Molecular identification of hemoparasites in ixodid ticks in Iran.

A total of 386 ticks were processed in order to investigate the occurrence of selected tick transmitted pathogens (i.e., Theileria, Babesia, Hepatozoon and Cytauxzoon) in ixodid ticks in six provinces of Iran (East Azerbaijan, Gilan, Kermanshah, Khuzestan, Sistan & Baluchestan and Tehran). Ticks identified as Dermacentor marginatus, Hyalomma aegyptium, Hy. anatolicum, Hy. asiaticum, Hy. marginatum, Ixodes ricinus, Rhipicephalus annulatus and R. sanguineus sensu lato were collected from sheep and cattle. Conventional PCR and sequencing results revealed DNA of Theileria ovis in three R. sanguineus sensu lato pools and one D. marginatus pool from sheep in Kermanshah and East Azerbaijan, T. annulata in one Hy. asiaticum pool from cattle in Kermanshah, and He. canis in an individual female Hy. aegyptium in Kermanshah. Data herein indicate the role of R. sanguineus complex and D. marginatus in the epidemiology of ovine theileriosis in western and northwestern Iran. Potential role of Hyalomma aegyptium in the transmission of He. canis is discussed. Considering non-principled movement of livestock across the country and increasing reports about the resistance of ticks to common acaricides, test-and-treatment of infected livestock, vaccination of the livestock against endemic tick-borne pathogens, and the use of non-chemical tick management strategies are recommended.

Genetic Characterization of Sandfly-Borne Viruses in Phlebotomine Sandflies in Iran.

Phleboviruses are classified into two main groups: the sandfly fever group (transmitted by sandflies and mosquitoes) and the Uukuniemi group (transmitted by ticks). Old World sandfly-borne viruses (SBVs) are classified into four main serocomplexes; sandfly fever Naples viruses (SFNVs), sandfly fever Sicilian viruses (SFSVs), Karimabad viruses (KARVs), and Salehabad viruses (SALVs). This study addresses current knowledge gaps on SBVs in Iran by focusing on identification and molecular epidemiology. We used PCR to examine DNA/RNA extracts to identify sandfly species and evaluate for SBV presence. We identified five specimens positive for phleboviruses: one Ph. sergenti for Tehran virus (TEHV), one Ph. papatasi for SFSV, and two Ph. papatasi and one Ph. perfiliewi for KARV. A phylogenetic tree indicated that the TEHV isolate from this study formed a cluster with previous isolates of TEHV, Zerdali virus, and Fermo virus. Meanwhile, the identified SFSV isolate fell in lineage I and was grouped with previous isolates of SFSVs and Dashli virus in Iran. Finally, the KARV isolates from this study formed a monophyletic clade in a sister relationship with other viruses in KARV lineages I and II. This comprehensive study on SBVs in Iran provided new insights into the molecular epidemiology of TEHV, SFSVs and KARVs in this country.

Investigation on Mosquitoes Fauna (Diptera: Culicidae) and Probable Vector of West Nile Virus in Lorestan Province, Western Iran.

Background: Fauna and larval habitat characteristics studies on mosquitoes are important tools to identify the breeding places of the vectors and management of the control strategies. This study was done to provide data on Culicidae fauna, larval habitat characteristics and identifying potential vectors of West Nile virus in Lorestan Province, west of Iran. Methods: Culicidae mosquitoes were collected at three counties and nine site stations from Lorestan Province, west of Iran in 2017. Adult mosquitoes were collected using human and animal bite collection methods, New Jersey and CDC light traps and pit shelters by aspirator. Larva were collected by dipping method. RT-PCR technique was employed for detection of the West Nile virus among mosquito's samples. Results: 4805 mosquitoes were collected from three counties and nine sites in Lorestan Province during June-October 2017, including 4363 adults and 442 larvae. The most abundant species collected from all counties in both adult and larval stages were Culex pipiens (49.10%), Cx. theileri (31.82%), Anopheles maculipennis (11.09%), An. superpictus (2.66%), An. stephensi (2.12%), Cx. perexiguus (1.89%), An. dthali (1.17%) and An. sacharovi (0.15%) respectively. West Nile virus was detected in none of mosquitoes examined. Conclusion: The results of this study revealed that arbovirus vectors such as Cx. pipiens along with Cx. theileri and Cx. perexiguus are well adapted to a broad range of habitats and different climatic conditions in Lorestan Province. That necessitates further routine surveillance of arboviral infections.

