Background: Drugs’ pharmacokinetics and pharmacodynamics can be affected by diverse genetic variations, within which simple nucleotide polymorphisms (SNPs) are the most common. Genetic variability is one of the factors that could explain questions like why a given drug does not have the desired effect or why do adverse drug reactions arise.

Methods: In this retrospective observational study, literature search limits were set within PubMed database as well as the epidemiological bulletins published by the Mexican Ministry of Health, from Jan 1st 2001 to Mar 31st 2017 (16 years).

Results: Metabolism of antiparasitic drugs and their interindividual responses are mainly modified by variations in cytochrome P450 enzymes. These enzymes show high frequencies of polymorphic variability thus affecting the expression of CYP2C, CYP2A, CYP2A6, CYP2D6, CYP2E6 and CYP2A6 isoforms. Research in this field opens the door to new personalized treatment approaches in medicine.

Conclusion: Clinical and pharmacological utility yield by applying pharmacogenetics to antiparasitic treatments is not intended as a mean to improve the prescription process, but to select or exclude patients that could present adverse drug reactions as well as to evaluate genetic alterations which result in a diversity of responses, ultimately seeking to provide a more effective and safe treatment; therefore choosing a proper dose for the appropriate patient and the optimal treatment duration. Furthermore, pharmacogenetics assists in the development of vaccines. In other words, the aim of this discipline is to find therapeutic targets allowing personalized treatments.

Background: Rhipicephalus sanguineus is the most widely distributed tick in the world, which is partly due to its biological flexibility and the global distribution of its major host, the domestic dog. In Mediterranean region it could be principal reservoir host for Leishmania infantum, usually transmitted by the phlebotomine sand flies. In this study, we evaluated the vector potential of R. sanguineus in transmitting L. infantum to uninfected dogs.

Methods: During 2014, five dogs with clinical manifestations of canine visceral leishmaniasis (CVL), high anti-Leishmania antibody titers and tick infestation, were selected from CVL endemic areas (Tehran and Alborz provinces). At least, twenty live ticks were removed from each infected dog. After morphological identification, the ticks were divided into two groups; ticks belonging to the first group were dissected for parasitological examinations and semi-nested PCR assay, and those of the second group were selected for the transmission of CVL caused by L. infantum to uninfected dogs. Following tick infestation, all uninfected dogs were kept for 9 months and examined monthly for clinical and serological tests.

Results: Nearly, 67% of ticks were infected by L. infantum using the semi-nested PCR. All other parasitological tests of ticks were negative. Clinical examinations and serological tests of the investigated dogs revealed negative results. Nested-PCR test results performed on splenic biopsy samples of dogs were also negative.

Background: Cystic echinococcosis (CE), as a zoonotic disease cause to health threat and economic losses. Despite implemented control programs, few countries have been able to decrease or eliminate this infection. Vaccination of the intermediate host offers an additional strategy to control the parasite transmission and EG95 antigen is considered more than the others in the vaccine issue. According to the high protection induced by the EG95 recombinant vaccine, this study was designed to construct recombinant plasmid formulation of EG95 antigen.

Methods: In 2015, the Echinococcus granulosus eggs were recovered from an infected dog in Parasitological laboratory of Tarbiat Modares University in Tehran, Iran. Following hatching, the oncospheres of E. granulosus were activated to increase the presence of the desired mRNA. The extracted mRNA was transcribed to the cDNA which used as template in RT-PCR. Then the EG95 gene cloned into pET28a vector and the recombinant plasmids expression was  investigated in prokaryotic and eukaryotic cells.

Results:  The recombinant plasmid encoding EG95 antigen was successfully constructed and identified by PCR, restriction enzyme digestion and sequencing. In vitro expression of the EG95 antigen was confirmed in prokary­otic and eukaryotic systems by SDS-PAGE and western blotting analysis.

Conclusion: Because of potential advantages of DNA vaccines, including ability to induce long-term immune responses, low production cost and stability in different temperatures, this study carried out to construct the EG95 gene into a vector. This recombinant vector can be evaluated in further studies as a DNA vaccine may provide new prospects for the development of a vaccine against cystic hydatid disease.

