Background & objectives: Leishmaniasis, known as a disease with high prevalence proportion throughout the world, is caused by protozoan parasites. Visceral leishmaniasis is the most severe form of this condition reported sporadically from all regions in Iran. Between different diagnostic tests, serodiagnosis of this infection is of utmost importance in both humans and dogs. Although rK39 ELISA test has been extensively validated in endemic areas, there are currently challenges regarding a more appropriate serodiagnostic test. Methods: A novel multi-epitope construct was designed consisting of highexposedB cell epitopes using eight important antigens of Leishmania infantum (Gp63, KMP-11, HSP70, CPA, H2A, H3, LACK and TRYP). Our artificial sequence, a Multi-epitope Recombinant Protein (MRP), was consequently produced and purified. Then, immunoreactivity was investigated by ELISA test and western blotting as well. Results: In the present study, the cutoff value (1.052) for the new MRP-ELISA was determined by receiver operator characteristic (ROC) curve analysis using 35 known positive and 20 known negative HVL sera previously tested for antibodies to L. infantum by DAT, showing a sensitivity of 93.1% and a specificity of 77.4%. The blotting test also showed a favorable band to detect visceral leishmaniasis. Interpretation & conclusion: According to the results, this new antigen had acceptable potential in detecting VL positive cases once western blotting was utilized, but the ELISA test did not proceed as expected for detecting true negative cases, probably due to some optimization issues.The present study is a promising start.

Wolbachia, known for its reproductive manipulation capabilities in insects, are being implemented to control dengue and chikungunya. To understand Wolbachia biology and its utility as a bio-control for vector mosquito’s populations, we investigated its dissemination pattern in field in collected Ae. albopictus along with its maternal transmission efficacy over generations in regions of endemic dengue (DENV) transmission. Field collected Ae. albopictus were subjected to PCR for Wolbachia screening. Overall mean Wolbachia infection frequency in Ae. albopictus was found out to be 87.3% wherein a trend was observed in the pattern of maternal transmission across generations. χ² for trend revealed a significant variation between Wolbachia infections and non-infections in Ae. albopictus generations. Linear regression analysis revealed the involvement of a strong negative correlation, implying that overall Wolbachia infection tends to decrease in places with high dengue cases.The reduction in Wolbachia infection frequency may be attributed to several environmental factors with the probability of being the cause for endemicity of dengue in the studied areas.This study reports on the transmission efficacy of naturally occurring Wolbachia in successive generations of Ae. albopictus and its correlation with dengue cases in clusters of Odisha, India. Studying the transmission trend of Wolbachia along with transovarial transmission of DENV might be indicative towards the interplay of Wolbachia infection in presence/absence of DENV.

Background & objectives: Malaria control strategies for a particular area largely depends on the correct identification of the mosquito species prevalent in that area. This study aimed to develop an android mobile app named ‘Pictorial Indian anophelines key’, which can be used to identify the 58 species of Indian female adult anophelines based on morphological characteristics. Methods: The pre-requisite to use this app requires a minimum basic knowledge of mosquito morphological characteristics and features. The user needs to view the collected mosquito under a dissecting binocular microscope and has to select the observations on the app presented in the form of labelled picture format. The app uses a divide and conquer algorithm which narrows the selection from genus level to final identification of species. Results: The app is user-friendly, quick and it accurately identifies the mosquito species in a maximum of six consecutive clicks. The result displays a complete image of identified mosquito along with its quick identification features. It can be used for both subgenus Cellia and Anopheles. Interpretation & conclusion: This pictorial key-based mobile app could be helpful for entomologists, malariologists, researchers, public health workers as well as students in the quick and correct identification of anopheline mosquitoes prevalent in India which in turn could be helpful in devising appropriate approaches towards malaria control and prevention. The app can be modified as per future requirements.

