Insights into factors sustaining persistence of high malaria transmission in forested areas of sub-Saharan Africa: the case of Mvoua, South Cameroon

Abstract Background In Mvoua, a village situated in a forested area of Cameroon, recent studies have reported high prevalence of Plasmodium falciparum infection among the population. In order to understand factors that can sustain such a high malaria transmission, we investigated the biology of Anopheles vectors and its susceptibility to insecticides, as well as long-lasting insecticidal net (LLIN) coverage, use and bio-efficacy. Methods A longitudinal entomological survey was conducted from July 2018 to April 2019. Adult mosquitoes were collected using the human landing catch (HLC) method and identified using morphological and molecular techniques. Anopheles gambiae (s.l.) larvae were sampled from several stagnant water pools throughout the village and reared to generate F1 adults. The presence of P. falciparum circumsporozoite antigen was detected in the heads and thoraces of mosquitoes collected as adults using an enzyme-linked immunosorbent assay. The insecticide susceptibility status of the local An. gambiae (s.l.) F1 population to the pyrethroid insecticides deltamethrin 0.5% and permethrin 0.75% was determined using World Health Organization-tube bioassays, while the frequency of the knockdown resistance (kdr) mutation was determined by PCR. Coverage, use and physical integrity of LLINs were assessed in households, then cone assays were used to test for their bio-efficacy on both the reference insecticide-susceptible Kisumu strain and on field F1 An. gambiae (s.l.) Results In total, 110 Anopheles mosquitoes were collected, of which 59.1% were identified as Anopheles funestus (s.l.), 38.18% as An. gambiae (s.l.) and 2.72% as An. ziemanii. Anopheles funestus was the most abundant species except in the long rainy season, when An. gambiae (s.l.) predominated (65.8%). In the dry seasons, vectors were principally endophagous (76% of those collected indoors) while they tended to be exophagous (66% of those collected outdoors) in rainy seasons. High Plasmodium infection was observed in An. gambiae (s.l.) and An. funestus, with a circumsporozoitic rate of 14.29 and 10.77%, respectively. Anopheles gambiae (s.l.) was highly resistant to pyrethroid insecticides (mortality rates: 32% for permethrin and 5% for deltamethrin) and harbored the kdr-L1014F mutation at a high frequency (89.74%). Of the 80 households surveyed, only 47.69% had achieved universal coverage with LLNs. Around 70% of the LLINs sampled were in poor physical condition, with a proportionate hole index > 300. Of the ten LLNs tested, eight were effective against the An. gambiae reference insecticide-susceptible Kisumu strain, showing mortality rate of > 80%, while none of these LLINs were efficient against local An. gamabie (s.l.) populations (mortality rates < 11.5%). Conclusion A combination of elevated P. falciparum infection in Anopheles vector populations, insufficient coverage and loss of effectiveness of LLINs due to physical degradation, as well as high resistance to pyrethroid insecticides is responsible for the persistence of high malaria transmission in forested rural area of Mvoua, Cameroon.

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Comparative assessment of insecticide resistance phenotypes in two major malaria vectors, Anopheles funestus and Anopheles arabiensis in south-eastern Tanzania

Malaria Journal ◽

10.1186/s12936-020-03483-3 ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Polius G. Pinda ◽

Claudia Eichenberger ◽

Halfan S. Ngowo ◽

Dickson S. Msaky ◽

Said Abbasi ◽

...

Keyword(s):

