Vector Borne Diseases

Strengthening of vector control in South-East Asia: Outcomes from a WHO regional workshop Bhupender Nath Nagpal, Tessa B Knox, Premarathane Risintha, Rajpal S Yadav, Susanta K Ghosh, Sreehari Uragayala, Neena Valecha, Eva-Maria Christophel, Mohammed Jamsheed Journal of Vector Borne Diseases 2018 55(4):247-257

Approaches for the successful isolation and cell culture of American Rickettsia species R Tello-Martin, K Dzul-Rosado, J Zavala-Castro, C Lugo-Caballero Journal of Vector Borne Diseases 2018 55(4):258-264 Rickettsia are intracellular vector-borne bacteria, which are the etiologic agent of severe infections that could inflict death to their host. The intracellular behaviour of Rickettsia makes the study of its genetics, proteomics and cellular processes very difficult. Hence, isolation remains an important experimental technique that permits the obtention of important yields of bacteria, useful for a broad range of experiments. Isolation of Rickettsia using passages in animals or embryonated eggs has been described for long time; however, it was until the 1990s that faster and more feasible approaches for cell culture were developed. Current isolation approaches are mainly based on shell vial culture, that varies according to the media, atmosphere or temperature conditions. These variations have allowed the establishment of isolates from different pathogenic and non-pathogenic Rickettsia species, using arthropod, animal or human samples. Purification method of bacteria has also witnessed changes alongside the quantification of its load in the resulting isolates, from the laborious and time consuming plaque assays, to the routinary use of real-time polymerase chain reaction (qPCR), which is faster and more accurate. This review discusses various approaches that have been used for the isolation and purification of different Rickettsia species along with the mention of some successful examples. It indicated that a successful strategy for the isolation of Rickettsia requires a careful selection of media, cell lines and culture conditions which now are not as time consuming as used to be.

Detection and distribution of Wolbachia endobacteria in Culex quinquefasciatus populations (Diptera : Culicidae) from Metropolitan Manila, Philippines Thaddeus M Carvajal, Jayson Dale R Capistrano, Kazuki Hashimoto, Kristin Joyce D Go, Maria Angeline Isabelle J Cruz, Monique Johanne Lourdee B Martinez, Vincent Stefano P Tiopianco, Divina M Amalin, Kozo Watanabe Journal of Vector Borne Diseases 2018 55(4):265-270 Background & objectives: Culex quinquefasciatus is a peridomestic mosquito known for its ability to transmit pathogenic diseases such as filariasis and Japanese encephalitis. The development and use of novel and innovative vector control measures such as the utilization of Wolbachia, along with the existing ones, are necessary to prevent the transmission of these diseases. Studies exploring the diversity of Wolbachia, particularly in Cx. quinquefasciatus are very limited in the Philippines. Thus, the aim of the study was to detect the presence, distribution, and phylogenetic relationship of Wolbachia infections in Cx. quinquefasciatus in Metropolitan Manila, Philippines. Methods: Adult Cx. quinquefasciatus mosquitoes were collected using a commercially available light-trap from May 2014–January 2015. Based on their sampling grids (n = 51), the adult mosquito abdomens were pooled and subjected to Wolbachia surface protein (wsp) gene amplification assay. Five selected wsp-positive samples were then sequenced and further analyzed to infer their phylogenetic relationship with known Wolbachia strains. Results: A total of 1090 adult Cx. quinquefasciatus mosquitoes were collected. Pooled abdomens (n = 53) were then sorted based on their sampling grids for subsequent screening of wsp gene. Wolbachia infection rate was 59% (31/53). These infections were located at 29 (57%) sampling grids, and were observed to be widely distributed in the study area. Phylogenetic analysis indicated that the sample sequences were Wolbachia pipientis isolated from known hosts, Cx. pipiens and Cx. quinquefasciatus belonging to supergroup B clade. Interpretation & conclusion: The study was able to demonstrate the prevalence and distribution of Wolbachia in Cx. quinquefasciatus in Metropolitan Manila, Philippines. The findings of this study are geared towards proposing a vector control program that utilizes the potential of Wolbachia as a biological control agent in preventing the transmission of Culex-borne diseases.

