Entomological Surveillance in Former Malaria-endemic Areas of Southern Italy

Malaria still represents a potential public health issue in Italy, and the presence of former Anopheles vectors and cases imported annually merit continuous surveillance. In areas no longer endemic, the concurrent presence of gametocyte carriers and competent vectors makes re-emergence of local transmission possible, as recently reported in Greece. In October 2017, due to the occurrence of four suspected introduced malaria cases in the province of Taranto (Apulia region), entomological investigations were performed to verify the involvement of local anopheline species. In 2019–2020 entomological surveys were extended to other areas historically prone to malaria between the provinces of Taranto and Matera and the province of Foggia (Gargano Promontory). Resting mosquitoes were collected in animal shelters and human dwellings, larvae were sampled in natural and artificial breeding sites, and specimens were both morphologically and molecularly identified. A total of 2228 mosquitoes were collected, 54.3% of which were anophelines. In all the investigated areas, Anopheles labranchiae was the most widespread species, while Anopheles algeriensis was predominant at the Gargano sites, and Anopheles superpictus and Anopheles plumbeus were recorded in the province of Matera. Our findings showed a potentially high receptivity in the surveyed areas, where the abundance of the two former malaria vectors, An. labranchiae and An. superpictus, is related to environmental and climatic parameters and to anthropic activities.

Blood meal sources of Anopheles vectors of human malaria in Malawi: Implications for malaria transmission and effectiveness of interventions

BackgroundSelection of blood meal hosts by mosquitoes is a key variable in the vectorial capacity of Anopheles mosquitoes for human malaria. Blood feeding on humans is likely to be modulated by use of different types of long-lasting insecticidal nets (LLINs) and the effectiveness of LLINs is impacted by the relative intensity of insecticide resistance. The aim of this study was to test the hypothesis that LLINs containing pyrethroid and the synergist piperonyl butoxide (PBO) would lead to a reduction of human host utilization than LLINs containing only pyrethroid and that blood feeding patterns of Anopheles in Malawi compromise malaria interventions.MethodsFemale Anopheles mosquitoes were sampled indoors from May 2019 through April 2020 by aspiration, pyrethrum spray catch, and CDC light trap in rural villages of Namanolo (conventional nets) and Ntaja (PBO nets) in Balaka and Machinga districts respectively. Anopheles species, blood meal sources, and infection with Plasmodium falciparum in the head and thorax of individual mosquitoes were determined with PCR.ResultsOf a total of 6,585 Anopheles females sampled indoors in 203 houses, 633 (9.6%) were blood-fed and consisted of An. arabiensis (44.1% (n = 279)), An. gambiae s.s (16.2% (n = 103)), An. funestus s.s (33.5% (n = 212)), An. parensis 0.3% (n = 2), and unidentified Anopheles spp (5.8% (n = 37)). Of the 541 mosquitoes (85.5%) successfully identified blood meals, 436 (81.0%) were solely human, 28 (5.2%) goat, 11 (2.0%) dog, 60 (11.1%) mixed goat-human, 5 (0.9%) dog-human, and 1 dog-goat. Human blood index and EIR was high in Namanolo than Ntaja (0.96 vs 0.89 (p = 0.001) and 0.11 vs 0.06 infective bites per person per year respectively) despite high net ownership (92%) and nightly use (75%) rates. Relative to host availability, non-human hosts were over selected in the two sites.ConclusionThe use of PBO nets was associated with lower HBI and EIR, however, the wide availability of LLINs was still associated with extensive successful human blood meals by the main malaria vectors in Malawi. The presence of a small fraction of mixed blood meals indicates constrained plasticity of Anopheles vectors to switch to non-human hosts and circumvent malaria control interventions.

The seasonal dynamics and biting behavior of potential Anopheles vectors of Plasmodium knowlesi in Palawan, Philippines

