key: cord-0968072-wukdvlgl authors: Bango, Zackary Austin; Tawe, Leabaneng; Muthoga, Charles Waithaka; Paganotti, Giacomo Maria title: ast and current biological factors affecting malaria in the low transmission setting of Botswana: A review date: 2020-07-12 journal: Infect Genet Evol DOI: 10.1016/j.meegid.2020.104458 sha: c88acc052ad2de05c76cd12ae3c49caa51cf3dc3 doc_id: 968072 cord_uid: wukdvlgl Malaria continues to be one of the top infectious agents contributing to morbidity and mortality in sub-Saharan Africa. Annually, Botswana accounts only for a small proportion of cases (<<1%). Despite significantly reduced incidence rate, the country still experiences sporadic outbreaks that hamper the goal of malaria elimination. This review evaluated previous and current biological factors that impact malaria in Botswana, specifically focussing on the vectors, the parasite and the host. This was accomplished via a literature review evaluating these variables in Botswana. Current literature suggests that Anopheles arabiensis is the main malaria vector in the country. Several other potential vectors have been found widely distributed throughout Botswana in high numbers, yet remain largely unstudied with regards to their contribution to the country's malaria burden. We also report the most up to date list of all Anopheles species that have been found in Botswana. Plasmodium falciparum is responsible for the vast majority of symptomatic malaria in the country and some drug resistance markers have been documented for this species. Plasmodium vivax has been reported in asymptomatic subjects, even though a large proportion of the Botswana population appears to be Duffy antigen negative. Very little is known about the true distribution of P. vivax and no point of care testing infrastructure for this species exists in Botswana, making it difficult to tailor treatment to address possible recrudescence or relapse. Due to a genetically diverse population with a substantial Khoisan contribution into the Bantu genetic background, several phenotypes that potentially impact prevalence and severity of malaria exist within the country. These include sickle cell trait, Glucose-6-Phosphate Dehydrogenase deficiency, and Duffy negativity. This review highlights the information that currently exists on malaria in Botswana. It also postulates that a comprehensive understanding of these aforementioned biological factors may help to explain malaria persistence in Botswana. Despite over a century of data driven education, research, and elimination efforts (N'ajera-Morrondo, (Thomson et al, 2005) . Although rainfall usually occurs between November and April, Botswana is vulnerable to intermittent and periodic drought (Thomson et al, 2005 ) associated with the El Niño Southern Oscillation (ENSO) pattern. Malaria transmission in Botswana is seasonal and highly unstable. Transmission typically occurs between November and April during the rainy season when extreme flooding creates prime breeding grounds for malarial vectors (Republic of Botswana, 2018) . Peak malaria transmission usually occurs in March-April due to increased rainfall and temperatures in the previous months (Thomson et al, 2005) . Spatial-statistical models have identified summer rainfall, mean annual temperature and altitude to be the main environmental predictors of malaria risk in Botswana, which explains the annual fluctuations registered in the last years (Craig et al, 2007) . These environmental variables make the more South and Southeastern regions prone to fluctuating levels of transmission and sporadic epidemics (Botswana Ministry of Health, 2010; Craig et al, 2007) . Whereas, malaria in the Northwestern region is considered endemic with sustained transmission annually (World Health Organization, 2015a). Malaria incidence in the region increases during La Niña Southern Oscillations episodes, and decreases with El Niño, despite some incongruences (Craig et al, 2007; Kgorobutswe, 2020 To date, relatively little has been published on malaria in Botswana. Here, we summarize those publications and consider current factors affecting malaria elimination in Botswana via a literature review over the past nine decades. We specifically considered studies involving malaria vector species, Two search engines, PubMed and Google Scholar, were utilized in the review of the widely available literature. All publications were included that matched the selection criteria published between 1 January 1937 and 5 May 2020. The criteria were defined as: any primary data or information in the context of Botswana involving the vectors of malaria, the parasite, or the host's interaction with malaria. The search query: "malaria Botswana OR malaria Bechuanaland" was used for PubMed. The search query: "allintitle: Botswana malaria" and "allintitle: Bechuanaland malaria" were utilized for Google Scholar. Each publication was examined for primary data or findings that matched at least one of our three selection criteria. If met, then it was included in the review. The titles of the references from these publications were also scanned for keywords matching our selection criteria and included if they met one of them. Authors included selected publications that did not meet selection criteria at their discretion in order to provide background information on a specific topic, or to further elaborate on a topic when no literature on Botswana were available for reference. Vectors within the Anopheles gambiae sensu lato (s.l.) complex are widely recognized as the primary contributors to malaria transmission in sub-Saharan Africa. Two species within this complex, An. 1972). Anopheles arabiensis accounts for the majority of malaria transmission within the SADC region (World Health Organization, 2015a). To various degrees, An. arabiensis when compared to similar vectors within the An. gambiae complex is described to as partially zoophilic, exophagic and exophilic. Wide variances in feeding and resting patterns have been recorded, mostly depending upon geographical location (Sinka et al, 2010) . These behavioral inconsistences may be controlled genetically rather than environmentally. Karyotype inversions in this species appear to alter resting behavior and anthropophily, allowing for rapid propagation of a trait if selective pressure is applied . They also found it to be highly prevalent at a larval stage (Chirebvu et al, 2014) . Additional studies in this village found samples to be primarily zoophagic, with only 27% of blood fed mosquitos testing positive for human blood (Chirebvu et al, 2014) . A study in seven other districts found a similar average human blood fed frequency of 28% for this species (Tawe et al, 2017) . Though, these mosquitoes were collected via pyrethrum spray catches (PSC), suggesting that the aforementioned frequency of 27-28% does not reflect the integration of anthropophily for the vector, given its known behavioral heterogeneity and the likelihood of blood fed mosquitos resting outdoors where PSC would fail to collect them. Additional studies examining the PSC collections, depending on the region, time of year, annual rainfall and human parasite reservoir (Tawe et al, 2017) . This delineates An. arabiensis as the only vector with confirmed Plasmodium infection in the country. This relatively limited prevalence (0-3%) of infected vectors by site contributes to high levels of malaria transmission during peak malaria season and could explain the epidemic nature of the disease in certain areas (Mbogo et al, 1995) , especially when considering that infected vectors have been shown to congregate in specific areas in other countries (Kulkarni et al, 2006) . Anopheles arabiensis has been described in all of Botswana's westernmost districts, placing over half of the country's populationat at risk to malaria due the presence of malaria infected vectors, is of particular concern due to documented insecticide resistance and for possibly causing the largest malaria epidemic in South Africa in over 50 years (Hargreaves et al, 2000) . Four studies from the past three decades: a PhD thesis published in 1995, a study from 1998, a survey completed in 2017, and a ( Figure 4 ). The study in 2017 confirmed the presence of An. parensis in 4 districts, An. longipalpis type C in 2 districts, as well as several non-vector species within the group (Tawe et al, 2017) ( Figure 4 ). Of the two species described in the 2017 study, only An. parensis was found to have taken a human blood meal. The most recent study in 2020 found An. longipalpus type C in one district, and An. Additional species of Anopheles are present in Botswana, though their capacity as vectors is not confirmed in Africa. However, disregarding these species in future vector studies should be considered erroneous due to the potential of emerging vectorial behavior or ability. In Box 1 we include the most up to date list of Anopheles species found in Botswana since 1935. Of the five species of Plasmodia generally accepted to exploit humans for schizogony ( . High levels of Duffy negativity in Africa, an antigen originally thought to be essential for erythrocyte invasion, initially led to P. vivax being discarded as a pathogen of major concern in the continent. However, international literature now points to P. vivax having the ability to This is noteworthy because many RDTs utilized within the country lack the capacity to identify species other than P. falciparum and only indicate a mixed infection. This makes it difficult for clinicians to tailor