Background: The objective of this study was to clone, express and characterize the gene encoding rhomboid 4 (ROM4) proteins, a vital gene in surface adhesion and host cell invasion process of tachyzoite of T. gondii in an appropriate expression vector and eukaryotic cell for production of recombinant protein.

Methods: Toxoplasma RNA was isolated from tachyzoites (RH strain) and complementary DNA was synthesized. Oligonucleotide primer pair was designed based on Toxoplasma ROM4 gene sequence with XhoI and EcoRI restriction sites at 5´ end of forward and reverse primers, respectively. ROM4 gene was amplified by PCR, cloned into pTG19-T vector and the recombinant plasmid was sequenced. The gene was subcloned into pcDNA3 plasmid and expressed in CHO cells as eukaryotic cell. SDS-PAGE and western blotting were performed for protein determination and verification.

Results: Cloning of ROM4 gene in pTG19-T vector was confirmed by colony-PCR and enzymatic digestion. The results of enzymatic digestion and gene sequencing confirmed successful cloning and subcloning procedures. The nucleotide sequence of the cloned ROM4 gene showed 99% homology compared to the corresponding sequences of original gene. SDS-PAGE and western blotting analyses of the purified protein revealed a single band having expected size of 65 kDa.

Background: We employed a highly sensitive loop-mediated isothermal amplification (LAMP) by targeting 18S rRNA gene to identify the rapid mass screening of Leishmania infections in captured sand flies of southwest Iran and In vitro culture.

Methods: One hundred fifty sand flies were collected from 11 sites adjacent to Iraqi’s borders in southern parts of Khuzestan Province by using sticky sheets of paper and CDC miniature light traps during late May 2014 to Nov 2015. Following morphological identification of sand flies species, the DNA of infected samples was extracted and amplified by PCR and LAMP assays by targeting ITS-rDNA and 18S rRNA genes. The PCR amplicons were directly sequenced to conduct the phylogenetic analysis

Results: Ten (6.6%) Leishmania infections were identified by LAMP assay (detection limit 0.01 parasites DNA) among infected Sergentomyia baghdadis, S. sintoni and Phlebotomus papatasi sand flies that was more sensitive than PCR (n=6.4%; (detection limit 10¹parasites DNA). LAMP can identify 10¹-10⁶promastigotes/100 µl RPMI 1640 while PCR recognized10⁴-10⁶ promastigotes. The majority infection rate of sand flies was confirmed to L. major inferred by phylogenetic analysis.

Conclusion: This is the first exploration characterized the Old World Leishmania infections by LAMP technique in both infected sand flies and In vitro conditions. The LAMP method because of its shorter reaction time, robustness, more sensitivity, lack of requirement of complicated equipment and visual discriminatory of positivity can be appeared a promising tool instead of PCR to identify low Leishmania loads and entomological monitoring of leishmaniasis in resource-limited endemic of the world.

Background: Acanthamoeba spp. is potentially pathogenic free-living amoeba that can exist in various water sources. The presence of this amoeba in water sources could be a health hazard as Acanthamoeba could lead to severe diseases such as Acanthamoeba keratitis and encephalitis. This study aimed to determine the genotypes of isolated Acanthamoeba spp. in raw wastewater samples in Tehran, Iran.

Methods: Overall, 90 raw wastewater samples were collected from water treatment facilities in west and south of Tehran, Iran during 2014-2016. Water samples were filtered and cultured on non-nutrient agar (NNA) medium enriched with Escherichia coli. Morphological and molecular analyses were done on positive strains. The pathogenic ability of the isolated strains was determined using physical assays.

Results: Totally, 6 out of 90 (6.7%) samples were positive for Acanthamoeba, according to morphological characteristics of double-walled cysts. Genotyping and sequencing of the positive strains showed Acanthamoeba belonging to T4 (83%) and T11 (17%) genotypes. In vitro pathogenicity tests were revealed that five isolates were classified as non-pathogenic strains and one strain belonging to T4 genotype was classified as the highly pathogenic amoebae.