Autochthonous Transmission of West Nile Virus by a New Vector in Iran, Vector-Host Interaction Modeling and Virulence Gene Determinants.

Using molecular techniques and bioinformatics tools, we studied the vector-host interactions and the molecular epidemiology of West Nile virus (WNV) in western Iran. Mosquitoes were collected during 2017 and 2018. DNA typing assays were used to study vector-host interactions. Mosquitoes were screened by RT-PCR for the genomes of five virus families. WNV-positive samples were fully sequenced and evolutionary tree and molecular architecture were constructed by Geneious software and SWISS-MODEL workspace, respectively. A total of 5028 mosquito specimens were collected and identified. The most prevalent species was Culex (Cx.) pipiens complex (57.3%). Analysis of the blood-feeding preferences of blood-fed mosquitoes revealed six mammalian and one bird species as hosts. One mosquito pool containing non-blood-fed Cx. theileri and one blood-fed Culex pipiens pipiens (Cpp.) biotype pipiens were positive for WNV. A phylogram indicated that the obtained WNV sequences belonged to lineage 2, subclade 2 g. Several amino acid substitutions suspected as virulence markers were observed in the Iranian WNV strains. The three-dimensional structural homology model of the E-protein identified hot spot domains known to facilitate virus invasion and neurotropism. The recent detection of WNV lineage 2 in mosquitoes from several regions of Iran in consecutive years suggests that the virus is established in the country.

Seroepidemiology of human fascioliasis in rural and nomad areas of Lorestan Province, western Iran, in 2016 and 2017.

According to previous studies in Lorestan Province, western Iran on human fascioliasis, we aimed to understand the epidemiology of the disease and to identify the cases in rural and nomad regions of this province. The studied population was a rural and nomadic population of nine districts of Lorestan province, of which 1053 were selected according to the population of each studied county based on random sampling in 2016-2017. Initially, a questionnaire was completed for each person, including age, gender, education, occupation, use of local native aquatic plants and history of travel to the northern provinces of the country where fasciolosis has been reported mostly. Then, 5 ml blood samples were taken and the samples were evaluated as for anti-Fasciola specific antibodies using ELISA technique. Overall, 1053 individuals were participated, of which 28 (2.66%) were infected with fasciolosis and 18 positive cases were female. The highest infection rate was in the age group of 20-29 years (23%) followed by 30-39 years of age (22%). There was no significant difference between the rate of infection in terms of gender (P = 0.89), age (P = 0.15), travel history to the northern provinces of the country (P = 0.089), history of aquatic plant consumption called Balmak natively (P = 0.48), history of surface water consumption (springs, streams) (P = 0.18), and occupation (P = 0.43). Considering the results of current and previous studies it seems that the disease in the Lorestan province is expanding and new foci in different parts of the province are formed or are being formed. Therefore, the preventive measures, control and treatment should be taken in areas with parasites transmission.

Morphological and molecular identification of Culicidae mosquitoes (Diptera: Culicidae) in Lorestan province, Western Iran.

Culicidae mosquitoes are main vectors of arboviruses that cause arboviral diseases in humans. Studies on fauna, ecology, biology, resting behaviors of Culicidae mosquitoes are important and greatly impacts the control of arboviral diseases that are transmitted by vectors. The aim of the present study was to determine fauna of mosquitoes (Diptera: Culicidae) based on morphological and molecular (genomic) identification and their habitats in Lorestan province, Western Iran. Meanwhile mosquito samples were examined for arbovirus infection. Culicidae mosquitoes were caught in 2015 and 2016 from human homes, animal dwellings, storehouses and pit shelters in Lorestan province, Western Iran, using an oral aspirator (hand catch), total catch, human and animal bait and light trap methods. The samples were identified on the genus and species. Six species of Culex and eight species of Anopheles were caught. One complex species (Cx. pipiens complex) and a hybrid between Cx. pipiens pipiens biotype pipiens and Cx. pipiens pipiens biotype molestus were identified. Among all of the trapped mosquitoes (4211), 94.68% were from genus Culex mosquitoes (3987), which indicate that this genus is the dominant in Lorestan province, Western Iran. Anopheles comprised of 201 individuals out of the total catch. Arboviruses were not detected in these samples.

The first molecular detection of a Theileria-like species (Apicomplexa: Piroplasmida) in Meriones persicus from western Iran.

