Molecular detection of dengue and chikungunya viruses in surveillance of wild-caught Aedes mosquitoes in Punjab, North India.

BACKGROUND: Dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses are transmitted mainly by Aedes mosquitoes and are responsible for a significant global healthcare burden. The current study aimed to detect arboviruses in the Aedes mosquitoes in close proximity of patients during the transmission season. METHODS: Both immature and adult mosquitoes were collected from in and around the patients' houses. Mosquito pools were homogenized and extracted RNA was subjected to reverse transcription polymerase chain reaction for arboviral detection. Transovarian transmission (TOT) was assessed by screening F0 adults. Mosquito positivity was correlated with the aetiological agents identified in patients. RESULTS: Of 46 pools, 19 consisted of wild Aedes, with arboviral positivity in 53% (10/19) of pools. Among wild A. aegypti pools, positivity of DENV mono-infection, CHIKV mono-infection and DENV+CHIKV co-infection was noted in four, two and three pools, respectively. One wild pool of Aedes albopictus was positive for DENV-1. Similarly, A. aegypti F0 (adult Aedes developed from immatures) pools showed 59.2% (16/27) positivity for arboviruses. F0 Aedes showed positivity in three, six and seven pools for DENV-2, CHIKV and DENV+CHIKV, respectively, suggestive of TOT. DENV serotypes and CHIKV from 24 patients' serum samples were matched with strains isolated from Aedes and correlation was observed in four instances. CONCLUSIONS: The study detected DENV and CHIKV from wild-caught Aedes and found evidence of DENV and CHIKV TOT in F0 adults.

First detection and genetic identification of Rickettsia spp. from ticks collected from rodents in north India.

BACKGROUND: Changing climatic conditions and invasion of ticks in urban areas have led to a greater number of cases of tick-borne diseases, thus, becoming a matter of increasing concern. Tick borne rickettsioses are one of the important emerging diseases worldwide. Knowledge of epidemiology of the vector and pathogen in the community is essential in order to understand and prevent the transmission of the disease to healthy population. METHODS: In our present study, we trapped rodents in selected areas of Chandigarh and Punjab in north India. The rodents were screened for the presence of ticks which were further screened for the presence of rickettsial agents. PCRs targeting 17 â€‹kDa and gltA genes were carried out followed by Sanger sequencing of the positive amplicons followed by phylogenetic analysis of the sequences. RESULTS: A total of 17 ticks were collected out of which one (Rhipicephalus sanguineus) was found to be harboring a Rickettsia sp. PCR targeting gltA and 17 â€‹kDa genes of rickettsia were put up and Sanger sequencing was performed. Phylogenetic analysis revealed the sequences to be closely related to Rickettsia rhipicephali. CONCLUSION: The current study establishes the presence of rickettsial agents in the community. Although Rickettsia rhipicephali is a non-pathogenic agent, the study encourages more vigorous community surveillance should be carried out in order to determine the exact burden of rickettsial agents in our community. To our knowledge, this is the first study reporting Rickettsia rhipicephali in India.

Malaria vector sibling species distribution in different endemic districts of Punjab, India.

BACKGROUND & OBJECTIVES: Malaria transmission in Punjab, India is mainly seasonal with variation in its endemicity that may be due to varying vector behaviour in different areas of the state, primarily attributed to the existence of sibling species complexes among the vector species. So far there is no report regarding the existence of malaria vectors sibling species in the state of Punjab, therefore, the present study was planned to investigate the status of sibling species of two main vectors of malaria viz. Anopheles culcifacies and Anopheles fluviatilis in different districts of Punjab. METHODS: Mosquito collections were made through hand catch in the morning hours. Malaria vector species An. culicifacies and An. fluviatilis were morphologically identified and man hour density was calculated. Both the vector species were subjected to molecular assays for sibling species identification through amplification of D3 domain of 28S ribosomal DNA by allele-specific PCR. RESULTS: Four sibling species of An. culicifacies, were identified viz. A, B, C and E. Species A was identified from Bhatinda district, species B, C and E from. S.A.S. Nagar and species C from Hoshiarpur. Two sibling species S and T of An. fluviatilis were identified from districts S.A.S. Nagar and Rupnagar. INTERPRETATION & CONCLUSION: Presence of four sibling species of An. culicifacies and two sibling species of An. fluviatilis in Punjab necessitates planning of longitudinal studies to ascertain their role in disease transmission so that appropriate interventions may be applied to achieve malaria elimination.

