Plasmodium parasites. However, de novo drug development is a costly and time-consuming process. An alternative strategy is to evaluate the anti-plasmodial activity of compounds that are already approved for other purposes, an approach known as drug repurposing. Here, we will review efforts to assess the anti-plasmodial activity of existing drugs, with an emphasis on the obligatory and clinically silent liver stage of infection. We will also review the current knowledge on the classes of compounds that might be therapeutically relevant against Plasmodium in the context of other communicable diseases that are prevalent in regions where malaria is endemic. Repositioning existing compounds may constitute a faster solution to the current gap of prophylactic and therapeutic drugs that act on Plasmodium parasites, overall contributing to the global effort of malaria eradication.Malaria remains one of the most prevalent infectious diseases worldwide, primarily affecting some of the most vulnerable populations around the globe. Despite achievements in the treatment of this devastating disease, there is still an urgent need for the discovery of new drugs that tackle infection by  Malaria remains one of the most prevalent infectious diseases worldwide, constituting a particularly major health concern across Africa, Asia, and the Americas . AccordiPlasmodium genus, which are transmitted to the mammalian host through the bite of an infected female Anopheles mosquito. Five different Plasmodium species can cause malaria in humans: P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. P. falciparum, which is responsible for most malaria cases and deaths, is prevalent in Africa, while P. vivax is more common in tropical areas outside of Africa, such as the Indo-Pacific and American regions. Although less deadly than P. falciparum, P. vivax and P. ovale have the ability to form hypnozoites, parasite forms that remain dormant in the liver and that can reactivate weeks, months, or even years after the initial mosquito bite, causing malaria relapses, which poses an additional challenge to disease eradication [Malaria is caused by apicomplexan parasites of the dication ,4.Plasmodium parasites is extremely complex, alternating between a mammalian and an invertebrate host, and comprising three developmental stages: the pre-erythrocytic or liver stage, the erythrocytic or blood stage, and the sporogonic or mosquito stage. When an infected female Anopheles mosquito takes a blood meal, Plasmodium sporozoites, the liver-infective form of the malaria parasite, are injected into the dermis of a mammalian host. Sporozoites use their gliding motility to reach the circulatory system and quickly access the liver [Anopheles mosquito feeds from an infected mammalian host, she ingests male and female gametocytes, initiating the sporogonic stage of the parasite’s life cycle. Gametocytes develop into gametes that fuse in the mosquito midgut, forming a zygote, which then transforms into an ookinete that develops into an oocyst [The life cycle of he liver ,5. Afterhe liver ,7. Durinhe liver ,5,8. Frehe liver . A propoPlasmodium infection make it an attractive target for pharmacologic prophylaxis. However, the specificities of sporozoite production, which requires access to an insectarium, limit the drug screening efforts against this step of infection, making them less frequent than those aimed at assessing blood stage activity. Furthermore, since drug development is costly and time-consuming, alternative approaches are necessary to streamline the process of discovery of novel malaria therapeutics. Drug repurposing, also known as drug repositioning, reprofiling, or re-tasking, is a strategy aimed at the identification of new uses for approved or investigational drugs that are outside the field of reference of the original medical indication [Plasmodium infection -approved drug, employed in the treatment of rheumatoid arthritis for 25 years, was identified in a High Throughput Screening (HTS) and repurposed for the treatment of human amoebiasis, a protozoan intestinal parasitic disease caused by per year ,17,18. Aper year .Thalidomide was originally marketed in Germany and England as a sedative, and was specifically prescribed to treat morning sickness in pregnant women . Taking Originally developed for cancer treatment in 1964, zidovudine (AZT), was approved for the treatment of Human Immunodeficiency Virus (HIV)/Acquired ImmunoDeficiency Syndrome (AIDS) in March 1987, few years after the first report of AIDS and its etiological infectious agent, HIV ,21. The