Background A small number of human cases of the zoonotic malaria Plasmodium knowlesi have been reported in Palawan Island, the Philippines. Identification of potential vector species and their bionomics is crucial for understanding human exposure risk in this setting. Here, we combined longitudinal surveillance with a trap-evaluation study to address knowledge gaps about the ecology and potential for zoonotic spillover of this macaque malaria in Palawan Island. Methods The abundance, diversity and biting behavior of human-biting Anopheles mosquitoes were assessed through monthly outdoor human landing catches (HLC) in three ecotypes representing different land use (forest edge, forest and agricultural area) across 8 months. Additionally, the host preference and biting activity of potential Anopheles vectors were assessed through comparison of their abundance and capture time in traps baited with humans (HLC, human-baited electrocuting net—HEN) or macaques (monkey-baited trap—MBT, monkey-baited electrocuting net—MEN). All female Anopheles mosquitoes were tested for the presence of Plasmodium parasites by PCR. Results Previously incriminated vectors Anopheles balabacensis and An. flavirostris accounted for > 95% of anophelines caught in longitudinal surveillance. However, human biting densities were relatively low (An. balabacensis: 0.34–1.20 per night, An. flavirostris: 0–2 bites per night). Biting densities of An. balabacensis were highest in the forest edge, while An. flavirostris was most abundant in the agricultural area. The abundance of An. balabacensis and An. flavirostris was significantly higher in HLC than in MBT. None of the 357 female Anopheles mosquitoes tested for Plasmodium infection were positive. Conclusions The relatively low density and lack of malaria infection in Anopheles mosquitoes sampled here indicates that exposure to P. knowlesi in this setting is considerably lower than in neighboring countries (i.e. Malaysia), where it is now the primary cause of malaria in humans. Although anophelines had lower abundance in MBTs than in HLCs, An. balabacensis and An. flavirostris were caught by both methods, suggesting they could act as bridge vectors between humans and macaques. These species bite primarily outdoors during the early evening, confirming that insecticide-treated nets are unlikely to provide protection against P. knowlesi vectors. Graphical abstract

The Seasonal Dynamics and Biting Behavior of Potential Anopheles Vectors of Plasmodium Knowlesi in Palawan, Philippines

Background: A small number of human cases of the zoonotic malaria Plasmodium knowlesi have been reported in Palawan Island, the Philippines. Identification of potential vector species and their bionomics is crucial for understanding of human exposure risk in this setting. Here, we combined longitudinal surveillance with a trap-evaluation study to address knowledge gaps about the ecology and potential for zoonotic spillover of this macaque malaria in Palawan Island.Methods: The abundance, diversity and biting behavior of human-biting Anopheles mosquitoes were assessed through monthly outdoor Human Landing Catches (HLC) in three ecotypes representing different land use (forest-edge, forest and agricultural area) across 8 months. Additionally, the host preference and biting activity of potential Anopheles vectors was assessed through comparison of their abundance and capture time in traps baited with humans (HLC, human-baited electrocuting net – HEN) or macaques (monkey-baited trap – MBT, monkey-baited electrocuting net – HEN). All female Anopheles mosquitoes were tested for presence of Plasmodium parasite by PCR.Results: Previously incriminated vectors Anopheles balabacensis and An. flavirostris accounted for >95% of Anophelines caught in longitudinal surveillance. However human biting densities were relatively low (An. balabacensis: 0.34 -1.20 per night, An. flavirostris: 0-2 bites per night). Biting densities of An. balabacensis were highest in the forest-edge while An. flavirostris was most abundant in the agricultural area. The abundance of An. balabacensis and An. flavirostris was significantly higher in HLC than MBT. None of the 357 female Anopheles mosquitoes tested for Plasmodium infection were positive.Conclusions: The relatively low density and lack of malaria infection in Anopheles mosquitoes sampled here indicates that exposure to P. knowlesi in this setting is considerably lower than neighbouring countries (Malaysia) where it is now the primary cause of malaria in humans. Although Anophelines had lower abundance in MBT than HLC, An. balabacensis and An. flavirostris were caught by both methods suggesting they could act as bridge vectors between humans and macaques. These species bite primarily outdoors and in early evening; confirming that Insecticide Treated Nets are unlikely to provide protection against P. knowlesi vectors.

Atlantic Forest Malaria: A Review of More than 20 Years of Epidemiological Investigation

In the south and southeast regions of Brazil, cases of malaria occur outside the endemic Amazon region near the Atlantic Forest in some coastal states, where Plasmodium vivax is the recognized parasite. Characteristics of cases and vectors, especially Anopheles (Kerteszia) cruzii, raise the hypothesis of a zoonosis with simians as reservoirs. The present review aims to report on investigations of the disease over a 23-year period. Two main sources have provided epidemiological data: the behavior of Anopheles vectors and the genetic and immunological aspects of Plasmodium spp. obtained from humans, Alouatta simians, and Anopheles spp. mosquitoes. Anopheles (K.) cruzii is the most captured species in the forest canopy and is the recognized vector. The similarity between P. vivax and Plasmodium simium and that between Plasmodium malariae and Plasmodium brasilianum shared between simian and human hosts and the involvement of the same vector in the transmission to both hosts suggest interspecies transfer of the parasites. Finally, recent evidence points to the presence of Plasmodium falciparum in a silent cycle, detected only by molecular methods in asymptomatic individuals and An. (K.) cruzii. In the context of malaria elimination, it is paramount to assemble data about transmission in such non-endemic low-incidence areas.