treatment to specific species, which can cause relapse. A recent report on drug resistance markers in P. falciparum demonstrated that the parasite harbors certain variation in several of the genes analyzed. In the study, 95.8% of samples showed N86 J o u r n a l P r e -p r o o f Journal Pre-proof polymorphism at Plasmodium falciparum multidrug resistance 1 gene (pfmdr1) known to confer sensitivity to 4-aminoquinolines (Howes et al, 2015) (chloroquine and amodiaquine) but also associated to a reduced sensitivity to lumefantrine (Wongsrichanalai et al, 2002) . This suggests that a similar situation to that seen in Malawi (Kublin et al, 2003) has unfolded in Botswana with regards to chloroquine. More importantly, P. falciparum may be generally tolerant to lumefantrine, which is one of the compounds utilized in first line treatment together with the highly efficacious artemether. The same study found two synonymous mutations -V555V and R513R -in the propeller domain of the P. falciparum Kelch13 gene (pfk13) which is implicated in the resistance to artemisinin. It should be noted that neither of these genetic variants were associated with a delayed parasite clearance phenotype. However, this indicates that molecular assessment and continuous surveillance of the pfk13 and pfmdr1 genes as drug resistance markers in Botswana is of paramount importance. No documented drug surveillance conducted by local Ministry of Health and Wellness for P. vivax has been reported in Botswana. Botswana is home to a population that arises from two distinct ancestries, the Bantu and Khoisan speaking peoples, with remarkably diverse genetic and linguistic backgrounds (Schlebusch et al, 2016) . . This is especially pertinent since the Bantu originated from a highly endemic malaria setting, whereas the Khoisan were likely only exposed to sporadic outbreaks that did not apply chronic selective pressure and therefore lack of malaria-resistance alleles (Maingard, 1937; J o u r n a l P r e -p r o o f Journal Pre-proof Schuster et al, 2010) . Additionally, the recent imposed sedentarism to the traditionally nomadic huntergatherers Khoisan communities of the Kalahari region of Botswana (Kent, 1995; Winters, 2015; Ikeya, 2018 ) may also have an impact on malaria acquisition risk. Recent reports have also indicated that this genetic diversity plays a significant role in pharmacogenetics pertaining also to antimalarial drugs used in Botswana . In Botswana, the peculiar genetic admixture between these two populations clusters specific genotypes more than other Southern African countries (Retshabile et al, 2018) . In particular, this genetic structure has potential implications on susceptibility and resistance to Of final note, the original occupants of the region, the Khoisan, appear to have been occasionally exposed to malaria "in exceptionally rainy seasons" (Maingard, 1937) and had traditional medicines to treat its symptoms. A report from the 1930s described a tribe of Khoisan that had lost many of its children to the disease during a flood year. This same group utilized the roots of Tarchonanthus camphoratus to treat the fevers and headaches associated with malaria (Maingard, 1937 ). In many African countries overall childhood mortality is high; but in children with sickle cell disease it is higher still (50-90%). Though, limited temporal data exists on the continent (Grosse et al, 2011) . The frequency and distribution of the haemoglobin S (HbS) allele, and its overlap with that of malaria, acts as the geographical confirmation of the malaria hypothesis for selection of this allele (Haldane, 1949) . In Botswana, the frequency of the HbS allele is estimated to be between 0 and 2%, though this is a very rough estimation given a lack of data for the region (Piel et al, 2010) . However, these limited data support a distribution of this allele that generally reflects the malaria endemicity in the country, with higher frequencies seen in the North and lower in the South. Lastly, a survey conducted in 1967 was unable to identify any sickled cells in a sample of Khoisan and Bantu individuals in the Kgalagadi (Jenkins et al, 1968) . Additional surveys would be needed in order to measure HbS allele frequency in J o u r n a l P r e -p r o o f Journal Pre-proof Botswana according to the geographical context and malaria endemicity. Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is common in Africa due to high levels of selective pressure from Plasmodium and conferred advantage against clinical malaria in heterozygous females (Sirugo et al, 2014) . In Botswana, data exist on the frequency of the African G6PD A-allele. Early publications from the 1960s and 1970s could only find relatively low G6PD deficiency in the Khoisan peoples they studied (1-5% with a higher percentage in the Northwest), and surprisingly no deficiency in the Bantu (Jenkins et al, 1968 ). This finding is somewhat congruent with a recent study that discovered the G6PD deficiency rate in a pattern that peaks in the Northwest (3.37%) and decreases steadily with latitude until reaching its minimum in the Southeast of the country . Interestingly, these latter results reflect the distribution of malaria endemicity in Botswana. Glucose-6-Phosphate Dehydrogenase testing should be considered if radical cure for P. vivax via primaquine is ever utilized in Botswana, especially with regards to the use of a point of care diagnostic tool for G6PD deficiency, prior to treatment. It is worth noting that single dose primaquine, that is gametocytocidal for P. falciparum, has been shown to be safe for G6PD deficient persons (World Health Organization, 2015b). Very little published data exists on the Duffy status of individuals in Botswana relative to their respective genetic backgrounds. In general, literature shows that there is a lack of the African-specific Southern Africa from a region between southern Nigeria and Cameroon (Nurse and Philippson, 2003) where malaria was (and is still) endemic, which correlates with having Duffy negativity at a very high rate. Thus, Duffy negativity rate in Botswana mostly depends from the admixture history between the two main populations. One survey based on 79 unrelated subjects showed that 75.9% of North-eastern J o u r n a l P r e -p r o o f Journal Pre-proof individuals from Botswana were Duffy negative (Fy(a-b-) ) (Chasko et al, 1979) . Another study by these authors found a similar frequency of the same phenotype in individuals from the Bakgalagadi tribe, which consists of peoples of mostly Bantu origin in the Southwest of the country (Jenkins et al, 1987) . A recent global report on Duffy, which summed data from several surrounding countries, predicted a gradual decline in the frequency of the negative phenotype in the Southwest direction, especially as one crosses the border into Namibia (Howes et al, 2011) . Given that the Bantu peoples As we write, the SARS-CoV-2 epidemic is ongoing in Botswana. The COVID-19 pandemic has forced countries to prioritize containment and mitigation measures such as social distancing and lockdowns as part of the primary response in reducing the number of cases. These measures have resulted in readjustment of resources that were initially allocated towards routine public health programs such as immunization and surveillance. Given that majority of malaria cases occur in Sub-Saharan Africa, one of the poorest regions in the world, the further readjusting of already limited resources is likely to put a strain on Governments in the provision of quality healthcare. J o u r n a l P r e -p r o o f Journal Pre-proof Furthermore, the similarity of the most symptoms associated between COVID-19 and malaria (such as fever and fatigue) has the potential to misdiagnose individuals. This places further strain on healthcare resources. It is important to note that all efforts towards malaria elimination should still continue in order to not erode all efforts and milestones that have been achieved. Botswana has made massive strides over the past 50 years with malaria control initiatives and has come remarkably close to elimination with the turn of the millennia. However, an increase of more than 80% background of Batswana should also be considered for insights into its complex interaction with malaria. Overall, these three major biological factors should be considered in order to craft an effective elimination strategy for the country. The authors declare that they have no competing interests. # P30 AI045008]. We would like to thank the entire joint University of Botswana-University of Pennsylvania molecular laboratory for their continuous support and dialogue. As well as staff from the Botswana-University of Pennsylvania Partnership and University of Botswana for their assistance. Finally, we would like to acknowledge all of the talented students that always brighten our lab. 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Accessed 3 Accessed 3 Published potential/known malaria vectors' distribution in Botswana An. funestus s.s., An. longipalpis, An. marshallii, An. parensis, An. pharoensis, An. rivulorum, An. squamosus, An. vaneedeni, An. ziemanni, An. nili, An. argentolobatus, An. caliginosus, An. coustani, An. distinctus, An. leesoni, An. listeri, An. pretoriensis, An. quadriannulatus, An. rhodesiensis, An. rufipes, An. seretsei, An. tchekedii, An. tenebrosus, An. walravensi, An. wellcomei ugandae, An. theileri Box 1. List of all 31 anophelines that have been identified in Botswana since 1935 Chirebvu et al J o u r n a l P r e -p r o o f