The results presented here show that, after five years of malaria vector control interventions, and in spite of reductions in the prevalence of human malaria on Bioko Island[1, 3], foci of persistent intensive malaria transmission remained. High human biting and sporozoite rates in Punta Europa, in the north-western part of the island, indicate that the human population in this area are potentially exposed to about 840 infective mosquito bites per year when indoor and outdoor biting is taken into account. The outdoor annual EIR was 922 infective mosquito bites per year; using the data from the March collections only the EIR was close to 1200. These rates are among the highest entomological inoculation rates published in the literature[19–21]. An annual EIR of 1,235 infective bites per person was reported in the early 1990’s in Sierra Leone, although this value was later revised to 884 by Hay et al.. All three sites where HLC were conducted in the present study had an annual EIR exceeding the calculated African average of 121 infective bites per year. An EIR of 1,030 was reported from Riaba on Bioko Island in the late 1990s, suggesting that Bioko Island had one of the (if not the) highest rates of malaria transmission when the control efforts started, and in spite of the intensive and extensive nature of these control efforts, transmission still persists, indicating the need for sustained and scaled up control. This underscores how important it is to have a long-term control framework in place, one that can sustain control over more than the typical project cycle, and one that can adapt to changing transmission realities.
The high EIR in Punta Europa in 2009 resulted from both extremely high HBR and high sporozoite rates. In a review by Hay et al. similar or higher HBRs than this were recorded only from Burundi and Cameroon. More recently, rainy season HBRs in Dakar, Senegal were recorded to be almost three times higher than those reported in the current study. The Punta Europa area is unique for Bioko Island in that it is a fast-growing industrial and commercial zone, where the country’s international airport is located. The area is unusually flat compared to the rest of Bioko Island; it has a low forest cover, relatively poor drainage, and consists of many construction sites, all of which contribute to providing excellent mosquito breeding habitats.
Mosquito density in the Punta Europa area has a complex relationship with the daily amount and intensity of rainfall. For example, the 2009 LTCs showed that the high mosquito density in early June followed high rainfall in early May, supplemented by moderate rainfall in the second half of May (Figure6). On the other hand, the sharp decline in mosquitoes on June 29 was likely due to one single heavy storm downpour on June 22 (included in the June 29 precipitation bar in Figure6), which presumably flushed larvae out of their breeding habitats. The mosquito densities in Arena Blanca and Riaba were not affected by precipitation in Punta Europa, probably because of a different rainfall pattern in those locations. It could also be due to the higher number of An. melas collected in these locations. Anopheles melas might be affected differently than An. gambiae s.s. by rainfall patterns, although this was not studied here.
The An. gambiae s.s. populations in Punta Europa also have a relatively high sporozoite rate averaging ~3% and ranging between 1.0% (± 1.7% 95% CI) and 3.9% (± 1.9% 95% CI) over the year (data by month not shown). In Riaba the sporozoite rate in this species was much higher, at least in the HLC, but a much lower human biting rate resulted in a considerably lower EIR in this location.
Malaria parasites in Punta Europa are highly prevalent in the human population[4, 5]. Although the high EIR values reported here indicate a high potential for transmission, this is only realized if the human population is not protected by effective control measures. We have previously reported high outdoor biting rates in this area, and given the high effective coverage with IRS and LLINs, a large fraction of the transmission between humans and mosquitoes probably occurs at outdoor venues, including bars and restaurants. Based on the data presented here outdoor biting seems to be prevalent throughout the island. The potential for malaria transmission is further aggravated by the observation that host seeking begins early in the evening. In the current HLCs, about 30-50% of indoor biting occurred before midnight when the protection from vector control measures is not optimal. About 35-40% of outdoor biting during HLC occurred before midnight, at a time when most people are active outside their houses and thus being greatly exposed to malaria mosquito bites. The potential for transmission in outdoor venues where people are not protected by IRS and LLIN is therefore considerable and underscores the need for the development of control methods that effectively target outdoor feeding populations in addition to IRS and LLINs.
A recent study from Bioko concluded that there is currently no epidemiological evidence of outdoor biting having an impact on malaria prevalence in children in Bioko. Although the study found that spending time outdoors at night was rare amongst children under 15 years, those who did spend the previous night outdoors did not have a higher prevalence of infection than those who stayed indoors. However, the power of that study to detect an impact of spending time outdoors was limited by the fact that few children slept under a bed net, and that the last IRS spray round was conducted 3–5 months prior to the survey, providing little protective effect to being indoors. Furthermore, adults are far more likely to stay outdoors at night than children and thereby potentially keeping parasites circulating in the general population. Further research is, therefore, required to determine the epidemiological impact of being outdoors in the evening and at night in both children and adults.
In 1998–1999, well before the implementation of the current control program, an annual EIR of 1,030 was reported for Riaba. Anopheles funestus was the main contributor (76%) to this EIR, whereas An. gambiae was responsible for the remaining 24%. It is quite clear that the entomological situation has changed drastically in Riaba in the thirteen years since the first report. The present results indicate that An. funestus was absent or, at most, occurring at extremely low densities in Bioko, including Riaba. Presumably, the continuous BIMCP vector control activities, particularly IRS, have nearly eliminated An. funestus on Bioko; something which was also observed following the first IRS rounds. Sporozoite rates are still high in Riaba among An. gambiae s.s. and An. melas, indicating that parasite transmission from human to mosquito is high in the area, despite relatively low biting rates compared to the rest of the island. The extreme peak in sporozoite rates in July and low HBRs could be explained by a shift in the age structure of the mosquito populations in this site. For example, if for some reason larval mortality was high for a period, this would result in fewer adult mosquitoes being collected, but they would be older and thus have a higher sporozoite rate.