Background & objectives: Alterations in plasma apolipoproteins in individuals with malaria infection and their potential roles in the pathogenesis are known but the link between the malaria parasite density and apolipoprotein A1 (apo-A1) level is insufficiently understood. This study was conducted to determine whether the plasma apo-A1 level is influenced by the degree of parasitaemia in malaria infections. Methods: In a case-control study, a convenient sample of children aged 2–10 years with uncomplicated malaria cases (UMC), asymptomatic parasitaemia cases (APC) and healthy children without parasitaemia (HCP) was recruited. The cases consisted of 61 UMC and 21 APC, while the controls consisted of 24 HCP. Levels of apo-A1 was determined using immunoturbidimetric assay and compared among the different degrees of parasite density. Results: Of the 82 participants with parasitaemia, density was ≤1000/μL in 12, 1001-10000/μL in 21 and >10000/μL in 49 children. There was significant difference among the mean values of apolipoprotein A1 of the three groups, viz: UMC [91.4 (95% CI: 81.3, 101.5) mg/dL], APC [67.0 (95% CI: 48.9, 84.9) mg/dL] and HCP [99.0 (95% CI: 76.6, 121.3) mg/dL], p=0.029. Post-hoc analysis revealed that the mean plasma level of apo-A1 in HCP was significantly higher than APC by 32.0±12.4 mg/dL and UMC by 7.5±4.2 mg/dL. However, there were no differences in the mean apolipoprotein A1 levels among the three groups of parasite density. Interpretation & conclusion: The presence of parasitaemia causes a remarkable reduction in apolipoprotein A1 level that was not influenced by the degree of parasitaemia.

Background & objectives: After launching of NFME and NSP with wide scale implementation of effective intervention tools during 2017, Odisha State of India observed a drastic reduction of malaria cases (81%) in 2018 and 88.6% in 2019, compared to 2017. The current study analysed the different factors causative for malaria reduction in Koraput district of Odisha State, India. Methods: The vector density and malaria incidences were assessed from January 2016 to December 2019 and human blood index, bio-efficacy and use rate of LLINs were assessed after distribution of long-lasting insecticidal nets. Results: The use rate of LLINs by the villagers ranged from 93.8% to 100% throughout the year. The mortality of Anopheles jeyporiensis remained 100.0% in both DuraNet and PermaNet LNs. The density of An. fluviatilis after distribution of LLINs reduced from 4.7 to 0.0 whereas, the density of An. culicifacies reduced from 12.2 to 9.3. The HBI of An. fluviatilis and An. culicifacies was 0.006 and 0.005, respectively after distribution of LLINs. The malaria incidences also drop from 14.2 to zero after distribution. Interpretation & conclusion: A significant reduction in malaria incidences was validated and the possible reasons for the reduction are discussed.

Background & objectives: Scrub typhus is a zoonotic rickettsial disease that is transmitted by the bite of the larval stage (chiggers) of trombiculid mites. The aim of this study was to determine the existence of scrub typhus in central and eastern Uttar Pradesh, India in patients with acute febrile illness (AFI) presenting to a super specialty tertiary level institute. Methods: This prospective hospital-based study was conducted for a period of one year, from August 2018 to July 2019. About 2–5 mL of blood samples, along with clinical, epidemiological, and demographic data from a total of 125 patients presenting with acute febrile illness to outpatient and inpatient departments, were collected. ELISA testing tested the sera from blood samples for IgM antibodies against scrub typhus. Samples were also tested for dengue, leptospirosis, malaria and typhoid. Results: During the study period, out of a total of 125 samples collected, 20% were found positive for IgM antibodies against scrub typhus. Demographically higher positivity was found in males, older age group, and in rural area. Rainfall was found to be important epidemiological parameter for presence of scrub typhus. Co-infection with dengue, leptospirosis and malaria was found. Interpretation & conclusion: Scrub typhus is found to be an important cause of acute febrile illness. It is necessary to include it in differential diagnosis of AFI cases even in absence of eschar. Diagnostic facilities of this as a screening test should be started in primary care centers or community health centers of rural areas of districts of central and eastern Uttar Pradesh, India.

Background & objectives: World Health Organization (WHO) revised its guidelines for classification and management of dengue in 2009. This revised system was found out to have good sensitivity and negative predictive value but poor specificity as well as positive predictive value. Methods: This retrospective study was carried out in a tertiary care hospital of Delhi, India to assess factors predicting the occurrence of severe dengue in children as per the revised classification. A total of 647 suspected dengue cases were admitted in the hospital in the year 2015. Detailed clinical and epidemiological data of 170 patients who were confirmed as dengue either by NS1 antigen test or by serology (Ig M positive) were recorded and statistically analyzed. Results: The number of laboratory-confirmed cases was 170 and included thirty (17.65%) dengue fever (DF), 106 (62.35%) dengue with warning signs (DWS) and 34 (20.0%) severe dengue (SD) patients. Regression analysis revealed that presence of vomiting, altered sensorium, shock, peri-orbital edema, hepatomegaly, splenomegaly, severe anemia, thrombocytopenia, elevated urea and creatinine, decreased total protein and globulin were significantly associated with occurrence of severe disease. Interpretation & conclusion: The addition of clinical features (peri-orbital edema and splenomegaly); and laboratory findings (elevated urea and creatinine, decreased serum protein and globulin) might help improve the sensitivity and specificity of the revised WHO dengue classification in predicting severe dengue.

