-stained thick blood films were examined under a light microscope for malaria parasites in 100 and 200 fields. DNA was extracted from blood samples and SnM-PCR based on the amplification of the small subunit ribosomal RNA [ssrRNA] gene sequences was applied. Microscopical examination showed that 48.5% [33.8% P. vivax and 14.7% P.falciparum] and 50% [35.3% P. vivax and 14.7% P.falciparum] of the samples were positive in 100 and 200 fields respectively. SnM-PCR showed the same results as the 200 field microscopy

Molecular Monitoring of Plasmodium vivax Infection after Radical Treatment in Southeastern Iran.

BACKGROUND: The aim was to evaluate the relapse risk of vivax malaria in patients who received radical treatment in Hormozgan Province, a malarious area located on southeast of Iran. METHODS: A total of 95 symptomatic vivax malaria infected patients were enrolled in urban health centers of Bandar-Abbas, Minab, Bandar-Jask and Bashagard districts of Hormozgan Province, southeast of Iran from January 2008 to March 2009 for consideration as a case- series study. DNA was extracted from parasite infected whole blood samples. A polymorphic region of Plasmodium vivax merozoite surface protein 1 (pvMSP1) was selected and a PCR method was employed for all the samples to amplify the specific variable gene fragment. The obtained fragments in primary and secondary samples were sequenced. Both nucleotide and amino acid sequences of the samples were investigated for returned patients. RESULTS: 3.2% of the patients experienced a second attack between 83-199 days after the initial episode of infection. Alignment of nucleotide and their deduced amino acid sequences between pair sequences of primary and secondary isolates revealed 8 and 6 dissimilarities respectively for the first case, and 9 and 7 dissimilarities for the second case. Although microscopical examination of recurrent thick blood smear of the third patient confirmed new P. vivax infection, the venous blood sample was accidentally missed. Sequencing results of primary and returned isolates 1P, 1S, 2P, 2S and 3P in this study showed an identity with BP13, T117, BP13, TC28 and Chesson genotypes respectively. CONCLUSION: The returned (secondary) isolates may account to be for the sake of reinfection.

Residual Effects of Deltamethrin WG 25% as a New Formulation on Different Surfaces against Anopheles stephensi, in Southeastern Iran.

BACKGROUND: Indoor residual spraying (IRS) is functioned as national interventions against malaria in southeastern foci of Iran and deltamethrin WP one of the insecticides have been used since past decade. In this study, the residual activity of the wettable granule (WG) was studied on different surfaces in hut scale trial against Anopheles stephensi in Iranshahr District, southeastern Iran. METHODS: Three dosages of 25, 40 and 50 mg a.i./m(2) of deltamethrin WG 25% formulation were applied on plaster, cement, mud, and wooden surfaces using Hudson(®) X-pert compression sprayer having 10 litters capacity. RESULTS: The residual effects of deltamethrin WG 25% on different surfaces was assessed based on reduction of mortality An. stepehnsi from 100% to about 70%. At 25, 40 and 50 mg a.i./m(2) the WG formulation of deltamethrin had a bioefficacy for about 2, 3 and 4 months respectively. CONCLUSION: There was an expectable fluctuation in mortality of An. stephensi at different sprayed surfaces as well as dosages. The proposed 50 mg/m2 WG is the longest activity for up to 4 months which needs to be applied for two spraying cycles per year at the climatically condition of southwestern Iran.

Monitoring of the therapeutic efficacy of chloroquine for the treatment of uncomplicated, Plasmodium falciparum malaria in Iran.

Between 2002 and 2004, the standardized 28-day protocol recently developed by the World Health Organization was used to explore the efficacy of chloroquine, in the treatment of uncomplicated, Plasmodium falciparum malaria, in five sentinel sites in southern Iran. All but 14 of the 158 patients enrolled (128, 28 and two from the provinces of Sistan-Baluchestan, Hormozgan and Kerman, respectively) were successfully followed-up. The overall frequency of treatment failure by day 28 was 78.5%, with 17.4% of the patients being classed as early treatment failures, 34.7% as late clinical failures, and 26.4% as late parasitological failures. There appeared to be no significant change in the frequency of treatment failure between the 2002-2003 and 2003-2004 transmission seasons, nor any significant between-site variation in the efficacy of chloroquine. Given these observations, the replacement of chloroquine, as the first-line drug for the treatment of uncomplicated, P. falciparum malaria in Iran, was inevitable. Artesunate-sulfadoxine-pyrimethamine is now the recommended first-line treatment, with artemether-lumefantrine used for second-line treatment. The efficacies of these combination therapies are currently being evaluated and monitored.

Monitoring of Plasmodium vivax sensitivity to chloroquine in vitro in Thailand.

The sensitivity of Plasmodium vivax to chloroquine in vitro was investigated in patients admitted to the Bangkok Hospital for Tropical Diseases, Thailand, between September 2001 and May 2002. Of 42 isolates, 34 were successfully tested for parasite sensitivity to chloroquine in vitro; the results showed a significant decrease in sensitivity compared with data published in 1989 and 1995: the IC50 and IC90 were 187.2 and 1217.9 ng/mL blood, respectively, an approximate 4-fold decrease in sensitivity in comparison with other data from the past 2 decades. A number of in vitro tests were performed simultaneously using both WHO microplates and our own laboratory-prepared pre-dosed microplates under the same conditions and there was no significant difference between the results.