Comparative efficacy of natural aquatic predators for biological control of mosquito larvae: A neglected tool for vector control.

Mosquitoes are a dominant fraction of dipteran fauna, occupying a variety of niches. The most common method deployed for their control is the use of insecticides. Throughout their life cycle they are exposed to a wide range of predators in different habitats, thus biological control of mosquitoes by using aquatic predators has been suggested. Therefore, the present study was carried out to explore the type of natural predators coexisting with the mosquito larvae in still water bodies and to determine their efficacy as predators for mosquito larvae. A coexistence of different predators with mosquito larvae was observed in 27 standing water bodies of Chandigarh, India. The predation efficiency of tadpoles of frog was comparable to Gambusia fish, as 97% of the mosquito larvae of all instars of the medically important mosquito genera Anopheles, Aedes, Culex and Armigeres were preyed. The toad tadpoles were found to be least effective and their predation rate was found to be negligible. Further studies on larval source management by frog tadpoles in combination with insecticides or stand-alone would be worthwhile.

Vector and rodent surveillance for Orientia tsutsugamushi in north India.

BACKGROUND & OBJECTIVES: Scrub typhus or chigger borne typhus, caused by Orientia tsutsugamushi is an emerging vector-borne disease as large numbers of cases have been reported in various tropical countries. It is transmitted to humans through bites of infected chiggers (larval mites). The knowledge about the vector, its distribution, density and habitat are important so as to understand the epidemiology of scrub typhus in a given area. To control rickettsial infections, regular rodent-vector surveillance should be planned in areas where the disease transmission is occurring and it will also help to strengthen the existing entomological data related to the vector of scrub typhus in northern India. METHODS: In the present study, rodent-vector surveillance was planned for one whole year, covering both mite active and non-active seasons (October 2019-December 2020) in selected areas of Chandigarh and Punjab in north India. Rodent tissues and mites were also examined for the presence of O. tsutsugamushi by nested PCR for 56 kDa gene and real-time PCR for 47 kDa outer membrane protein gene. 18S gene PCR was performed for molecular identification of mites. RESULTS: In the surveillance, three types of ectoparasite, viz. mites, fleas and ticks were obtained in rodents. All mites found were of Laelapidae family. None of the pooled rodent tissue samples as well as mite samples were found positive for O. tsutsugamushi by nested PCR for rickettsial DNA. INTERPRETATION & CONCLUSION: In the present study, we did not get any evidence of carriage of O. tsutsugamushi in either mites or rodents collected and sampled in selected regions in Chandigarh and Punjab. We need to strengthen the entomological surveillance over a broader region and increase the frequency of trapping rodents to increase clarity on vector-reservoir dynamics in this geographical region.

Surveillance-based estimation of the malaria disease burden in a low endemic state of Punjab, India, targeted for malaria elimination.