Somewhat puzzling, Cano et al. conducted malaria mosquito collections in 1998–99 on Bioko Island but did not identify any An. melas specimens, although this species was found at considerable frequency in the current collections in both Riaba and Arena Blanca. Anopheles melas was also present in Riaba in the 2003–2004 pre-spray BIMCP collections with very high sporozoite rates. Possibly, most of the collection sites of Cano et al. were located further inland, away from the coast where An. melas breeding sites are located.
In Arena Blanca, An. melas was the major malaria vector. This site is close to the town of Luba, but the environmental and socioeconomic conditions differ markedly from Luba. Arena Blanca is a small fishing village comprised of approximately 50 houses located directly on the beach. The majority of people living there are Annobonese fishermen with a culture distinctly different from the rest of the island. Arena Blanca is also a popular weekend beach resort for people from around the island. Although the calculated EIR is the lowest on the island, people, including weekenders, are still exposed to an average of about 22 outdoor biting malaria mosquitoes during the first half of the night, of which ~1% might be infected.
Our results indicate that the S form of An. gambiae s.s. was eliminated from the island while the M form remains. Prior to the BIMCP, 36% of the An. gambiae s.s. mosquitoes collected on Bioko consisted of the M form, but by the end of the third spray round this had increased to 80%. Successive analyses showed that the M-form increased steadily from 80% in 2005 (n = 189), 98% in 2006 (n = 467), 94% in 2007 (n = 79), and 100% in 2008 (n = 149) (BIMCP unpublished data). In the 2009 collections, none of the 3,043 mosquitoes analyzed (consisting of a proportional sample across all sites and time points) belonged to the S form of An. gambiae s.s. A possible explanation for the apparent disappearance of the S form could be differences in the frequencies of kdr mutation between the two molecular forms. Knockdown resistance allele frequencies among An. gambiae on Bioko Island are unusual in that the L1014F allele was at high frequency in the M form, rather than the S form early on. This contrasts with the situation on the West African mainland, where this allele first spread through S form populations, prior to introgression into the M form. This could potentially have led to the persistence of the M form and the disappearance of the S form on Bioko.
On the other hand, a recent modeling study [Kiszewski et al. in review] indicated that kdr alleles limit the efficacy of IRS only to a certain degree making it hard to see how this difference in kdr frequency resulted in the disappearance of the S form. Likewise, this does not explain the continued decline of the S form after the BIMCP switched to using carbamates in IRS instead of pyrethroids. In the equatorial rain forest of Central Africa, the M and S forms are widely sympatric and no niche differentiation has been detected. Another possibility could be that the M and S forms differed in their propensity to feed and/or rest indoors, making the M form less susceptible to IRS and LLINs, although an earlier rather limited survey found no evidence of outdoor feeding An. gambiae s.l. on Bioko Island. In addition, or alternatively, the M form may have been less susceptible to the two classes of insecticides used in the IRS and LLIN campaign due to kdr and more efficient detoxification.
The L1014F mutation was the only insecticide resistance allele detected among the An. gambiae mosquitoes on Bioko. The frequency of L1014F allele varied but exceeded 40% in most locations. The highest frequency was observed in the urban populated areas in the north of the island; reaching nearly 90% in Semu. This suggests that insecticide use from previous and present pyrethroid-based control campaigns, but most likely household insecticide use as well, has resulted in increased frequency of the L1014F allele. Large businesses and companies often have their own mosquito control programs independent from the BIMCP, further reinforcing selection pressure for insecticide resistance. Agricultural use of insecticides is not common in Equatorial Guinea[31, 32] and is therefore unlikely to impact kdr allele frequencies.
Following an initial spray round with a pyrethroid, the BIMCP switched to bendiocarb, a carbamate insecticide, in the IRS. This decision was based on the presence of kdr and absence of ace-1
mutations. As the frequency of kdr mutation was low in An. gambiae s.s. in Riaba and the mutation was absent in An. melas in Arena Blanca, these locations are favorable for a change in insecticide used in IRS. The NMCP of Equatorial Guinea has recently adopted the WHO Guidelines for Insecticide Resistance Management (GPIRM) and decided to switch to pyrethroids on a rotational basis for the whole of Bioko Island.
Although four years of control on Bioko was successful in greatly reducing child mortality and having a dramatic impact on the effective population size of An. gambiae and An. melas entomological indicators indicate that, even if the force of transmission has been substantially reduced relative to pre-intervention levels, it still remains high on the island relative to other African contexts, and that there remain foci of very high transmission. As the current twice-a-year spray rounds with bendiocarb have insufficient residual duration to provide effective control on Bioko, the IRS intervention needs to be supported by much higher LLIN coverage and supplemented with an additional set of focally administered control measures. The BIMCP is currently piloting such a stratified focal approach in Punta Europa, combining IRS with universal LLIN distribution, Focal Mass Screening and Treatment (FMST), reinforced IEC/BCC and source reduction through larval source management. Future focal efforts might also evaluate the efficacy of infrastructural modifications and environmental management, along with testing and possible introduction of other transmission barriers (e.g. insecticide treated materials) and personal protection methods. Collaboration between BIMCP, the airport administration, and local businesses is also a strategy that should be explored to reduce mosquito breeding in these areas through infrastructural modification and environmental management. Although the precise impact of outdoor biting on malaria prevalence still needs to be fully clarified, the fact that extensive outdoor biting is occurring on Bioko Island and other parts of Africa suggests that alternative and innovative control options should be developed and implemented to address this problem.