Genetic diversity and structural analysis of 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE) from Plasmodium falciparum Kavita Kadian, Sonam Vijay, Ritu Rawal, Jagbir Singh, Anup Anvikar, Veena Pande, Arun Sharma Journal of Vector Borne Diseases 2018 55(4):271-281 Background & objectives: Plasmodium parasite harbours unique methylerythritol phosphate (MEP) pathway which is obligatory for the biosynthesis of isoprenoids. In malaria parasites, the isoprenoids are indispensable during hepatic, erythrocytic and gametocytic stages. Owing to the criticality of MEP pathway and the potential of its enzymes to act as antimalarial drug target, this study comprehensively investigated the genetic diversity and structural composition of 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE), fourth enzyme of MEP pathway in Indian Plasmodium falciparum (PfIspE). Methods: The study employed sequencing, modeling and bioinformatics approaches to examine the genetic diversity and associated structural polymorphism in the PfIspE gene amplified from the clinical blood samples collected from seven malaria endemic geographical regions of India. Results: The sequence analysis showed that PfIspE gene is highly conserved with 100% sequence identity among all the P. falciparum Indian isolates as well as with the PfIspE gene of reference strain 3D7. Phylogenetic analysis suggested that PfIspE is highly evolved and differ sufficiently from human orthologue mevalonate kinase gene. Structural modeling studies revealed that PfIspE has conserved ATP and CDPME-binding domains. The active site was observed to be relatively rigid in architecture with >60% β-pleated sheets. Interpretation & conclusion: The results of genetic, phylogeny and modeling studies strengthen the potential of PfIspE enzyme as a promising antimalarial drug target.

Larvicidal activity of Ricinus communis extract against mosquitoes Nisha Sogan, Neera Kapoor, Himmat Singh, Smriti Kala, A Nayak, BN Nagpal Journal of Vector Borne Diseases 2018 55(4):282-290 Background & objectives: Vector control strategies play significant role in reducing the transmission of malaria, dengue and other vector-borne diseases. The control of vector population using synthetic insecticides has resulted in development of insecticide resistance and negative effects on humans and environment. The present investigation evaluated the larvicidal potential of methanol, dichloromethane and hexane extracts of leaves and seeds of Ricinus communis (castor) plant against the early IV instar larvae of the dengue vector, Aedes aegypti, and malaria vector, Anopheles culicifacies. Methods: Plant extracts were screened for their efficacy against Ae. aegypti and An. culicifacies using WHO standard larval susceptibility test method. Dose response bioassay was performed to get lethal concentrations. Further, gas chromatography-mass spectroscopy (GC-MS) analysis was carried out to identify the bioactive chemical constituents of the extracts of R. communis. Toxicity of the extracts towards non-target organism, Poecilia reticulata was also evaluated. Results: The leaf and seed extracts of R. communis showed significant mortality against the larvae of Ae. aegypti and An. culicifacies at concentrations of 31.25, 62.5, 125, 250, 500 ppm; and 2, 4, 8, 16, 32, 64 ppm, respectively. At 24 h of the exposure period, the larvicidal activities were highest for the methanol extract of seeds with LC50 15.52 and 9.37 ppm and LC90 45.24 and 31.1 ppm for Ae. aegypti and An. culicifacies, respectively. The methanol extract of seeds and leaves was found to be safe towards non-target organism, P. reticulata. The GC-MS profile showed that seed extracts were having higher concentration of stigmasterol (7.5%), β-sitosterol (11.48%), methyl linoleate (2.5%), vitamin E (11.93%), and ricinoleic acid (34%) than the leaf extracts. Interpretation & conclusion: The seed extract of R. communis has better larvicidal activity than the leaf extract and can be used as an effective larvicide against mosquitoes. The non-toxicity of the extracts towards P. reticulata further suggests that these plant extracts could be used along with predatory fishes in integrated vector control approaches.