The archived blood of rodents species such as Meriones, Mus, and Microtus species was investigated in order to detect any piroplasms species. In this study 18S rRNA target gene of piroplasm parasite was amplified by PCR in a Meriones persicus; so, the Locus 1 and Locus 2 of 18S rRNA were sequenced, successfully. A Theileria-like taxa was suspected in accordance with the BLAST analysis of 18S rRNA L1 and L2 with 96% and 91% sequence homology, respectively. The present study was the first report of a Theileria-like species in M. persicus from Iran.

Efficiency of Two Capture Methods Providing Live Sand Flies and Assessment the Susceptibility Status of Phlebotomus papatasi (Diptera: Psychodidae) in the Foci of Cutaneous Leishmaniasis, Lorestan Province, Western Iran.

BACKGROUND: The aims of this study were to evaluate the efficiency of two capture methods for providing live sandflies used for determining the susceptibility level of Phlebotomus papatasi, the main vector of zoonotic cutaneous leishmaniasis in Lorestan Province, west of Iran. METHODS: The sand flies were collected from indoor and outdoor by hand-catch and baited traps during the peak of seasonal activity. The susceptibility level of sand flies was assessed using insecticide-impregnated papers against DDT 4%, bendiocarb 0.1%, permethrin 0.75%, deltamethrin 0.05%, and cyfluthrin 0.15%. RESULTS: A total of 2486 live sandflies were caught from both indoor and outdoor places. Totally 849 sand flies were caught from outdoors with a sex ratio(SR) 0.1 versus 1637 sand flies collected from indoor using the hand-catch method with SR= 0.6. The dominant species of sand flies was Ph. papatasi in the study area. Mortality rates of outdoor-collected sand flies were exposed to DDT 4%, deltamethrin 0.05%, permethrin 0.75%, and bendiocarb 0.1%, and mortality rate ranged from 92.0-97.9% and for indoor-collected sand flies were 87.7-96.8%. Both outdoor and indoor collected sand flies were susceptible to cyfluthrin 0.15% that caused 100% mortality. CONCLUSION: Based on the findings, the most appropriate method for collecting the live female Ph. papatasi is the baited traps due to providing enough females is necessary for conducting the susceptibility tests. The finding indicated that Ph. papatasi was resistant to DDT, under 'verification required' status to deltamethrin, permethrin, bendiocarb, and susceptible to cyfluthrin.

Molecular detection of Coxiella (Gammaproteobacteria: Coxiellaceae) in Argas persicus and Alveonasus canestrinii (Acari: Argasidae) from Iran.

BACKGROUND: Coxiella burnetii and non-C. burnetii bacteria or endosymbiotic Coxiella-like were reported in various tick species. We aimed to detect C. burnetii within soft tick species, Argas persicus and Alveonasus canestrinii. METHODS: Argasid ticks were collected from different counties of Lorestan province, west of Iran. Partial fragments of 16S rRNA, IS1111 insertion sequence, com1, htpB, and icd genes related to Coxiella genus were sequenced. RESULTS: A partial 16S rRNA and com1 gene fragment as well as IS1111 was detected in four Ar. persicus and twelve Al. canestrinii pools. Moreover, partial htpB and icd gene was only detected in one pool of Ar. persicus. CONCLUSIONS: Detection of C. burnetii in tick samples was failed due to the occurrence of Coxiella-like endosymbionts and leads to misidentification. Thus, the house-keeping genes should be designated to distinguish C. burnetii within Coxiella-like endosymbionts.

Correction: DNA barcodes corroborating identification of mosquito species and multiplex real-time PCR differentiating Culex pipiens complex and Culex torrentium in Iran.

[This corrects the article DOI: 10.1371/journal.pone.0207308.].

Seroepidemiological study of Q fever, brucellosis and tularemia in butchers and slaughterhouses workers in Lorestan, western of Iran.

Most zoonoses are occupational diseases. Q fever, brucellosis and tularemia are major zoonotic diseases for butchers and slaughterhouse workers. However, little information is available about these infectious diseases in such professional populations in western of Iran. The aim of this study was to investigate the seroprevalence and risk factors associated with these three zoonoses among butchers and slaughterhouse workers in the Lorestan province of Iran. In 2017, 289 individuals (144 butchers or slaughterhouse workers, and 145 people from the general population) were enrolled in 11 different counties of this province. Collected serum samples were tested by ELISA for detection of IgG antibodies against Coxiella burnetii, Brucella spp. or Francisella tularensis antigens. The seroprevalence of Q fever, brucellosis and tularemia among all participants were 23.5%, 31.8% and 3.8%, respectively. The seroprevalence of brucellosis and Q fever among butchers and slaughterhouse workers (43.7% and 29.8%, respectively) were significantly higher (p < 0.05) than those of the general population (20% and 17.2%, respectively). A contact history with small ruminants (sheep and goats) was associated with a higher risk of positive serology for all three studied zoonoses. The high seroprevalence for Q fever and brucellosis we found among butchers and slaughterhouse workers suggests that both diseases are common in these populations of the Lorestan province. Since these two infectious diseases are clinically unspecific, they must be systematically included in the etiological diagnosis of infectious diseases occurring in these at-risk populations. In addition, we recommend specific training programs as well as the use of personal protective equipment in these occupational groups to reduce the occurrence of these zoonotic diseases.