Background: This study is the first phylogenetic genotype analysis of Giardia lamblia in Iran. The main objective was to determine genotyping and identify the sub-assemblages of Giardia lamblia isolates involved in the transmission of giardiasis in Fars Province, south of Iran, in 2012.

Methods: Forty G. lamblia isolates were collected from the patient’s fecal samples with gastrointestinal discomfort referred to the health centers and hospitals in Shiraz, Fars Province, south of Iran. Purification of G. lamblia cysts from fecal samples and DNA extraction were performed using monolayer of sucrose density gradient and Phenol-Chloroform-Isoamylalcohol (PCI) respectively. Semi-nested PCR and sequence analysis were then performed using the primers (GDHeF, GDHiF, and GDHiR) which amplified a 432-bp fragment of Giardia glutamate dehydrogenase (gdh) gene. Phylogenetic analysis was carried out using a neighbor-joining tree composed of the nucleotide sequences of G. lamblia isolates obtained in this study and the known sequences isolates published in GenBank.

Results: G. lamblia sub-assemblage AII was the most prevalent genotype with 80% of the cases and 20% of the cases belong to sub-assemblage BIII and BIV based on the DNA sequence of the gdh. G. lamblia isolates at Fars Province were widely distributed within assemblage A cluster (sub-assemblage AII) and the remaining isolates were dispersed throughout the assemblage B cluster (sub-assemblage BIII and BIV).

Conclusion: PCR Sequencing and phylogenetic analysis was a proper molecular method for genotyping and discriminating of the of G. lamblia sub-assemblages in fecal samples, using the glutamate dehydrogenase gene that suggests a human contamination origin of giardiasis.

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. 

Background: Cutaneous leishmaniasis (CL) is associated with a broad and complex clinical spectrum of diseases. The objectives of this study were to assess the clinical features and identification of the causative agents of CL in a well-known focus of anthroponotic CL (ACL) caused by Leishmania tropica, southeast Iran.

Methods: This study was performed randomly as a descriptive cross-sectional survey to evaluate 2000 CL patients by active and passive case-detection approaches in Kerman Province from 1994 to 2014. The ACL patients were confirmed by direct smear and 600 cases by one or a combination of intrinsic methods.

Results: Children aged <10 yr old were the most infected patients (P<0.001). The majority of the CL lesions were located in hands (46.3%), face (34.1%), legs (14.3%), and other parts of the body (5.3%). The mean number of lesions was 1.5 and most of the patients had single lesion (65%).Typical clinical lesions included papule (36.8%), followed by ulcerated nodule (20.7%), plaque (18.4%), and ulcerated plaque (18.5%). While among atypical clinical features, leishmaniasis recidivans (LR) (4.7%) and leishmanid (0.3%) were the dominant forms, followed by diffuse, disseminated, sporotrichoid, and erysipeloid types, 0.1% each, and then lymphedematous, lymphadenic, hyperkeratotic, paronychial, and mutilating types, 0.05% each. Based on various intrinsic methods the parasites isolated from the lesions were characterized as L. tropica.

Background: This cross-sectional investigation aimed to evaluate the prevalence of IgM and IgG anti- Toxoplasma gondii antibodies and the associated risk factors among healthy blood donors in Khorasan Razavi Province, northeast of Iran from Nov 2014 to May 2015.

Methods: Overall, 491 serum samples from apparently healthy blood donors referred the six biggest blood centers in Razavi Khorasan, Iran, were screened for IgG and IgM anti-T. gondii antibodies by enzyme-linked immunosorbent assay (ELISA). A structured questionnaire was used to obtain information on risk factors for T. gondii infection. Nested PCR was also used to detect DNA of T. gondii in the IgM-positive samples by using of B1 and RE (Repetitive Element) as marker for amplifying fragment size of 531 bp and 164 bp in PCR method.