Background & objectives: Visceral and cutaneous leishmaniasis are endemic in Iran. The aim of this study was to model the changing suitability patterns of five confirmed and one suspected leishmaniasis vector Phlebotomus species resident in the country. Methods: The potential present and future suitability patterns of the sandfly species in Iran were modelled using climate envelope forecasting method for the reference period 1970–2000 and the future period 2041–2060. Results: The reference period climate of Iran seemed to be the most suitable for Phlebotomus perfiliewi and Phlebotomus tobbi and less suitable for Phlebotomus simili, while Phlebotomus neglectus, Phlebotomus papatasi and Phlebotomus sergenti showed intermediate values among the studied sandfly species. The modelled changes in the suitability values show a similar pattern in the case of the six species, even the exact magnitude of the modelled values varied. The model results indicate that climate change could decrease the sandfly habitability in the present-day arid regions in Central Iran. The Iranian sandfly populations will move to higher elevation regions, and the suitability values of the sandfly species are predicted to increase in the foothills of the mountainous regions in the northern and the western part of the country. Interpretation & conclusion: The increase of the maximally suitable areas in Iran was found which was predicted to be accompanied by the parallel shrinkage of the sandfly-inhabited areas in the arid regions of the country. Topographical conditions could strongly influence the suitability patterns of the vectors in Iran.

Background & objectives: Entomopathogenic fungi are being investigated for the biological control of a range of mosquitoes. Metarhizium conidiospores (conidia) effectively kill Aedes aegypti larvae and could be deployed as an alternative to chemical insecticides. Conidial yield and virulence of fungi when cultured on three different types of solid media, was investigated. Methods: Three culture media were tested: a) Sabouraud dextrose agar (SDA); b) rice flour yeast agar (RYA) and c) rice grains. Conidia produced using these substrates were tested for virulence against Ae. aegypti larvae obtained from field collected eggs. Larvae (2^nd – 3^rd instar) were exposed to aqueous conidial suspensions and survival monitored over 7 days. Survival analysis was performed using Log-Rank and Kaplan Meier tests, while fungal growth and conidial yields were analyzed using a two-way ANOVA. Results: There were only small differences between growth rates on RYA and SDA; however, ESALQ 818 showed the highest conidial yield on rice. Conidia produced on rice grains were more virulent, rapidly reducing survival rates of mosquito larvae. ESALQ 818 conidia produced on rice grains, RYA and SDA killed 100% of the larvae on the 2^nd, 3^rd and 4^th day of exposure, respectively. IP 46 virulence of was consistently lower than ESALQ 818 for all the media tested. Interpretation & conclusion: The choice of culture media can influence the virulence of fungal conidia to Ae. aegypti larvae, demonstrating the importance of not only selecting the most virulent isolate but also standardizing growth conditions when screening for virulence.

Background & objectives: The incidence of Borreliosis, Anaplasmosis, Babesiosis and other tick-borne diseases acquired from private residential/peri-domestic areas has increased over the decades. However, tick activity and proportion of private residential properties with established tick populations remain unknown. The purpose of the current study was to determine the predictors of tick activity in peri-domestic areas. Methods: In a cross-sectional study design, we used snowball-sampling with cold-calling techniques to collect free-living ticks, sociodemographic, and microclimatic data from June to November 2018 from a total of 96 private residential areas in south-central Indiana, USA. Results: Thirty-eight percent of peri-domestic areas sampled had tick activity, and of these, 50% had established tick populations. Nymphal ticks were the most abundant life stage. Self-reported TBD diagnosis was 16%. Amblyomma americanum [Linnaeus (lone star tick)] was the most abundant tick species collected. Other tick species identified include: Ixodes scapularis [Say (black-legged/deer tick)] and Dermacentor variabilis [Say (American dog tick)]. Increasing temperature was positively associated with tick activity, while elevation was negatively associated with tick abundance. Interpretation & conclusion: Our study results reveal that the proportion of peri-domestic areas in Indiana with established tick populations is high. Amblyomma americanum tick is the most predominant tick species in peri-domestic areas of south-central Indiana. Active surveillance of ticks in peri-domestic areas is necessary for informing decisions by households and communities about where to target tick exposure and tick-borne disease prevention efforts.