BACKGROUND: The state of Punjab in India qualifies for malaria elimination because the number of cases reported through routine surveillance is in decline. However, surveillance system prevalence mainly provides malaria trends. Therefore, a prospective epidemiological study was designed to estimate the malaria burden in the state. METHODS: District-wise annual parasite incidence (API) was used for identification of three strata, representing high, moderate and low API zones. A total of 0.9 million people from nine districts was under malaria surveillance for 1 y. The weighted estimates of API for the three regions was calculated and combined to give an estimate of API for the total population of the state. RESULTS: Based upon the primary data generated, malaria cases from high, moderate and low malaria-endemic areas were estimated to be 3727, 904 and 106, respectively. Further, the total number of malaria cases in the state was estimated to be 4737 (95% CI 4006 to 5469) cases per annum. CONCLUSION: Actual burden of malaria in the state of Punjab, India, is about seven to eight times higher than that reported by routine surveillance activities. However, the state still qualifies for malaria elimination but needs vigorous efforts to strengthen the active surveillance and reporting system along with implementation of effective control strategies to achieve malaria elimination.

Prevalence of dengue vectors, larval breeding habitats, Stegomyia indices and their correlation with dengue cases in urban and rural areas of Punjab, India.

BACKGROUND & OBJECTIVES: The state of Punjab, India is highly endemic for dengue fever as high number of confirmed dengue cases have been reported since 2013. A better understanding of vectors distribution and their seasonal variation is necessary to control the disease. Therefore, the present study was conducted in both rural and urban areas of 11 out of 22 districts of Punjab to highlight seasonal prevalence of Aedes vector mosquitoes. METHODS: Entomological surveys were carried out in different seasons and all kinds of indoor and outdoor breeding habitats were examined and Aedes immatures were collected. The Stegomyia indices were calculated and compared from urban and rural areas in different seasons. RESULTS: Both vectors of dengue, i.e. Aedes aegypti and Ae. albopictus were recorded to be prevalent. Ae. aegypti mosquitoes were observed in all districts surveyed while Ae. albopictus were found only in seven districts of Punjab. The Stegomyia indices were significantly high during monsoon as compared to pre- and post- monsoon periods. Occurrence of dengue cases were found to be correlated with the Stegomyia indices. INTERPRETATION & CONCLUSION: This is the first detailed study of prevalence of dengue mosquito vectors in Punjab showing the presence of Ae. aegypti and Ae. albopictus in both urban and rural areas of the state, thereby demonstrating wide distribution of this vector. Different breeding habitats identified in the study should be subjected to targeted intervention such as source reduction in order to achieve effective control of dengue cases.

Utilizing larvicidal and pupicidal efficacy of Eucalyptus and neem oil against Aedes mosquito: An approach for mosquito control.

BACKGROUND AND OBJECTIVES: Plant-based products can provide safe and biodegradable mosquito control agents. The essential oils have a strong odor due to complex secondary metabolites and exhibit lower density than that of water, which renders them suitable to form a thin layer above the water surface. The present study was designed to evaluate the larvicidal, pupicidal activity of Eucalyptus and neem oils against Aedes aegypti and Aedes albopictus. MATERIALS AND METHODS: We evaluated the activity of commercially available Eucalyptus (Eucalyptus globulus) and neem (Azadirachta indica) oils against larvae and pupae of A. aegypti and A. albopictus for their larvicidal and pupicidal activity, stability in different water types, dependence on volume and surface area of the water body, and residual efficacy. RESULTS: Eucalyptus oil was found to be more effective against larvae and pupae at lower concentrations, i.e., concentration at which 50% is observed (LC50) for larvae and pupae was 93.3 and 144.5 parts per million (ppm) and concentration at which 90% is observed (LC90) was 707.9 and 741.3 ppm, respectively, while for neem oil, LC50 for larvae and pupae was 7852 and 19,054 ppm and LC90 was 10,092 and 19,952 ppm, respectively. The efficacy of Eucalyptus oil depended on surface area rather than volume of water, and the residual efficacy of Eucalyptus oil was up to 8 days. CONCLUSIONS: Eucalyptus oil was more effective against mosquito larvae at lower concentration as compared to neem oil. It can, therefore, be utilized in the community in artificial and small temporary water bodies as an eco-friendly vector control measure in the era of increasing resistance to chemical insecticides.