Host-feeding patterns of Culex mosquitoes in Iran.

BACKGROUND: Different mosquito-borne pathogens are circulating in Iran including Sindbis virus, West Nile virus, filarioid worms and malaria parasites. However, the local transmission cycles of these pathogenic agents are poorly understood, especially because ecological data on vector species are scarce and there is limited knowledge about the host range; this understanding could help to direct species-specific vector control measurements or to prioritize research. METHODS: In the summers of 2015 and 2016, blood-fed mosquitoes were collected at 13 trapping sites on the coast of the Caspian Sea in northern Iran and at an additional trapping site in western Iran. Mosquitoes were generally collected with either a Biogents Sentinel trap or a Heavy Duty Encephalitis Vector Survey trap installed outside. A handheld aspirator was used at the trapping site in western Iran, in addition to a few samplings around the other trapping sites. On average, eight trapping periods were conducted per trapping site. The sources of blood meals were identified using a DNA barcoding approach targeting the cytochrome b or 16S rRNA gene fragment. RESULTS: The source of blood meals for 580 blood-fed mosquito specimens of 20 different taxa were determined, resulting in the identification of 13 different host species (9 mammals including humans, 3 birds and 1 reptile), whereby no mixed blood meals were detected. Five mosquito species represented more than 85.8% of all collected blood-fed specimens: Culex pipiens pipiens form pipiens (305 specimens, 55.7% of all mosquito specimens), Cx. theileri (60, 10.9%), Cx. sitiens (51, 9.3%), Cx. perexiguus (29, 5.3%) and Anopheles superpictus (25, 4.6%). The most commonly detected hosts of the four most abundant mosquito species were humans (Homo sapiens; 224 mosquito specimens, 40.9% of all mosquito specimens), cattle (Bos taurus; 171, 31.2%) and ducks (Anas spp.; 75, 13.7%). These four mosquito species had similar host-feeding patterns. The only exceptions were a relatively high proportion of birds for Cx. pipiens pipiens f. pipiens (23.2% of detected blood meal sources) and a high proportion of non-human mammals for Cx. theileri (73.4%). Trapping month, surrounding area, or trapping method had no statistically significant impact on the observed host-feeding patterns of Cx. pipiens pipiens f. pipiens. CONCLUSIONS: Due to the diverse and overlapping host-feeding patterns, several mosquito species must be considered as potential enzootic and bridge vectors for diverse mosquito-borne pathogens in Iran. Most species can potentially transmit pathogens between mammals as well as between mammals and birds, which might be the result of a similar host selection or a high dependence on the host availability.

DNA barcodes corroborating identification of mosquito species and multiplex real-time PCR differentiating Culex pipiens complex and Culex torrentium in Iran.

Identifying mosquito species is a fundamental step in risk assessment and implementation of preventative strategies. Moreover, Culex pipiens is the most widespread mosquito vector in several regions of Iran and is the main vector for transmission of West Nile virus (WNV). Mosquitoes were collected at 14 sites in northern regions of Iran in 2015 and 2016. A subset of mosquito specimens was selected for identification confirmation using a DNA-barcoding technique. Construction of a phylogenetic tree showed clustering of mosquito sequences into three main genera: Aedes, Anopheles and Culex with individuals of a single species clustered closely together, regardless of where and when they were collected. Cx. pipiens complex and Cx. torrentium were identified and differentiated using multiplex real-time PCR targeting the gene locus for acetylcholinesterase 2 (ace2) to discriminate between Cx. pipiens pipiens and Cx. torrentium. The CQ11 microsatellite locus was used for discrimination between Cpp. biotypes. The predominant mosquito species in investigated regions were Cx. pipiens pipiens biotype pipiens, but we also detected Culex pipiens pipiens biotype molestus and hybrids of the two pipiens biotypes, as well as Cx. torrentium. The results of this study represent the first certain evidence of the presence of Cx. pipiens pipiens biotype molestus and hybrids between pipiens and molestus forms, and Cx. torrentium in Iran through a molecular identification approach. This report of a potentially important bridge vector for WNV might have key influence in the risk projections for WNV in Iran.