Insecticide Resistance ◽

Malaria Transmission ◽

Anopheles Arabiensis ◽

Pyrethroid Resistance ◽

Anopheles Funestus ◽

Indoor Residual Spraying ◽

Sub Saharan Africa ◽

World Health ◽

Malaria Vectors ◽

South Eastern

Abstract Background Long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) have greatly reduced malaria transmission in sub-Saharan Africa, but are threatened by insecticide resistance. In south-eastern Tanzania, pyrethroid-resistant Anopheles funestus are now implicated in > 80% of malaria infections, even in villages where the species occurs at lower densities than the other vector, Anopheles arabiensis. This study compared the insecticide resistance phenotypes between the two malaria vectors in an area where pyrethroid-LLINs are widely used. Methods The study used the World Health Organization (WHO) assays with 1×, 5× and 10× insecticide doses to assess levels of resistance, followed by synergist bioassays to understand possible mechanisms of the observed resistance phenotypes. The tests involved adult mosquitoes collected from three villages across two districts in south-eastern Tanzania and included four insecticide classes. Findings At baseline doses (1×), both species were resistant to the two candidate pyrethroids (permethrin and deltamethrin), but susceptible to the organophosphate (pirimiphos-methyl). Anopheles funestus, but not An. arabiensis was also resistant to the carbamate (bendiocarb). Both species were resistant to DDT in all villages except in one village where An. arabiensis was susceptible. Anopheles funestus showed strong resistance to pyrethroids, surviving the 5× and 10× doses, while An. arabiensis reverted to susceptibility at the 5× dose. Pre-exposure to the synergist, piperonyl butoxide (PBO), enhanced the potency of the pyrethroids against both species and resulted in full susceptibility of An. arabiensis (> 98% mortality). However, for An. funestus from two villages, permethrin-associated mortalities after pre-exposure to PBO only exceeded 90% but not 98%. Conclusions In south-eastern Tanzania, where An. funestus dominates malaria transmission, the species also has much stronger resistance to pyrethroids than its counterpart, An. arabiensis, and can survive more classes of insecticides. The pyrethroid resistance in both species appears to be mostly metabolic and may be partially addressed using synergists, e.g. PBO. These findings may explain the continued persistence and dominance of An. funestus despite widespread use of pyrethroid-treated LLINs, and inform new intervention choices for such settings. In short and medium-term, these may include PBO-based LLINs or improved IRS with compounds to which the vectors are still susceptible.

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Comparative assessment of insecticide resistance phenotypes in two major malaria vectors, Anopheles funestus and Anopheles arabiensis in south-eastern Tanzania

10.21203/rs.3.rs-19762/v3 ◽

2020 ◽

Author(s):

Polius Gerazi Pinda ◽

Claudia Eichenberger ◽

Halfan S Ngowo ◽

Dickson S Msaky ◽

Said Abbasi ◽

...

Keyword(s):

Insecticide Resistance ◽

Malaria Transmission ◽

Anopheles Arabiensis ◽

Pyrethroid Resistance ◽

Anopheles Funestus ◽

Indoor Residual Spraying ◽

Sub Saharan Africa ◽

World Health ◽

Malaria Vectors ◽

South Eastern

Abstract BackgroundLong-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) have greatly reduced malaria transmission in sub-Saharan Africa, but are threatened by insecticide resistance. In south-eastern Tanzania, pyrethroid-resistant Anopheles funestus are now implicated in > 80% of malaria infections, even in villages where the species occurs at lower densities than the other vector, Anopheles arabiensis. This study compared the insecticide resistance phenotypes between the two malaria vectors in an area where pyrethroid-LLINs are widely used.MethodsThe study used the World Health Organization (WHO) assays with 1×, 5× and 10× insecticide doses to assess levels of resistance, followed by synergist bioassays to understand possible mechanisms of the observed resistance phenotypes. The tests involved adult mosquitoes collected from three villages across two districts in south-eastern Tanzania and included four insecticide classes.FindingsAt baseline doses (1×), both species were resistant to the two candidate pyrethroids (permethrin and deltamethrin), but susceptible to the organophosphate (pirimiphos-methyl). Anopheles funestus, but not An. arabiensis was also resistant to the carbamate (bendiocarb). Both species were resistant to DDT in all villages except in one village where An. arabiensis was susceptible. Anopheles funestus showed strong resistance to pyrethroids, surviving the 5× and 10× doses, while An. arabiensis reverted to susceptibility at the 5× dose. Pre-exposure to the synergist, piperonyl butoxide (PBO), enhanced the potency of the pyrethroids against both species and resulted in full susceptibility of An. arabiensis (>98% mortality). However, for An. funestus from two villages, permethrin-associated mortalities after pre-exposure to PBO only exceeded 90% but not 98%.ConclusionsIn south-eastern Tanzania, where An. funestus dominates malaria transmission, the species also has much stronger resistance to pyrethroids than its counterpart, An. arabiensis, and can survive more classes of insecticides. The pyrethroid resistance in both species appears to be mostly metabolic and may be partially addressed using synergists, e.g. PBO. These findings may explain the continued persistence and dominance of An. funestus despite widespread use of pyrethroid-treated LLINs, and inform new intervention choices for such settings. In short and medium-term, these may include PBO-based LLINs or improved IRS with compounds to which the vectors are still susceptible.