Plasmodium vivax malaria in Southeast Iran in 1999-2001: establishing the response to chloroquine in vitro and in vivo.

Chloroquine-resistant Plasmodium vivax is emerging in Oceania, Asia and Latin America. The drug sensitivity of P. vivax to chloroquine both in vivo and in vitro in the southern part of Iran was assessed; chloroquine-resistant Plasmodium falciparum has already been documented in this area. The in vitro sensitivity of 39 P. vivax isolates was assessed: the mean IC50 and IC90 were 189 ng/ml and 698 ng/ml blood respectively; for in vivo testing, all 39 vivax malaria patients were treated with a standard regimen of chloroquine and followed-up at 28 days: the mean parasite clearance time was 67.2 +/- 22.5 hours. The in vitro development of young parasites to mature schizonts in standard test medium was compared with that obtained in McCoy's 5A medium: no significant difference was observed. Synchronization of the blood-stage parasites was performed according to Lambros' method: the method was not suitable because it was detrimental to the parasites. A number of in vitro tests were performed using both our own laboratory-predosed microplates and WHO microplates: there was no significant difference between the results.

Cyfluthrin (EW 050)-impregnated bednets in a malaria control program in Ghassreghand (Baluchistan, Iran).

In a study carried out in the Ghassreghand Division (Baluchistan, Iran) from March through November 1995, efficacy of cyfluthrin-impregnated bednets was compared to that of untreated nets, in relation to malaria control. Ten villages with a total population of 4,572 and 3 villages with a total population of 1,935 were used as treatment and control, respectively. The collection, impregnation (target dosage of 40 mg active ingredient [AI]/m2), and redistribution of the nets (9% nylon, 52% light cotton, 30% medium cotton, and 9% heavy cotton), carried out in mid-April, were done by local health workers, supervised by the senior research staff. Anopheles culicifacies was considered to be the main vector of malaria in the named area. This species is mainly zoophilic, endophilic, and exophagic. The initial uptake of the insecticide was lower than the target dosage, with high variation (nylon, 12.5 +/- 5.4 mg AI/m2; light cotton, 33.3 +/- 26.1 mg AI/m2; medium cotton, 25.9 +/- 20 mg AI/m2; heavy cotton, 17.6 +/- 12.5 mg AI/m2). The use of impregnated mosquito nets (used primarily outside) had no significant effect on the incidence of malaria. No difference was detected in the parasite density of patients with positive slides. No significant effect was observed in the parous rate, human blood index, and sporozoite rate of anopheline vectors. Only the indoor resting densities of An. culicifacies and other malaria vectors were drastically reduced after the introduction of the cyfluthrin-impregnated nets into the treatment villages. The residual activity of cyfluthrin was lower than expected. The mortality of anophelines brought in contact with the treated nets for 3 min in bioassays dropped to less than 55% in 3 months. The loss of chemical activity was greatest for the light cotton nets, followed by the medium cotton nets. Cyfluthrin-treated nets were mildly irritating to host-seeking female anophelines in the laboratory. The protective rate of impregnation (all fabric kinds included) in preventing female mosquitoes from biting through the impregnated nets was initially 5-6 times that of the nonimpregnated nets. The study did not detect any significant difference between the use of untreated versus impregnated bednets in the Ghassreghand area. In planning future medium-scale trials, comparison of new compounds and formulations to the more widely used pyrethroids such as permethrin and deltamethrin is highly recommended.

Development of halofantrine resistance and determination of cross-resistance patterns in Plasmodium falciparum.

Intermittent exposure to halofantrine (HF) of both chloroquine-susceptible (T9.96) and chloroquine-resistant (K1) isolates of Plasmodium falciparum in vitro resulted in a rapid reduction in susceptibility to HF. After 6 months, the 50% inhibitory concentration (IC50) of HF, determined with [G-3H]hypoxanthine incorporation as a marker, increased ninefold for the chloroquine-resistant (K1) isolate and threefold for the cloned chloroquine-susceptible (T9.96) isolate, the derived isolates being termed the K1HF3 and T9.96HF4 isolates, respectively. By microscopic examination of cultured erythrocytes, we determined that there was a fivefold increase in the IC50 for isolate T9.96HF4. The responses of the parental isolates and the HF-resistant isolates to chloroquine, mefloquine, quinine, amodiaquine, qinghaosu, and pyrimethamine were determined. In comparison with the parental K1 isolate, HF-resistant isolate K1HF3 was significantly more susceptible to the action of chloroquine and exhibited a significantly reduced susceptibility to quinine and mefloquine. The other HF-resistant isolate, T9.96HF4, showed no alteration in susceptibility to amodiaquine or chloroquine but a significantly decreased susceptibility to mefloquine. Resistance was stable in the two isolates, both in the absence of drug pressure or when kept frozen in liquid nitrogen. In contrast, continuous exposure to HF had no effect on the susceptibility of the parasites to this drug above HF concentrations of 3.2 x 10(-9) M.