Seroepidemiological study of Q fever in Lorestan province, western Iran, 2014.

BACKGROUND AND OBJECTIVES: Q fever is a zoonotic disease and farm animals serve as the main reservoir of the disease. The aim of this study was to evaluate the seroprevalence of Q fever in sheep, in Lorestan province in western Iran. MATERIALS AND METHODS: In this cross-sectional study, 330 blood samples were collected from sheep, from each county in Lorestan province. The samples were tested by ELISA for the presence of immunoglobulin (IgG) against Coxiella burnetii. RESULTS: Among the samples tested, 45 samples (13.64%) were seropositive. Of 35 studied herds, 21 (60%) had a history of infection. In terms of number of positive samples, there was no significant difference between the three geographical regions (central, west and east) (p=0.687). There was no statistically significant difference between age groups (p =0.604). Gender also had no effect on infection rates, in female and male sheep (p =0.814). No significant difference was observed between the number of lactation and positive serology (p =0.376). The rate of infection with Q fever and abortion also had no statistically significant difference (p =0.152). CONCLUSION: The findings of this study showed that sheep in Lorestan were infected by Q fever and the cycle of disease transmission had been established between animals and ticks.

The Potential Use of Methotrexate in the Treatment of Cutaneous Leishmaniasis: In Vitro Assays against Sensitive and Meglumine Antimoniate-resistant Strains of Leishmania tropica.

BACKGROUND: The present study aimed to evaluate the effect of methotrexate (MTX) alone and in combination with meglumine antimoniate (MA, Glucantime) against sensitive and MA-resistant Leishmania tropica stages in vitro. METHODS: The present study was carried out in 2014 in Leishmaniasis Research Center at School of Medicine, Kerman University of Medical sciences, Kerman, Iran. The effects of MTX alone and along with MA on promastigote and amastigote stages of sensitive (SS) and MA-resistant (RS) L. tropica strains have been evaluated using a colorimetric MTT assay and in a macrophage model, respectively. In addition, the inhibitory effect of MTX on the Leishmania invasion of murine macrophages was assessed in promastigotes of both strains of L. tropica. Sensitive and MA resistant L. tropica are referred to those isolates that are responsive or non-responsive to one or two courses of treatment by MA systemically and/or intralesionally, respectively. RESULTS: The findings of OD and IC50 showed that MTX plus MA (SS: 16.1 µg/ml, RS: 39.8 µg/ml) had a higher anti-leishmanial effect than MA (SS: 52.2 µg/ml, RS: 170 µg/ml) or MTX alone (SS: 22.2 µg/ml, RS: 51.4 µg/ml) on promastigotes of both strains of L. tropica. The MTX plus MA caused a significant decrease (P<0.05) in the mean infection rate (MIR) and the mean number of amastigotes in each macrophage compared with positive control. Infectivity of promastigotes is significantly (P<0.05) reduced when it was preincubated with MTX. CONCLUSION: This study indicated high potency and a synergistic effect of MTX on MA in inhibiting the growth rateof promastigote and amastigote stages of sensitive and meglumine antimoniate-resistant L. tropica. Further works are needed to evaluate the anti-leishmanial effects of MTX on L. tropica using a clinical setting.

Control of Cutaneous Leishmaniasis Using Deltamethrin Treated Nets in Comparison to Indoors Residual Spraying in a Rural Area of Iran.

BACKGROUND: Cutaneous leishmaniasis (CL) is an important vector-borne disease in Iran. The present study aimed to evaluate the efficacy of Deltamethrin treated nets in comparison to Indoors Residual Spraying (IRS) for the control of leishmaniasis in a new focal area of Iran. METHODS: The study was carried out in 5 villages in the west of Iran during 2012-2013. One village was selected to the application of Insecticide Treated Nets (ITNs, 25 mg/m2 deltamethrin) among residents. Four other villages were considered for IRS using deltamethrin emulsion, 2.5% EG and Aicon 10WP. Spraying was repeated after 3 month. Other control methods e.g. environmental managements, and treatment of positive cases were carried out in all villages. RESULTS: After 1 yr of trial, the cases of disease reduced from 50 to 2 and 128 to 5 for ITNs distributed nets and use of IRS in the villages, respectively. CONCLUSION: Reduction of CL cases in both methods were significant (P<0.001).