Besides the drugs that have already been successfully repurposed, several other candidates are presently under consideration. Such is the case of chlorcyclizine, an over-the-counter drug for allergy symptoms first approved in 1940, which may be repurposed for the treatment of Hepatitis C Virus (HCV) infection ,22. The Plasmodium parasites, as will be described in detail below. In fact, doxycycline is currently recommended by the Centers for Disease Control and Prevention (CDC) as a chemoprophylaxis option for travelers, as well as for the treatment of uncomplicated malaria in combination with other anti-plasmodial drugs, but it is contraindicated in pregnant women and in children under the age of 8 [Plasmodium infection have been screened for their anti-plasmodial activity. Their impact on the liver stage of infection and/or on other parasite stages and their consequent potential for repurposing towards malaria will be the focus of this review.The anti-plasmodial strategies toolbox has also benefitted from a successful case of drug repurposing, the tetracycline antibiotic doxycycline, which impacts both pre-erythrocytic and erythrocytic age of 8 . BesidesPlasmodium infection that takes place in the mammalian host is a major bottleneck of the malaria parasite’s life cycle. Hepatic infection obligatorily precedes the blood stage of infection, where disease takes place, providing an opportunity for targeted prophylaxis [Plasmodium species, the liver also constitutes a reservoir of dormant hypnozoites that can reactivate weeks to years after the primary infection, posing an additional burden to the global effort of malaria eradication [Plasmodium parasite species. Currently, only two drugs, primaquine and tafenoquine, have been approved for the elimination of hypnozoites in the treatment of malaria relapses [Plasmodium must be urgently fulfilled. One possible approach to this challenge lies in exploiting the possibility of repurposing approved drugs from various therapeutic categories to bridge this gap  compounds, employed in the treatment of HIV infection, have been assessed for their activity against Plasmodium liver stage in vitro and in vivo, in both rodent and human models of malaria. Among the classes of compounds employed in ARV therapy, protease inhibitors (PIs) have been the most extensively evaluated against Plasmodium parasites. In particular, the PIs saquinavir, lopinavir, ritonavir, and indinavir were shown to act upon the hepatic stages of the rodent parasites P. yoelii [P. berghei [P. falciparum parasite at clinically-relevant concentrations [P. yoelii parasites [P. berghei’s intra-hepatic development in a human hepatoma cell line. The field combinations recommended by the WHO, specifically efavirenz + AZT + lamivudine, efavirenz + tenofovir + emtricitabine, and nevirapine + tenofovir + emtricitabine [Plasmodium parasites have also been tested in vitro and/or in vivo against the ensuing blood stage of infection of P. berghei, P. cynomolgi, P. knowlesi, P. vivax, and P. falciparum [P. falciparum parasites [P. falciparum transmission blocking activity [P. berghei, while ritonavir, saquinavir, and indinavir were shown to inhibit this parasite’s oocyst development [Plasmodium parasites has been suggested to be mediated by the inhibition of their aspartyl proteases, some of which localize to the food vacuole and are involved in the degradation of haemoglobin [P. falciparum revealed that the latter is not effective [P. berghei infection showed only a modest impact in the control of parasitaemia [Plasmodium parasite’s life cycle, ARV compounds have also been assessed in the field, in both children and adult populations, as briefly reviewed by Hobbs et al. [Plasmodium infection sets the stage for their repositioning as possible multistage anti-plasmodial strategies. The co-endemicity of HIV and malaria further supports the need to deeply understand the effect of ARV compounds on Plasmodium infection. Such knowledge may be employed to adjust the current ARV treatment recommendations to gain a bonus anti-plasmodial effect without compromising the control of HIV infection.. yoelii  and/or P berghei . Lopinavtrations . Lopinavarasites , whereasarasites . In anotcitabine , were adcitabine . All PIslciparum ,39,40,43arasites . This waactivity . Lopinavelopment . The actmoglobin ,135,136.ffective ,34. Furtsitaemia . Besidess et al. . The broPlasmodium infection has been extensively investigated. Among the antibiotics most widely studied in this regard are tetracyclines, macrolides, quinolones, fluoroquinolones, sulfones, and