IgG antibody response against Anopheles salivary gland proteins in asymptomatic Plasmodium infections in Narino, Colombia

Background The humoral immune response against Anopheles salivary glands proteins in the vertebrate host can reflect the intensity of exposure to Anopheles bites and the risk of Plasmodium infection. In Colombia, the identification of exposure biomarkers is necessary due to the several Anopheles species circulating. The purpose of this study was to evaluate risk of malaria infection by measuring antibody responses against salivary glands extracts from Anopheles (Nyssorhynchus) albimanus and Anopheles (Nys.) darlingi and also against the gSG6-P1 peptide of Anopheles gambiae in people residing in a malaria endemic area in the Colombian Pacific coast. Methods Dried blood spots samples were eluted to measure the IgG antibodies against salivary gland extracts of An. albimanusstrains STECLA (STE) and Cartagena (CTG) and An. darlingi and the gSG6-P1 peptide by ELISA in uninfected people and microscopic and submicroscopic Plasmodium carriers from the Colombia Pacific Coast. A multiple linear mixed regression model, Spearman correlation, and Mann-Whitney U-test were used to analyse IgG data. Results Significant differences in specific IgG levels were detected between infected and uninfected groups for salivary glands extracts from An. albimanus and for gSG6-P1, also IgG response to CTG and gSG6-P1 peptide were positively associated with the IgG response to Plasmodium falciparum in the mixed model. Conclusion The CTG and STE An. albimanus salivary glands extracts are a potential source of new Anopheles salivary biomarkers to identify exposure to the main malaria vector and to calculate risk of disease in the Colombian Pacific coast. Also, the gSG6-P1 peptide has the potential to quantify human exposure to the subgenus Anopheles vectors in the same area.

IgG antibody response against Anopheles salivary gland proteins in asymptomatic Plasmodium infections in Narino, Colombia

Background The humoral immune response against Anopheles salivary glands proteins in the vertebrate host can reflect the intensity of exposure to Anopheles bites and the risk of Plasmodium infection. In Colombia, the identification of exposure biomarkers is necessary due to the several Anopheles species circulating. The purpose of this study was to evaluate risk of malaria infection by measuring antibody responses against salivary glands extracts from Anopheles (Nyssorhynchus) albimanus and Anopheles (Nys.) darlingi and also against the gSG6-P1 peptide of Anopheles gambiae in people residing in a malaria endemic area in the Colombian Pacific coast. Methods Dried blood spots samples were eluted to measure the IgG antibodies against salivary gland extracts of An. albimanusstrains STECLA (STE) and Cartagena (CTG) and An. darlingi and the gSG6-P1 peptide by ELISA in uninfected people and microscopic and submicroscopic Plasmodium carriers from the Colombia Pacific Coast. A multiple linear mixed regression model, Spearman correlation, and Mann-Whitney U-test were used to analyse IgG data. Results Significant differences in specific IgG levels were detected between infected and uninfected groups for salivary glands extracts from An. albimanus and for gSG6-P1, also IgG response to CTG and gSG6-P1 peptide were positively associated with the IgG response to Plasmodium falciparum in the mixed model. Conclusion The CTG and STE An. albimanus salivary glands extracts are a potential source of new Anopheles salivary biomarkers to identify exposure to the main malaria vector and to calculate risk of disease in the Colombian Pacific coast. Also, the gSG6-P1 peptide has the potential to quantify human exposure to the subgenus Anopheles vectors in the same area.

IgG antibody response against Anopheles salivary gland proteins in asymptomatic infections in Narino – Colombia.