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Implication of Anopheles funestus in malaria transmission in the city of Yaoundé, Cameroon

Parasite ◽

10.1051/parasite/2020005 ◽

2020 ◽

Vol 27 ◽

pp. 10 ◽

Cited By ~ 1

Author(s):

Landre Djamouko-Djonkam ◽

Diane Leslie Nkahe ◽

Edmond Kopya ◽

Abdou Talipouo ◽

Carmene Sandra Ngadjeu ◽

...

Keyword(s):

Anopheles Gambiae ◽

Malaria Transmission ◽

Anopheles Funestus ◽

Abundant Species ◽

Enzyme Linked Immunosorbent Assay ◽

Anopheles Coluzzii ◽

Urban District ◽

Human Landing ◽

Control And Prevention ◽

The City

The contribution of Anopheles funestus to malaria transmission in the urban environment is still not well documented. The present study assesses the implication of An. funestus in malaria transmission in two districts, Nsam and Mendong, in the city of Yaoundé. Adult mosquitoes were collected using Centers for Disease Control and Prevention miniature light traps (CDC-LT) and human landing catches from April 2017 to March 2018 and were identified morphologically to the species level. Those belonging to the Anopheles gambiae complex and to the Anopheles funestus group were further processed by PCR to identify members of each complex/group. Anopheline mosquitoes were analysed to determine their infection status using an enzyme-linked immunosorbent assay. Bioassays were conducted with 2–5-day-old female Anopheles funestus and An. gambiae s.l. to determine their susceptibility to permethrin, deltamethrin and dichlorodiphenyltrichloroethane (DDT). Six anopheline species were collected in the peri-urban district of Mendong: Anopheles gambiae, An. coluzzii, An. funestus, An. leesoni, An. ziemanni and An. marshallii; only four out of the six were recorded in Nsam. Of the two members of the Anopheles gambiae complex collected, An. coluzzii was the most prevalent. Anopheles coluzzii was the most abundant species in Nsam, while An. funestus was the most abundant in Mendong. Both Anopheles funestus and An. gambiae s.l. were found to be infected with human Plasmodium at both sites, and both were found to be resistant to DDT, permethrin, and deltamethrin. This study confirms the participation of An. funestus in malaria transmission in Yaoundé and highlights the need to also target this species for sustainable control of malaria transmission.

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An increasing role of pyrethroid-resistant Anopheles funestus in malaria transmission in the Lake Zone, Tanzania

Scientific Reports ◽

10.1038/s41598-021-92741-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nancy S. Matowo ◽

Jackline Martin ◽

Manisha A. Kulkarni ◽

Jacklin F. Mosha ◽

Eliud Lukole ◽

...

Keyword(s):

Malaria Transmission ◽

Anopheles Funestus ◽

Sub Saharan Africa ◽

North West ◽

Vector Population ◽

Lake Zone ◽

Sub Saharan ◽

Long Lasting Insecticidal Nets ◽

Study District

AbstractAnopheles funestus is playing an increasing role in malaria transmission in parts of sub-Saharan Africa, where An. gambiae s.s. has been effectively controlled by long-lasting insecticidal nets. We investigated vector population bionomics, insecticide resistance and malaria transmission dynamics in 86 study clusters in North-West Tanzania. An. funestus s.l. represented 94.5% (4740/5016) of all vectors and was responsible for the majority of malaria transmission (96.5%), with a sporozoite rate of 3.4% and average monthly entomological inoculation rate (EIR) of 4.57 per house. Micro-geographical heterogeneity in species composition, abundance and transmission was observed across the study district in relation to key ecological differences between northern and southern clusters, with significantly higher densities, proportions and EIR of An. funestus s.l. collected from the South. An. gambiae s.l. (5.5%) density, principally An. arabiensis (81.1%) and An. gambiae s.s. (18.9%), was much lower and closely correlated with seasonal rainfall. Both An. funestus s.l. and An. gambiae s.l. were similarly resistant to alpha-cypermethrin and permethrin. Overexpression of CYP9K1, CYP6P3, CYP6P4 and CYP6M2 and high L1014S-kdr mutation frequency were detected in An. gambiae s.s. populations. Study findings highlight the urgent need for novel vector control tools to tackle persistent malaria transmission in the Lake Region of Tanzania.

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Pyrethroid and Etofenprox Resistance in Anopheles gambiae and Anopheles coluzzii from Vegetable Farms in Yaoundé, Cameroon: Dynamics, Intensity and Molecular Basis

Molecules ◽

10.3390/molecules26185543 ◽

2021 ◽

Vol 26 (18) ◽

pp. 5543

Author(s):

Michael Piameu ◽

Philippe Nwane ◽

Wilson Toussile ◽

Konstantinos Mavridis ◽

Nadja Christina Wipf ◽

...