Mosquito abundance and pig seropositivity as a correlate of Japanese encephalitis in human population in Assam, India Aditya Baruah, Razibuddin A Hazarika, Nagendra N Barman, Saidul Islam, Baldev R Gulati Journal of Vector Borne Diseases 2018 55(4):291-296 Background & objectives: Assam is the most vulnerable state for Japanese encephalitis (JE) in India. The situation warrants characterization of epidemiological patterns of JE in vectors, pigs and human population. This investigation was aimed to determine the relative abundance of mosquito species and seroprevalence of JE in pigs in order to draw an epidemiological association with reported human JE cases in Assam. Methods: Pig sera and mosquitoes from selected farms in Sivasagar and Kamrup districts of Assam were collected fortnightly for one year during June 2015–May 2016. Pig sera were tested for JE antibodies by haemagglutination and virus neurtralization tests. Mosquito species were identified microscopically following the taxonomic keys. The results were analyzed with data on confirmed human JE cases in the selected districts. Results: Culex gelidus (26.07%) and Cx. tritaeniorhynchus (24.07%) were the most abundant species in collected mosquitoes (n = 997). A total of 22.99% of pigs (n = 335) were JEV seropositive and 45.65% of human acute encephalitis syndrome cases (n = 230) were positive for JE virus (JEV) infection. Relative mosquito abundance, pig positivity and human cases were highest during monsoon (June–September) and least during winter (December–February). Rise in mosquito population was observed during pre-monsoon season (March–May) and concurrently higher number of human cases and pig seropositivity were recorded. A good correlation was observed between mosquito number and JEV positivity in pigs/human, and between pigs and human cases (p < 0.05). Human population in Sivasagar was at higher risk for JE infection (OR: 6.46, p < 0.0001) than in Kamrup rural district. Interpretation & conclusion: This study indicates that a seasonal correlation exists between mosquito abundance and JEV seroconversion in pigs with concurrent human JEV outbreaks under field conditions in Sivasagar and Kamrup rural districts of Assam and that monitoring mosquito abundance/density and pig JEV seropositivity may help in predicting JEV outbreak in human population in the region.

Modeling spatial distribution of Rhombomys opimus as the main reservoir host of zoonotic cutaneous leishmaniasis in northeastern Iran A Sofizadeh, AA Hanafi-Bojd, HR Shoraka Journal of Vector Borne Diseases 2018 55(4):297-304 Background & objectives: Zoonotic cutaneous leishmaniasis (ZCL) is among the most endemic zoonotic diseases in Golestan Province of Iran. The aim of this study was to find the high risk areas of this infection by considering the distribution of reservoirs and human infection. Methods: A cross-sectional study was conducted, in which Rhombomys opimus (Gerbils) were captured from different collection sites across the Golestan Province, Iran. records about the occurrence of Rh. opimus in the Province was obtained from earlier studies and were gathered in a database. Furthermore, records about the disease existence were also obtained from the health system database of Golestan Province. Villages with at least three cases of ZcL were considered endemic foci and used as presence sites for Leishmania major. ArcGIS and MaxEnt model were used to map and predict the best ecological niches for both reservoir and parasite. Results: According to the MaxEnt model, the area under Roc curve for Rh. opimus and L. major was 0.92 and 0.89, respectively. The probability of presence for both species in the northeastern part of Golestan Province was more than the other parts. The Jackknife test indicated that factors like temperature and altitude plays significant role in predicting the environmental suitability for ZcL reservoir and parasite, respectively. Interpretation & conclusion: this modeling approach predicted the areas suitable for reservoir host and circulation of parasite to human. These findings can be used in proper mapping, surveillance and control of the CL.

First field evidence infection of Culex perexiguus by West Nile virus in Sahara Oasis of Algeria S Benbetka, A Hachid, KE Benallal, C Benbetka, A Khaldi, I Bitam, Z Harrat Journal of Vector Borne Diseases 2018 55(4):305-309 Background & objectives: West Nile virus (WNV) is considered one of the most widely distributed arboviruses in the world which is transmitted by several mosquito species including the Culex genus. Culex pipiens is the major vector of this virus in Europe and USA whereas in African countries, other species such as Cx. perexiguus is considered as an important vector. This paper aimed to study the mosquito species involved in WNV transmission in Aougrout, one of the highly populated Oasis of Timimoun Province in Algeria where human WNV neuroinvasive diseases are prevalent. Methods: CDC light-traps were installed in animal and human shelters for three nights. Collected mosquitoes were pooled and real-time PCR was performed to detect and identify WNV lineages 1 and 2 in the samples. Results: CDC light-traps collected 270 mosquitoes belonging to three genera. Culex genus was predominant with Cx. perexiguus as main species followed by Aedes and Anopheles genus. A total of 33 pools were tested; one pool containing Cx. perexiguus was found positive for WNV lineage 1. Interpretation & conclusion: This study reports for the first time a WNV natural infection of Culex perexiguus in the study region indicating that species other than Cx. pipiens should be taken into consideration in WNV surveillance, especially in specific environments like Saharan Oasis ecosystem.