Results: Totally, 200 (40.7%) samples were seropositive for anti-T. gondii antibodies; 184 (37.5%) donors tested seropositive for only IgG antibody, 8 (1.6%) tested seropositive for both IgM and IgG and 8 (1.6%) were positive for IgM antibody alone. Several risk factors significantly related to T. gondii seropositivity in the univariate analysis at P<0.05 included age (P<0.001), and raw/half-cocked meat consumption (P=0.015). T. gondii DNA was found in all sixteen IgM-positive samples.

Conclusion: T. gondii infection was present among healthy blood donors in northeast of Iran. Thus, it is suggested to design screening programs for preventing transfusion-transmitted toxoplasmosis.

Background: This work studied the natural infection of Neospora caninum during the first gestation of heifers in a dairy farm in animals consuming a ration contaminated naturally with Zearalenone (ZEA), and to find out effect of mycotoxin in the levels of estrogen (E) and progesterone (P4) and that`s relation to the infection to N. caninum and in the abortions.

Methods: The study was conducted in a dairy farm located in El Llano municipality, in Aguascalientes, Mexico, in 2013. Two groups were formed, the group “A” with 20 seronegative animals to N. caninum, and group “B” with 20 seropositive. Once a month was determined the levels of total IgG to N. caninum, the serum concentration of E and P4, and the level of ZEA in the ration; in cases of abortion, fetal brain samples were taken to identify the presence of N. caninum DNA.

Results: In group “A”, was observed two subgroups: seronegative (60%) and seroconverted (40%), and three abortions. In group “B”, all animals maintain their serostatus, and three animals aborted. All abortions were positive for N. caninum DNA. The level of ZEA in the ration has an average of 426 µg/kg; during the gestation did not identify that animals suffer any alteration in the levels of E or P4. No statistical differences among the studied variables (levels of E and P) in time (nine months of gestation) were detected. It was not identified any interaction with the natural exposure to ZEA intake in any of the groups under study.

Conclusion: The chronic ingestion of ZEA does not affect serum concentrations of E and P4 during gestation of heifers under study and cannot be related to the infection for N. caninum and the abortion.

Background: Rodents are considered as reservoirs of various zoonotic diseases including helminthic infections. The current study aimed to evaluate the prevalence of helminth infections in rodents, in Boyer-Ahmad district, Southwestern Iran.

Methods: Overall, 52 rodents were captured from various areas of the district by Sherman live traps. The animals were then euthanized and dissected. During necropsy, each organ was examined macroscopically for presence of any cyst or visible parasite. The gastrointestinal tract was removed and their contents were evaluated for larva or adult worms. Trichinella larvae in the rodents’ muscles were investigated by both digestion and pathological methods.

Results: Twenty-eight (53.8%) of the trapped rodents were male. The rodents were including 25 (48.1%) Meriones persicus, 1(1.9%) Calomyscus bailwardi, 1 (1.9%) Arvicola terresterris, 7 (13.5%) Rattus rattus, 8 (15.4%) R. norvegicus, and 10 (19.2%) Apodemus sylvaticus. Of them, 38 (73.0%) were infected with at least one helminth. Collected rodents were infected with Hymenolepis diminuta (50%), Hymenolepis nana fraterna (28.8%), Skrjabinotaenia sp. (15.4%), Anoplocephalidae sp. (15.4%), Cysticercus fasciolaris (5.8%), Trichuris muris (36.5%), Aspiculuris tetraptera (15.4%), Syphacia sp. (5.7%), Rictularia sp. (15.4%), Trichostrongylus sp. (3.8%), and Gongylonema sp. (3.8%). M. persicus was the most (84%) infected rodent, yet the differences between rodent genus and helminth infectivity were not statistically significant (P>0.05).

Background: Blastocystis is a common protozoon that inhabits human intestinal tract and has a worldwide distribution. This study aimed to determine subtype (ST) distribution of Blastocystis among school-aged children in a western city of Turkey between Mar and Jun 2014.

Methods: This cross-sectional study was conducted among primary school children in Mugla between Mar and Jun 2014. Overall, 468 stool samples from children were examined by direct microscopy and inoculated into Jones medium. Blastocystis partial small subunit ribosomal RNA gene (SSU-rDNA) was amplified and sequenced from culture positive isolates. Subtypes were determined according to closest or exact match at GenBank and Blastocystis ST (18S) database.