Keyword(s):

Anopheles Gambiae ◽

Mortality Rates ◽

World Health ◽

Anopheles Coluzzii ◽

Cross Sectional ◽

Metabolic Genes ◽

Anopheles Gambiae S.L ◽

Underlying Mechanisms ◽

Health Organization ◽

The City

Previous studies have indicated widespread insecticide resistance in malaria vector populations from Cameroon. However, the intensity of this resistance and underlying mechanisms are poorly known. Therefore, we conducted three cross-sectional resistance surveys between April 2018 and October 2019, using the revised World Health Organization protocol, which includes resistance incidences and intensity assessments. Field-collected Anopheles gambiae s.l. populations from Nkolondom, Nkolbisson and Ekié vegetable farms in the city of Yaoundé were tested with deltamethrin, permethrin, alpha-cypermethrin and etofenprox, using 1× insecticide diagnostic concentrations for resistance incidence, then 5× and 10× concentrations for resistance intensity. Subsamples were analyzed for species identification and the detection of resistance-associated molecular markers using TaqMan® qPCR assays. In Nkolbisson, both An. coluzzii (96%) and An. gambiae s.s. (4%) were found together, whereas only An. gambiae s.s. was present in Nkolondom, and only An. coluzzii was present in Ekié. All three populations were resistant to the four insecticides (<75% mortality rates―MR1×), with intensity generally fluctuating over the time between mod-erate (<98%―MR5×; ≥98%―MR10×) and high (76–97%―MR10×). The kdr L995F, L995S, and N1570Y, and the Ace-1 G280S-resistant alleles were found in An. gambiae from Nkolondom, at 73%, 1%, 16% and 13% frequencies, respectively, whereas only the kdr L995F was found in An. gambiae s.s. from Nkolbisson at a 50% frequency. In An. coluzzii from Nkolbisson and Ekié, we detected only the kdr L995F allele at 65% and 60% frequencies, respectively. Furthermore, expression levels of Cyp6m2, Cyp9k1, and Gste2 metabolic genes were highly upregulated (over fivefold) in Nkolondom and Nkolbisson. Pyrethroid and etofenprox-based vector control interventions may be jeopardized in the prospected areas, due to high resistance intensity, with multiple mechanisms in An. gambiae s.s. and An. coluzzii.

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Insecticide resistance in Anopheles stephensi in Somali Region, eastern Ethiopia

10.21203/rs.2.18020/v3 ◽

2020 ◽

Author(s):

Solomon Yared ◽

Araya Gebressielasie ◽

Lambodhar Damodaran ◽

Victoria Bonnell ◽

Karen Lopez ◽

...

Keyword(s):

Insecticide Resistance ◽

Anopheles Stephensi ◽

Mortality Rates ◽

Resistance Mechanisms ◽

World Health ◽

Malaria Vectors ◽

Resistance Locus ◽

Eastern Ethiopia ◽

Pyrethroid Insecticides ◽

Pirimiphos Methyl

Abstract Background The movement of malaria vectors into new areas is a growing concern in the efforts to control malaria. The recent report of Anopheles stephensi in eastern Ethiopia has raised the necessity to understand the insecticide resistance status of the vector in the region to better inform vector-based interventions. The aim of this study was to evaluate insecticide resistance in An. stephensi in eastern Ethiopia using two approaches: 1) World Health Organization (WHO) bioassay tests in An. stephensi; and 2) genetic analysis of insecticide resistance genes in An. stephensi in eastern Ethiopia. Methods Mosquito larvae and pupae were collected from Kebri Dehar. Insecticide susceptibility of An. stephensi was tested withmalathion 5%, bendiocarb 0.1%, propoxur 0.1%, deltamethrin 0.05%, permethrin 0.75%, Pirimiphos-methyl 0.25% and DDT 4%, according to WHO standard protocols. In this study, the knockdown resistance locus (kdr) in the voltage gated sodium channel (vgsc) and ace1R locus in the acetylcholinesterase gene (ace-1) were analysed in An. stephensi. Results All An. stephensi samples were resistant to carbamates, with mortality rates of 23% and 21% for bendiocarb and propoxur, respectively. Adult An. stephensi was also resistant to pyrethroid insecticides with mortality rates 67% for deltamethrin and 53% for permethrin. Resistance to DDT and malathion was detected in An. stephensi with mortality rates of 32% as well as An. stephensi was resistance to pirimiphos-methyl with mortality rates 14%. Analysis of the insecticide resistance loci revealed the absence of kdr L1014F and L1014S mutations and the ace1R G119S mutation. Conclusion Overall, these findings support that An. stephensi is resistant to several classes of insecticides, most notably pyrethroids. However, the absence of the kdr L1014 gene may suggest non-target site resistance mechanisms. Continuous insecticide resistance monitoring should be carried out in the region to confirm the documented resistance and exploring mechanisms conferring resistance in An. stephensi in Ethiopia.