Background & objectives: Acute kidney injury associated with scrub typhus is an emerging health problem in the tropics including India. This study intended to find out the incidence, clinical outcome, cytokine response and genotypes of Orientia tsutsugamushi associated with AKI patients in Odisha, a state in eastern India. Methods: Acute febrile illness or history of acute fever with various degrees of kidney involvement admitted to SCB Medical College Hospital, Cuttack were included in the study. A detailed demographic characteristics and clinical features were recorded with pre-tested questionnaire at the time of admission. Scrub Typhus was detected by ‘IgM ELISA’ test (OD > 0.5) and PCR. Routine urine, haematological and biochemical tests were performed. Genotyping of the Orientia tsutsugamushi was done using 56-kDa gene for Orientia species and phylogenetic tree by neighbor-joining method. The plasma level of the IFN-γ (pro-inflammatory cytokine) and IL10 (anti-inflammatory) were measured by commercially available ELISA kit. The statistical analysis was performed using Graph Pad Prism software (version 4). Results: Out of 140 acute febrile illness or history of acute febrile illness patients with AKI admitted to hospital, 32.14% were confirmed to be scrub typhus positive; eschar was seen in 17.8% of them. Of the total scrub typhus positive cases, 24.4% were having multi organ dysfunction. Majority of the AKI patients (60%) were in the “failure” category under RIFLE criteria. The mortality rate was 20.0%. Risk of dialysis requirement and mortality increases with RIFLE classification. “Karp” was the predominant circulating genotype. IFN-γ and IL10 level was high among the scrub typhus associated AKI patients. Interpretation & conclusion: The study shows a high incidence of scrub typhus associated AKI and high case fatality rate. Hence, emphasis should be given on differential diagnosis. RIFLE classification is applicable with increment risk of dialysis requirement and death. An in-depth study is required to determine the role of O. tsutsugamuchi KARP strain and INF-γ/ IL-10 in disease severity so as to identify a prognostic marker.

Background & objectives: Phlebotomus argentipes is the main vector of visceral leishmaniasis in Bangladesh and is controlled using deltamethrin, a synthetic pyrethroid, through indoor residual spraying (IRS). A mutation at L1014 (leucine at codon 1014) of the voltage-gated sodium channel (VGSC), known as a knockdown resistance (kdr) gene, is thought to be an important pyrethroid resistance mechanism. This study detected mutations at codon 1014, and at codons 1011, 1016, and 1020, which are kdr sites in other insects. The kdr relationship with deltamethrin resistance in P. argentipes from an IRS-targeted site in Bangladesh was also evaluated. Methods: Sand flies were collected from Magurjora village, Mymensingh district, Bangladesh in November 2012. A WHO cone bioassay test using deltamethrin was conducted and specimens were grouped as ‘live’ or ‘dead’. After morphological identification, genomic DNA was used to genotype a partial VGSC gene from P. argentipes. The kdr/ pyrethroid resistance relationship was evaluated using Fisher’s exact test. Results: Targeted codons were genotyped from 8 ‘live’ and 63 ‘dead’ P. argentipes. All ‘live’ specimens had mutant alleles (L1014F and L1014S) at codon 1014. The mutant allele rate was 94% for ‘live’ specimens and 55% for ‘dead’ specimens. The mutant allele survival odds were higher for the wild-type L1014L allele, and L1014F odds were lower for L1014S. There were no mutations at codons 1011, 1016, and 1020. Interpretation & conclusion: The L1014 mutations suggested that pyrethroid resistance had appeared in Bangladesh. Further research on kdr mutations in P. argentipes is important for the appropriate IRS.