Results: The positive rate of Blastocystis was 7.4% (n=35) with xenic in-vitro culture (XIVC). The subtypes could be identified for 33 (94.2%) isolates; 12 (34.2%) were ST3, 11 (31.4%) were ST1, 9 (25.7%) were ST2, one was (2.8%) ST7. No relationship was found between Blastocystis infected and non-infected cases in terms of gastrointestinal symptoms. Additionally, none of the possible risk factors was related to Blastocystis infection.

Conclusion: Subtypes in children was similar to those reported in most of the studies that found ST3 as the most common subtype. 

Background: Curcumin is the major active ingredient of Curcuma longa L., traditionally known as turmeric and has been shown to exhibit a wide range of pharmacological activities including anti-parasitic effect. However, it is found to be water-insoluble and has low bioavailability. The aim of this study was to explore the potential role of turmeric solved in olive oil either alone or in combination with praziquantel (PZQ) in treatment of schistosomiasis mansoni.

Methods: The whole turmeric powder suspended in olive oil (as a solvent) is indicated to S. mansoni-infected mice aiming to study its potential therapeutic role, either alone or in combination with PZQ.

Results: Turmeric significantly reduced S. mansoni worm burden and complete absence of adult worms achieved in mice treated with combination of turmeric and PZQ. Turmeric has slight non-significant effect on the oogram pattern in all examined S. mansoni infected mice. Turmeric and PZQ found to exert a significant reduction of granuloma size in comparison with control. However, turmeric has a non-significant effect on granuloma number. On the other hand, turmeric or/and PZQ treated mice showed obvious improvement of pathology with mild cloudy swelling and less hydropic degeneration.

Background: Theileria equi is a tick borne protozoan parasite which causes piroplasmosis among equines worldwide. The present study was aimed to determine seroprevalence of T. equi in donkeys, horses, and mules from two equine populated districts (Peshawar and Charsadda) of Khyber Pakhtunkhwa (KPK), Pakistan.

Methods: A total of 393 equine (195 horses, 194 donkeys and 4 mules) serum samples were collected from five and four randomly selected localities in Charsadda (n = 193) and Peshawar (n = 200), respectively. The presence of antibodies to T. equi was determined using a commercially available competitive enzyme-linked immunosorbent assay.

Results: An overall seroprevalence of 38.2% (n=150) was observed among all the tested animals suggesting a higher seropositivity among equids belonging to Charsada (50.3%) as compared to Peshawar (27.5%). Binary logistic regression analysis revealed that being a donkey (OR 2.94), having tick infestation (OR 4.32), history of voiding red (i.e., blood containing) urine (OR 3.97) and anemia (OR 2.1) were the factors significantly associated with the seroprevalence of T. equi. For animals with higher anti-T. equi antibody titers, a strong association of seroprevalence for T. equi was recorded with species, age, sex, tick infestation, anemia and history of hematuria.

Conclusion: The present study indicates a high level of exposure of working equids to T. equi in KPK region, Pakistan. Future studies should focus on tick vector identification and other factors responsible for spread of the disease. 

Abstract

Background: Stray dogs are considered potential reservoirs for zoonotic diseases. Previous helminthic surveys in Iran, have accounted for mainly species of nematodes and cestodes, and rarely digeneans.

Methods: We accessed 42 car-crashed stray dogs from the Farah Abad Region in the Mazandaran Province (North Iran) between Oct 2012 and Dec 2013, to be inspected for parasites. Helminths were collected from the intestine and they were morphologically studied.

Results: We found five adult digeneans from the family Brachylaimidae, identified as Brachylaima sp. Worms were assigned to the genus based on the shape of the body, the position of genital pore, cirrus sac and testes, and the extension of the vitellarium. Absence of additional information on the developmental stages of the parasite precluded its specific identification. As the geographic distribution of species of Brachylaima is restricted to the Mediterranean region, we raise the hypothesis that dogs may become infected with parasites through the consumption of helicid snails when searching for food on the street.