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Insecticide Resistance in Anopheles stephensi in Somali Region, Eastern Ethiopia

10.21203/rs.2.18020/v2 ◽

2020 ◽

Cited By ~ 1

Author(s):

Solomon Yared ◽

Araya Gebressielasie ◽

Lambodhar Damodaran ◽

Victoria Bonnell ◽

Karen Lopez ◽

...

Keyword(s):

Insecticide Resistance ◽

Anopheles Stephensi ◽

Mortality Rates ◽

Resistance Mechanisms ◽

World Health ◽

Malaria Vectors ◽

Eastern Ethiopia ◽

Pyrethroid Insecticides ◽

Pirimiphos Methyl ◽

Health Organization

Abstract Background: The movement of malaria vectors into new areas is a growing concern in the efforts to control malaria. The recent report of Anopheles stephensi in eastern Ethiopia has raised the necessity to understand the insecticide resistance status of the vector in the region to better inform vector-based interventions. The aim of this study was to evaluate insecticide resistance in An. stephensi in eastern Ethiopia using two approaches: 1) World Health Organization (WHO) bioassay tests in An. stephensi and 2) genetic analysis of insecticide resistance genes in An. stephensi in eastern Ethiopia. Methods: Mosquito larvae and pupae were collected from Kebridehar. Insecticide susceptibility of An. stephensi was tested with malathion 5%, bendiocarb 0.1%, propoxur 0.1%, deltamethrin 0.05%, permethrin 0.75%, Pirimiphos-methyl 0.25% and DDT 4%, according to WHO standard protocols. Results: All An. stephensi samples were resistant to carbamates, with mortality rates 23% and 21% for bendiocarb and propoxur, respectively. Adult An. stephensi was also resistant to pyrethroid insecticides with mortality rates 67% for deltamethrin and 53% for permethrin. Resistance to DDT and malathion was detected in An. stephensi with mortality rates of 32% as well as An. stephensi was resistance to pirimiphos-methyl with mortality rates 14%. Analysis of the voltage gate sodium channel gene (vgsc) revealed the absence of kdr L1014 mutations. Conclusion: Overall, these findings support that An. stephensi is resistant to several classes of insecticides, most notably pyrethroids. However, the absence of the kdr L1014 gene may suggest non-target site resistance mechanisms. Continuous insecticide resistance monitoring should be carried out in the region to confirm the documented resistance and exploring mechanisms conferring resistance in An. stephensi in Ethiopia.

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Spatial Distribution of Anopheles gambiae and Anopheles funestus and Malaria Transmission in Suba District, Western Kenya

International Journal of Tropical Insect Science ◽

10.1017/s1742758400023584 ◽

2003 ◽

Vol 23 (03) ◽

pp. 187-196 ◽

Cited By ~ 4

Author(s):

Josephat I. Shililu ◽

Charles M. Mbogo ◽

Clifford M. Mutero ◽

James T. Gunter ◽

Chris Swalm ◽

...

Keyword(s):

Spatial Distribution ◽

Anopheles Gambiae ◽

Malaria Transmission ◽

Anopheles Funestus ◽

Western Kenya

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Anopheles arabiensis and Anopheles funestus s.s. biting pattern in Dielmo, an area of low level exposure to malaria vectors.

10.21203/rs.3.rs-24165/v1 ◽

2020 ◽

Author(s):

Souleymane Doucoure ◽

Omar Thiaw ◽

Amélé N. Wotodjo ◽

Charles Bouganali ◽

Nafissatou Diagne ◽

...