The relationship between skin rash and outcome in dengue Ajay Kumar Mishra, Anu Anna George, K.P.P. Abhilash Journal of Vector Borne Diseases 2018 55(4):310-314 Background & objectives: Dengue fever (DF) is a common cause of acute febrile illness. Skin involvement is seen in more than half of the patients. This study was aimed to compare the clinical profile and outcome in DF patients with or without skin involvement. Methods: This study included all the patients with DF from the acute febrile illness database of a tertiary care health centre in south India. These patients were further subgrouped into SP and SN (skin involvement positive and negative) based on the presence and absence of skin rash. Differences in clinical presentation, laboratory parameters, disease course, morbidity and outcome among patients with DF with or without skin rash were recorded and analysed statistically. Results: In total 387 patients (>16 yr) with DF were enrolled into the study. Among these 55 patients had evidence of skin rash. Presence of history of overt bleeding (OR = 4.96, p = 0.027) including gum bleeding (OR = 1.17, p = 0.23), epistaxis (OR = 5.52, p = 0.04), and haematuria (OR = 6.41, p = 0.01) were more among patients with SP as compared to SN. The SP patients were found to have lower levels of platelets during the disease course. Patients with SP had a higher percentage of platelet transfusion which was statistically significant. There was no difference in organ dysfunction and mortality among both the groups. Interpretation & conclusion: Cutaneous involvement, though common, is not pathognomonic and can help in dengue diagnosis. Adult patients with skin rash can develop worsening thrombocytopenia requiring platelet transfusion. However, there are limited data to suggest that such patients have a worse outcome and higher mortality.

High resolution melting analysis as an accurate method for identifying Leishmania infantum in canine serum samples Ahmad Hosseini-Safa, Mehdi Mohebali, Homa Hajjaran, Behnaz Akhoundi, Zabih Zarei, Kourosh Arzamani, Afshin Davari Journal of Vector Borne Diseases 2018 55(4):315-320 Background & objectives: Leishmania (L.) infantum is the principal agent of visceral leishmaniasis (VL) in the Mediterranean and American regions. So far different molecular methods including high resolution melting (HRM) analysis have been developed for detecting and identifying L. infantum infection. HRM assay is an automted molecular method which detects and identifies different genus and species of infectious agents. This study aimed to diagnose and identify Leishmania infection caused by L. infantum species using real-time PCR coupled with HRM assay in the serum samples in comparison with anti-L. infantum antibodies obtained using direct agglutination test (DAT), in domestic and wild canines of northeastern Iran. Methods: Serum samples of 15 foxes, 14 jackals, seven domestic dogs and three wolves were collected in some villages around Shirvan and Bojnourd districts from the northeast regions of Iran during 2014–15. Initially, all the collected serum samples were tested by DAT for the detection of anti-L. infantum antibodies. Afterwards, genomic DNA was extracted from the samples and tested by real-time PCR–HRM analysis targeting hsp70, ITS1 and gp63 genes. The level of agreement between DAT and HRM assay were analysed statistically. Results: Out of the 39 serum samples, eight showed anti-L. infantum antibodies at titre 1: 80 while only one of them showed anti-L. infantum antibodies at titre 1 : 160. All the nine seropositive samples showed positive results with HRM analysis. Additionally, three DAT negative serum samples were also found positive in the HRM technique. Altogether, 12 out of the 39 DNA samples showed positive results in HRM analysis. Among the three gene sequences used, gp63 was best for separation and identification of species. Interpretation & conclusion: HRM analysis targeting hsp70, ITS1 and gp63 genes can be used as a highly sensitive technique for the screening and early detection of L. infantum infection in the wild and domestic canines. It has higher accuracy than DAT and allows detection and discrimination of different Leishmania species responsible for the Leishmaniases.