Conclusion: This is the second report of a species of Brachylaima in Iran and the third digenean species from stray dogs in the area. We want to raise the attention of researchers to helminthic surveys in potential zoonotic reservoirs like stray dogs. 

Background: Cystic Echinococcosis (CE) is one of the most common parasitic zoonosis caused by Echinococcus granulosus worldwide. This study investigated genotype diversity of Echinococcus granulosus isolated from stray dogs and golden jackals in Ilam province, West of Iran.

Methods: Adult worms were collected from the small intestine of the stray dogs and golden jackals from Ilam Province roads during 2012-2014. DNA was extracted from the adult worms and the partial mitochondrial NADH dehydrogenase subunit1 (nad1) was amplified by PCR then the products were digested by using HpaII, Rsa1, and Alu1 restriction enzymes. In order to confirm RFLP results, a number of PCR products were bi-directionally sequenced.

Results: Totally, 20 stray dogs out of 75 (26.66%) and two out of 73 (2.74%) golden jackal showed infection with E. granulosus. Amplified PCR product for all isolates was a band of approximately 550bp. Alu1 digested the product into two bands of approximately 160bp and 390bp fragments, while the Rsa1 cut the product into 320bp and 230bp fragments and the HpaII had no effect on the PCR product for both dog and jackal samples. The isolate sequences of mtDNAnad1 gene indicated 100% homology with references G1, G2 and G3 sequences in the GenBank database.

Conclusion: The genotype of adult E. granulosus was similar to larval stage genotypes of parasite (G1-G3 complex).

Background: The aim of present study was to evaluate antileishmanial effects of Lavandula angustifolia (L. angustifolia) and Rosmarinus officinalis (R. officinalis) medicinal plants essential oils and nano-emulsions on Leishmania major (L. major).

Methods: The present study was performed in Leishmaniasis Reference Lab at Mazandaran University of Medical Sciences, Iran during 2016-2017. The IC50 values were calculated in both the promastigote and amastigote stages in J774 macrophage in comparison with meglumine antimoniate (MA) as positive control. In addition, cytotoxicity effects of essential oils and nano-emulsions prepared from both plants against macrophages were evaluated.

Results: Both essential oil and nano-emulsion of Lavander and Rosemary showed anti-leishmania activity on promastigote with IC₅₀=0.11 μl/mL, IC₅₀=0.26 μl/mL, and IC₅₀=0.08 μl/mL respectively. Moreover, during amastigote assay, Lavander and Rosemary essential oils and nano-emulsion were effective at least in concentration of 0.12 μl/mL and 0.06 µl/mL (P=0.0001) respectively, on mean infected macrophages (MIR) and amastigotes in macrophages (P=0.0001). Additionally, cytotoxicity assay against macrophage revealed no toxicity on the host cells at IC₅₀ concentrations.

Background: Leishmaniasis manifests as visceral (VL), cutaneous (CL) or a dermal sequel of VL, known as Post kala-azar dermal leishmaniasis (PKDL). The aim of the study was to analyze the clinical and laboratory features of cases diagnosed with leishmaniasis.

Methods: This hospital-based retrospective study included all cases of VL, PKDL, and CL diagnosed between Jan 2011 to Jan 2016 at All India Institute of Medical Sciences, New Delhi. Clinical and laboratory profile of the diagnosed cases were analyzed in detail. All diagnosed cases were mapped according to the state and the district from which the cases originated.

Results: A total of 91 VL cases and 4 PKDL cases were reviewed. Only one case of CL (1 female) and mucocutaneous leishmaniasis (1 female) were observed during the study period. Majority of the cases of VL (75/91) originated from Bihar. The most common presenting symptoms in all our patients were fever (97.8%), weight loss (40.6%) and abdominal discomfort (17.6%) while the most common presenting signs were hepatosplenomegaly (45.8%), isolated splenomegaly (23.1%) and skin pigmentation (11%). The most common laboratory abnormality was anaemia followed by thrombocytopenia and leucopenia.

Conclusion: VL is globally recognized as a neglected tropical disease. Even after continued effort to bring down its transmission in India, it continues to affect the endemic states with reports from new pockets.