Keyword(s):

Malaria Transmission ◽

Anopheles Funestus ◽

Molecular Techniques ◽

Malaria Vectors ◽

Bed Nets ◽

Low Level ◽

Biting Rate ◽

Outdoor Biting ◽

Night And Day ◽

Biting Behaviour

Abstract Background: In Dielmo, Senegal, the widespread use of long lasting insecticidal nets has decreased both the incidence of malaria and the density of the anopheles population. However, persistent low-level malaria transmission may hamper efforts to eliminate the disease. Therefore, continuous monitoring of the vector population is needed in order to improve knowledge of anopheles biting behaviour and to readjust control interventions.Methods: In 2015, anopheles were collected every month for a whole year and each specimen was identified using morphological and molecular techniques. The biting pattern of each species was analysed according to night (7pm–7am) and day (7am–11am) periods, the place of biting and the season. The ELISA CSP technique was used to evaluate the entomological inoculation rate (EIR).Results: An. arabiensis and An. funestus s.s. were found to be the main vectors biting human in Dielmo. Overall, the biting rate was low, at 3.84bites per night (bpn) and 1.27 bites per day (bpd), respectively. The EIR was 2.51 and 5.03 infected bites per year during the night and day, respectively. During the night, the An. arabiensis and An. funestus biting rate was 1.81 bpn and 1.71 bpn, respectively (p=0.80). During daylight hours, their aggressiveness decreased to 0.51 bpd and 0.73 bpd for An. arabiensis and An. funestus, respectively (p=0.30). During the night, depending on the seasons, only An. funestus displayed a significant outdoor biting behaviour both in the rainy and dry seasons, while during the day, no specific trend of indoor or outdoor biting was observed, regardless of the season for both vectors.Conclusion: This study highlighted low level anopheles nocturnal and diurnal biting and the associated risk of malaria transmission. It showed also the influence of the season on the indoor and outdoor biting pattern, indicating that the human population could be exposed all year round to a low level of anopheles bites. Control programs should increase awareness of the use of bed-nets throughout the year, particularly shortly after dawn when people are still indoors.

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Plasmodium ovale: Parasite and Disease

Clinical Microbiology Reviews ◽

10.1128/cmr.18.3.570-581.2005 ◽

2005 ◽

Vol 18 (3) ◽

pp. 570-581 ◽

Cited By ~ 125

Author(s):

William E. Collins ◽

Geoffrey M. Jeffery

Keyword(s):

Public Health Problem ◽

Molecular Techniques ◽

Sub Saharan Africa ◽

Enzyme Linked Immunosorbent Assay ◽

Developmental Cycle ◽

Major Public Health Problem ◽

Plasmodium Ovale ◽

Malaria Parasites ◽

Sub Saharan

SUMMARY Humans are infected by four recognized species of malaria parasites. The last of these to be recognized and described is Plasmodium ovale. Like the other malaria parasites of primates, this parasite is only transmitted via the bites of infected Anopheles mosquitoes. The prepatent period in the human ranges from 12 to 20 days. Some forms in the liver have delayed development, and relapse may occur after periods of up to 4 years after infection. The developmental cycle in the blood lasts approximately 49 h. An examination of records from induced infections indicated that there were an average of 10.3 fever episodes of ≥101°F and 4.5 fever episodes of ≥104°F. Mean maximum parasite levels were 6,944/μl for sporozoite-induced infections and 7,310/μl for trophozoite-induced infections. Exoerythrocytic stages have been demonstrated in the liver of humans, chimpanzees, and Saimiri monkeys following injection of sporozoites. Many different Anopheles species have been shown to be susceptible to infection with P. ovale, including A. gambiae, A. atroparvus, A. dirus, A. freeborni, A. albimanus, A. quadrimaculatus, A. stephensi, A. maculatus, A. subpictus, and A. farauti. An enzyme-linked immunosorbent assay has been developed to detect mosquitoes infected with P. ovale using a monoclonal antibody directed against the circumsporozoite protein. Plasmodium ovale is primarily distributed throughout sub-Saharan Africa. It has also been reported from numerous islands in the western Pacific. In more recent years, there have been reports of its distribution on the Asian mainland. Whether or not it will become a major public health problem there remains to be seen. The diagnosis of P. ovale is based primarily on the characteristics of the blood stages and its differentiation from P. vivax. The sometimes elliptical shape of the infected erythrocyte is often diagnostic when combined with other, subtler differences in morphology. The advent of molecular techniques, primarily PCR, has made diagnostic confirmation possible. The development of techniques for the long-term frozen preservation of malaria parasites has allowed the development diagnostic reference standards for P. ovale. Infections in chimpanzees are used to provide reference and diagnostic material for serologic and molecular studies because this parasite has not been shown to develop in other nonhuman primates, nor has it adapted to in vitro culture. There is no evidence to suggest that P. ovale is closely related phylogenetically to any other of the primate malaria parasites that have been examined.

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