Prevalence of submicroscopic malaria in low transmission state of Punjab: A potential threat to malaria elimination.

BACKGROUND & OBJECTIVES: Submicroscopic malaria infections with low parasite density serve as a silent reservoir for maintaining residual transmission in the population. These infections should be identified and targeted to be eliminated for sustained malaria control. The conventional methods of diagnosis such as light microscopy and rapid diagnostic kits often fail to detect low density infections. Therefore, the more sensitive molecular techniques should be employed to detect low density infections. The objectives of the study was to explore the prevalence of sub-microscopic infections in low transmission areas of Punjab using highly sensitive molecular tool. METHODS: A total of 1114 finger prick blood samples were collected through active surveillance and tested for malaria diagnosis using light microscopy, RDT and PCR. Nested PCR amplification was performed using a pair of Plasmodium genus-specific primers from the 18S rRNA small subunit gene (18S rRNA). The amplified PCR products were analysed using a 2% agarose gel, stained with ethidium bromide and observed under transilluminator. RESULTS: Test positive rate (TPR) by microscopy, RDT and PCR was 4.4, 3.95 and 5.75%, respectively. Microscopy and RDT failed to detect mixed infections whereas 0.26% cases were found to be mixed infection in PCR. Compared to LM and RDT, PCR has detected 1.3% additional positive cases. However, of the total positive cases detected by PCR, 23.4% infections were found to be submicroscopic, which could not be detected by conventional methods of diagnosis. INTERPRETATION & CONCLUSIONS: The molecular study revealed the existence of submicroscopic malaria cases in the study population which would have remained undetected by conventional methods of diagnosis. This is particularly important because Punjab state is in malaria elimination phase and targeted to achieve elimination in 2021. However, such undetected parasite positive cases may pose bigger problem any time due to continued transmission. Therefore, application of more sensitive diagnostic tools like PCR and LAMP with conventional methods may be much more useful in case detection particularly in low transmission settings for malaria elimination.

Rapid identification of medically important mosquitoes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: Accurate and rapid identification of dipteran vectors is integral for entomological surveys and is a vital component of control programs for mosquito-borne diseases. Conventionally, morphological features are used for mosquito identification, which suffer from biological and geographical variations and lack of standardization. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for protein profiling of mosquito species from North India with the aim of creating a MALDI-TOF MS database and evaluating it. METHODS: Mosquito larvae were collected from different rural and urban areas and reared to adult stages. The adult mosquitoes of four medically important genera, Anopheles, Aedes, Culex and Armigerus, were morphologically identified to the species level and confirmed by ITS2-specific PCR sequencing. The cephalothoraces of the adult specimens were subjected to MALDI-TOF analysis and the signature peak spectra were selected for creation of database, which was then evaluated to identify 60 blinded mosquito specimens. RESULTS: Reproducible MALDI-TOF MS spectra spanning over 2-14 kDa m/z range were produced for nine mosquito species: Anopheles (An. stephensi, An. culicifacies and An. annularis); Aedes (Ae. aegypti and Ae. albopictus); Culex (Cx. quinquefasciatus, Cx. vishnui and Cx. tritaenorhynchus); and Armigerus (Ar. subalbatus). Genus- and species-specific peaks were identified to create the database and a score of > 1.8 was used to denote reliable identification. The average numbers of peaks obtained were 55-60 for Anopheles, 80-100 for Aedes, 30-60 for Culex and 45-50 peaks for Armigeres species. Of the 60 coded samples, 58 (96.67%) were correctly identified by MALDI-TOF MS with a score > 1.8, while there were two unreliable identifications (both Cx. quinquefasciatus with scores < 1.8). CONCLUSIONS: MALDI-TOF MS appears to be a pragmatic technique for accurate and rapid identification of mosquito species. The database needs to be expanded to include species from different geographical regions and also different life-cycle stages to fully harness the technique for entomological surveillance programs.