Background and objectives: Insecticide resistance in malaria vectors has been a major challenge to vector control programs and updated information to the commonly used insecticides is essential for planning appropriate vector control measures. Anopheles culicifacies and An. fluviatilis are the two main vectors prevalent in Jharkhand state of India and role of An. minimus is contemplated in the transmission of malaria in this state. All the districts in the state are predominantly inhabited by the tribal population and are endemic for malaria. A study was undertaken in 12 districts of Jharkhand state to determine the insecticide susceptibility status of the 3 prevalent primary vector species, An. culicifacies, An. fluviatilis, and An. minimus. Methods: Wild-caught adult female An. culicifacies, An. fluviatilis, and An. minimus, mosquitoes were collected from stratified ecotypes from different localities of 12 tribal districts of Jharkhand state during 2018 and 2019. Susceptibility tube tests were conducted following the WHO method using test kits. Mosquitoes were exposed to WHO impregnated papers with the prescribed discriminatory dosages of DDT - 4%, malathion - 5%, deltamethrin - 0.05%, permethrin - 0.75%, cyfluthrin - 0.15% and lambda cyhalothrin - 0.05%. Results: Results indicated that An. culicifacies has developed multiple insecticide resistance in all the 12 districts of Jharkhand state. An. fluviatilis was reported resistant for the first time to DDT in all the districts but was susceptible to malathion, deltamethrin, and permethrin whereas in one district it showed possible resistance to malathion. An. minimus was studied in Noamundi CHC of West Singhbhum district, showed possible resistance against DDT but was susceptible to malathion, deltamethrin, and permethrin. Interpretation & conclusion: The development of multiple insecticide resistance in An. culicifacies including to pyrethroids, has been a concern for malaria control programmes for effective vector management but a report of resistance to DDT for the first time in An. fluviatlis in all the districts in the state is alarming, An. minimus was found in possible resistance category to DDT in one district and both the species were reported susceptible to malathion, deltamethrin, and permethrin. The result of the present study indicates a need for regular monitoring to assess the insecticide susceptibility to formulate effective vector control measures and resistance management.

Crimean-Congo hemorrhagic fever is a tick-borne disease with high fatality rate that is endemic in some parts of Asia, Africa and Europe. Rapid diagnostics of Crimean-Congo hemorrhagic fever (CCHF) is necessary for appropriate clinical management of this disease and also can be useful in preventing of secondary spread from human-to-human, though, common tests which are used to diagnose Crimean-Congo hemorrhagic fever have some limitations. Here we review 1) common diagnostic tests for CCHF, 2) limitations in laboratories methods of CCHF and 3) biosensor researches for detection of CCHF. It is necessary to design and develop an effective, rapid, and also low-cost tool such as biosensor to detect Crimean-Congo hemorrhagic fever. Based on the key role of rapid detection of CCHF in the control of infection, development of a biosensor as a rapid tool seems very major in the diagnosis of CCHF, though, there are limited studies on this field and more researches are needed in this issue.

Background & objectives: In India, vector-borne diseases, dengue and chikungunya are major public health concerns. In recent decades, dengue outbreaks have been reported in almost every part of India. In 2016, India recorded 101388 dengue cases and 210 deaths, including 4337 cases and six deaths in Delhi, whereas Chikungunya outbreaks were reported from several states in 2006, with 1.3 million cases. The Dengue virus (DENV) and Chikungunya virus (CHIKV) are both transmitted by the same Aedes mosquito species. DENV and CHIKV co-infections have been reported in 13 of 98 countries, with both viruses being transmitted locally. The reasons for the sudden upsurge in cases of these diseases are undetermined. Methods: From March to December 2016, a study was carried out in 66 localities of Delhi in collaboration with the Municipal Corporation of Delhi. Localities were selected on the basis of confirmed dengue cases reported during the last five years and the study area was visited once a month. A door-to-door entomological survey was conducted to identify Aedes breeding in all water-filled containers in and around houses. Both immature and mature stages of Aedes mosquitoes were collected. Mosquitoes were pooled (n≤10 each for male and female) breeding site-wise and stored in Trizol at -80°C. The Chikungunya and dengue viruses were detected using a multiplex RT-PCR. Results: A total of 981 Aedes mosquitos were distributed among 146 Pools, and DENV and CHIKV were detected using Multiplex Reverse Transcriptase-PCR. Chikungunya virus was identified in 19 pools of females captured adults, whereas dengue virus was found in 8 pools of females captured adults. There was no evidence of coinfection in any of the pools. Interpretation & conclusion: In endemic areas, continuous surveillance for both dengue and Chikungunya viruses is required to identify and characterize these viral pathogens. This information will also help implement effective strategies to combat outbreaks produced by these emerging viral pathogens.

Myiasis, infestation of the human body by larva of flies is an under-recognized and ignored medical condition. Not only is this condition unsightly and extremely distressing to the patient, but it also generates a ghastly feeling in care givers and health care workers as well. The authors report extensive myiasis of the leg in a patient with filarial lymphedema from southern India, which is rarely illustrated in published literature. Treatment of myiasis is described in brief. There is a need for strengthening morbidity management in filariasis elimination program.