West Nile Virus lineage-2 in Culex specimens from Iran.

OBJECTIVE: Screening of mosquitoes for viruses is an important forecasting tool for emerging and re-emerging arboviruses. Iran has been known to harbour medically important arboviruses such as West Nile virus (WNV) and dengue virus (DENV) based on seroepidemiological data. However, there are no data about the potential mosquito vectors for arboviruses in Iran. This study was performed to provide mosquito and arbovirus data from Iran. MATERIALS AND METHODS: A total of 32 317 mosquitos were collected at 16 sites in five provinces of Iran in 2015 and 2016. RT-PCR for detection of flaviviruses was performed. The PCR amplicons were sequenced, and 109 WNV sequences, including one obtained in this study, were used for phylogenetic analyses. RESULTS: The 32 317 mosquito specimens belonging to 25 species were morphologically distinguished and distributed into 1222 pools. Culex pipiens s.l. comprised 56.429%. One mosquito pool (0.08%), containing 46 unfed Cx. pipiens pipiens form pipiens (Cpp) captured in August 2015, was positive for flavivirus RNA. Subsequent sequencing and phylogenetic analyses revealed that the detected Iranian WNV strain belongs to lineage 2 and clusters with a strain recently detected in humans. No flaviviruses other than WNV were detected in the mosquito pools. CONCLUSION: Cpp could be a vector for WNV in Iran. Our findings indicate recent circulation of WNV lineage-2 strain in Iran and provide a solid base for more targeted arbovirus surveillance programs.

Prevalence of HIV, HBV, and HCV and Related Risk Factors amongst Male Homeless People in Lorestan Province, the West of Iran.

BACKGROUND: Homeless people are at a higher risk of blood-born infectious diseases. The aim of this study was to estimate the prevalence of HIV, HBV, HCV and related risk factors among male homeless people. STUDY DESIGN: A cross-sectional study. METHODS: This study was conducted in Khorramabad City, western Iran from January to June 2015. A pre-designed validated questionnaire was used to collect the data on behavioral and other potential risk factors. Blood samples were taken in order to diagnose HIV, hepatitis B and C infections. The prevalence of HIV, hepatitis B, C and related risk factors was reported with a 95% confidence interval (CI). RESULTS: The participants were 307 male homeless people with a mean (±SD) age of 35.86 (±9.62) yr. The prevalence of HIV, HBs Ag, and HCV Ab positive cases was 6.51% (95% CI: 4.23, 9.90), 0.98% (95% CI: 0.31, 3.00), and 31.27% (26.31, 36.71), respectively. The prevalence of co-infections of HIV and HCV Ab+ was 5.76% (95% CI: 1.34, 8.51). The most common recently used drugs were heroin, methamphetamine, and opium with a prevalence of 44.30%, 41.04%, and 24.76%, correspondingly. CONCLUSIONS: According to this study, prevalence of HIV and hepatitis C among homeless people was considerable. Abusing heroin, methamphetamine, and industrial drugs was also significant. Considering the association between drug abuse, HIV, and hepatitis C infections, planning for effective control and preventive interventions is important in homeless people.

Short and long term evaluation of the efficiency of PermaNet® 2.0 bed net against environmental factors and washing using bioassay tests.

The aim of the present study was to examine the resistance of PermaNet® 2.0 bed nets against repeated washing and environmental factors by using bioassay tests. After 5, 15 and 21 washings with detergents and by using bioassay tests, the resistance of 40 PermaNet® 2.0 bed nets was compared with that of 40 bed nets conventionally treated with one K-O tablet. To examine the long-term resistance, 31 PermaNet® 2.0 bed nets were also distributed among villagers, and were re-collected to perform bioassay tests after 1, 2 and 5 years. In the first phase of this study, the insecticidal effect of the conventionally-treated nets significantly decreased due to repeated washings (P < 0.001); however, it was not significant regarding PermaNet® 2.0 bed nets (P = 0.92 in continuous exposure and P = 0.12 in mortality tests). In the long-term phase of this study, the time required for knockdown of PermaNet® 2.0 increased over the first 2 years and then decreased. In addition, the mortality rate decreased over the first 2 years and then increased. In conclusion, it seems that the technique used by the manufacturer for impregnation of PermaNet® 2.0 bed nets has an acceptable efficiency in comparison with conventional techniques.