Background: The humoral immune response against Anopheles salivary glands proteins in the vertebrate host can reflect the intensity of exposure to Anopheles bites and the risk of Plasmodium infection. In Colombia, the identification of exposure biomarkers is necessary due to the several Anopheles species circulating. The purpose of this study was to evaluate risk of malaria infection by measuring antibody responses against salivary glands extracts from An. (Nys.) albimanus and An. (Nys.) darlingi and also against the gSG6-P1 peptide of An. gambiae in people residing in a malaria endemic area in the Colombian Pacific coast. Methods: We eluted dried blood spots samples to measure the IgG antibodies against salivary gland extracts of An. (Nys.) albimanus strains STECLA (STE) and Cartagena (CTG) and An. (Nys.) darlingi and the gSG6-P1 peptide by ELISA in uninfected people and microscopic and submicroscopic Plasmodium carriers from the Colombia Pacific Coast. A multiple linear mixed regression model, Spearman correlation, and Mann-Whitney U-test were used to analyze IgG data. Results: Significant differences in specific IgG levels were detected between infected and uninfected groups for salivary glands extracts from An. (Nys.) albimanus and for gSG6-P1, also IgG response to CTG and gSG6-P1 peptide were positively associated with the IgG response to P. falciparum in the mixed model. Conclusion: The CTG and STE An. (Nys.) albimanus salivary glands extracts are a potential source of new Anopheles salivary biomarkers to identify exposure to the main malaria vector and to calculate risk of disease in the Colombian Pacific coast. Also, the gSG6-P1 peptide has the potential to quantify human exposure to the subgenus Anopheles vectors in the same area.

Humoral immune response against Anopheles bites as a measure of exposure to Plasmodium falciparum in asymptomatic infections in a malaria endemic area of Colombia

Background The humoral immune response against Anopheles salivary glands proteins in the vertebrate host can reflect the intensity of exposure to Anopheles bites and the risk of Plasmodium infection. In Colombia, the identification of exposure biomarkers is necessary due to the several Anopheles species circulating. The purpose of this study was to evaluate human exposure to Anopheles bite by measuring antibody responses against salivary glands extracts from An. albimanus and An. darlingi and also against the gSG6-P1 peptide of An. gambiae in asymptomatic P. falciparum infections in the Colombian Pacific coast.Methods We eluted dried blood spots samples to measure the IgG antibodies against salivary gland extracts of An. albimanus and An. darlingi and the gSG6-P1 peptide by ELISA in uninfected people and microscopic and submicroscopic Plasmodium carriers from the Colombia Pacific Coast. A multiple linear mixed regression model, Spearman correlation, and Mann-Whitney U-test were used to analyze IgG data.Results Significant differences in specific IgG levels were detected between infected and uninfected groups for salivary glands extracts from An. albimanus and for gSG6-P1, also IgG response to CTG and gSG6-P1 peptide were positively associated with the IgG response to P. falciparum in the mixed model.Conclusion The CTG and STE An. albimanus salivary glands extracts are a potential source of new Anopheles salivary biomarkers to identify exposure to the main malaria vector in the Colombian Pacific coast. Also, the gSG6-P1 peptide has the potential to quantify human exposure to the subgenus Anopheles vectors in the same area.

Comparison of CDC Bottle Bioassay with WHO Standard Method for Assessment Susceptibility Level of Malaria Vector, Anopheles stephensi to Three Imagicides

Background: The detection of insecticide resistance in natural populations of Anopheles vectors is absolutely necessary for malaria control. CDC bottle bioassay as a new tools has been employed for detecting the insecticide resistance. For a limit number of mosquito vectors, diagnostic doses and diagnostic times for some insecticides have already been deter­mined using this new assay. For the first time in the area, susceptibility levels of Anopheles stephensi was done with DDT, deltamethrin, and bendiocarb using CDC bottle bioassay and compared results with WHO standard test method. Methods: Anopheles stephensi were collected in larvae stage from the cisterns of drinking water in Chabahar port which considered as old malaria foci, Sistan and Baluchistan province. The field collected larvae were colonized at the insectary of School of Public Health (SPH), Tehran University of Medical Science. The susceptibility tests were carried out on sugar fed female mosquitoes aged 2–3 days, against DDT 4%, bendiocarb 1% and deltamethrin 0.05% using WHO and CDC susceptibility methods. The mortality and knockdown rates, as well as the parameters of regression analysis, includ­ing LT50 and LT90, was calculated separately for the WHO and CDC methods. Results: The 24h mortality rates of An. stephensi were 28.6% and 25.6% for DDT, 60.8% and 64.6% for bendiocarb and 100% for deltamethrin using both WHO and CDC assay at 30 and 60min respectively. The 50% lethal times (LT50) were estimated 44.9 and 66.2min, 38.9 and 81.8min and 0.7 and 15.0min respectively using both WHO and CDC susceptibility tests. Conclusion: The similar results of susceptibility levels were shown for DDT, bendiocarb and deltamethrin. The lethal times (LT50) showed significant difference using both WHO and CDC bioassay methods.