sulfonamides.The repurposing of various classes of antibiotics employed in the management of bacterial infections to be used against the liver stage of Plasmodium infection. Early in vivo studies demonstrated that demeclocycline extended the prepatent period of P. cynomolgi infection in rhesus monkeys, while further displaying blood schizonticidal activity [P. cynomolgi in vivo and/or the liver stage of P. berghei in vitro [P. berghei and P. yoelii parasites, and was shown to reduce hepatic parasite numbers and development in vitro, and completely prevent the ensuing parasitemia in vivo [P. falciparum apicoplast [The broad-spectrum antibiotic tetracyclines have been shown to impact the liver stage of activity . Terramyin vitro ,46,47. Tin vitro ,49,50,51in vitro . Neverthin vitro ,139. Sin in vivo . Further in vivo . Doxycycicoplast ,56, impaicoplast ,55,57,58P. yoelii infection [P. falciparum [P. vivax [P. falciparum parasites, but both compounds were equally active in a P. vivax-endemic region [P. falciparum, azithromycin may be useful to treat individuals with contraindications to the administration of doxycycline, such as pregnant women and children under the age of 8. In fact, the combination of azithromycin with chloroquine has been proposed as an alternative for intermittent preventive treatment of malaria in pregnancy, as reviewed by Chico et al. [P. berghei infection revealed that azithromycin did not impact the number nor the development of intrahepatic parasites, but instead rendered the liver-derived merozoites non-infectious [P. berghei or P. falciparum [Plasmodium infection may benefit from the combination with faster acting compounds, an option that has been exploited in clinical trials, as reviewed by Rosenthal et al. [P. berghei development in the insect vector [The potential application of the macrolide antibiotic azithromycin as a prophylactic agent was initially proposed based on the protection observed in a mouse model of nfection . This aclciparum ,143,144 P. vivax . Azithroc region . Despiteo et al. . Furtherfectious . Azythrolciparum ,61,62,63lciparum ,147. Conl et al. . BesidesP. yoelii hepatic infection, revealing that some members of this class of broad-spectrum antibiotics have potential use as prophylactic agents for malaria. Among the 25 compounds tested, grepafloxacin, norfloxacin, piromidic acid, trovafloxacin, cinoxacin, ciprofloxacin, rufloxacin, sparfloxacin, ofloxacin, temafloxacin, pefloxacin, and clinafloxacin displayed variable anti-plasmodial activity, with IC50 values ranging between 4.4 and 90.2 µg/mL. Grepafloxacin, piromidic acid, and trovafloxacin were among the most active compounds, impacting not only parasite numbers but also its development and morphology. These compounds were additionally shown to display a similar activity profile against the in vitro infection of primary human hepatocytes by P. falciparum. Furthermore, all 25 compounds, most notably grepafloxacin, trovafloxacin, and ciprofloxacin, displayed in vitro activity against the blood stage of chloroquine-sensitive and chloroquine-resistant P. falciparum strains [P. yoelii infection, ciprofloxacin only modestly decreased parasitaemia, but prevented mortality [A panel of 25 quinolones and fluoroquinolones was screened in vitro for activity against  strains . These o strains ,67,68,69ortality . Ciproflortality ,69,71 anortality .P. yoelii in a mouse model of infection, as estimated from the appearance of ensuing blood parasitemia [The sulfone antibiotic dapsone, also known as diaminodiphenyl sulfone, which is used in combination in the treatment of leprosy, was shown to exhibit mild activity against the liver stage of asitemia . Dapsoneasitemia ,150. Besasitemia .P. falciparum malaria, in HIV-infected patients. In vitro, TMP-SMX inhibited the intra-hepatic development of rodent P. berghei and P. yoelii parasites and reduced the numbers of hepatic P. falciparum parasites. Accordingly, this drug impacted the P. yoelii liver load in a mouse model of infection, preventing the subsequent appearance of parasitemia [P. knowlesi parasites in rhesus macaque livers [P. falciparum infection in vitro [P. yoelii [P. berghei [P. berghei and P. cynomolgi in vivo as early as 1951 [P. falciparum blood stages in combination with pyrimethamine, sulfadiazine was inferior to sulfadoxine [Co-trimoxazole, a combination of the dihydrofolate reductase inhibitor trimethoprim and the sulfonamide antibiotic sulfamethoxazole (TMP-SMX), is employed in the prevention of opportunistic infections, including clinical episodes of asitemia . TMP-SMXe livers . TMP-SMXin vitro ,75. In hin vitro  and by Gin vitro . Another. yoelii  and P. b berghei  in vivo. as 1951 ,78. Whenfadoxine , which ifadoxine .Plasmodium parasites. Their action may either be stage-specific or may impact the parasite throughout infection of the mammalian host. Given the limited availability of Plasmodium sporozoites, many studies have solely focused on the identification of antibiotics with blood stage activity. In the future, the screening of those drugs against Plasmodium hepatic infection will likely identify additional antibiotics with multistage anti-plasmodial activity.As illustrated by the successful case of doxycycline, antibiotics have great potential for repurposing against P. berghei in vitro [P. falciparum sporozoite injection. At the dose and regimen employed, ivermectin did not impact pre-patency [Plasmodium infection added up to its previously known impact against this parasite’s life cycle from the mammalian host through to the insect vector. In the former, ivermectin impacted the asexual blood stages of P. falciparum [Plasmodium infection make it a very promising tool in the prevention, treatment, and blocking of parasite transmission.Avermectins are a family of macrocyclic lactones with antiparasitic and insecticidal properties that have already been proposed for the reduction of the incidence of vector-borne diseases . Within in vitro ,80. Iverin vitro . The app-patency . The actlciparum ,82 and, lciparum . In the lciparum ,162,163,lciparum ,169,170.lciparum ,172,173 lciparum ,176,177.Trypanosoma brucei gambiense and T. b. rhodesiense parasites, which are transmitted by tsetse flies. This disease poses a threat to 36 countries in sub-Saharan Africa and is, therefore, co-endemic with malaria. HAT is characterized by two disease stages, for which there are alternative treatments. For the first stage, when parasites replicate in subcutaneous tissues, blood, and lymph, treatment recommendations include pentamidine and suramin. In the second stage, when parasites have crossed the blood–brain barrier to infect the central nervous system, treatment recurs to melarsoprol, eflornithine, and nifurtimox. In 2019, fexinidazole was included in the WHO Essential Medicines List (EML) and WHO’s treatment guidelines for both first stage and non-severe second stage HAT cases [P. falciparum invasion of these hepatocytes with an IC50 of 50 µM [P. falciparum invasion of erythrocytes, possibly by binding to and preventing the proteolytic cleavage of merozoite surface protein-1 (MSP1) [P. berghei parasitaemia in vivo, as well as P. falciparum intraerythrocytic development in vitro [P. falciparum targets. Specifically, it was shown to inhibit the catalytic activity of a parasite aldolase [P. falciparum diadenosine tetraphosphate hydrolase [P. berghei in vivo in a dose-dependent manner, as assessed by the presence or absence of an ensuing parasitaemia. In this model, DFMO had no impact on the parasite’s erythrocytic stages [P. berghei-infected human hepatoma cells [P. berghei in vivo [P. falciparum, DFMO impacted parasitaemia in the millimolar range [P. berghei in the Anopheles stephensi vector [Plasmodium infection. Nonetheless, it exhibited nanomolar-ranged activity against in vitro P. falciparum blood stage [P. falciparum-infected erythrocytes, where it bound to the toxic ferriprotoporphyrin IX generated during hemoglobin digestion, inhibiting its crystallization into the non-toxic hemozoin [P. berghei blood stages in vitro, although at higher concentrations than against P. falciparum [Plasmodium hepatic and/or blood stage infection.Human African trypanosomiasis (HAT), also known as sleeping sickness, is caused by protozoan AT cases ,179. Surof 50 µM . Besides1 (MSP1) . Suraminin vitro . Besidesaldolase , as wellaldolase , both ofaldolase . Finallyydrolase . Howeverydrolase . This coc stages . DFMO’s c stages ,92,93,94 in vivo ,95,96, aar range ,101,102,ar range ,101. Mori vector . Pentamiod stage ,110,111.hemozoin . Pentamilciparum . MelarsoPlasmodium parasites, other, perhaps less obvious, candidates include medicines that are employed in the treatment of non-communicable or chronic diseases. These comprise antidiabetic drugs, antihistamine agents, immunosuppressive compounds, proton pump inhibitors, antihypertensive agents, an ovulation inducer, anticancer drugs, an antirheumatic agent, and an antianginal agent.Although anti-infective medicines have been extensively tested for repurposing against P. berghei mouse model, where it decreased both parasite numbers and intra-hepatic parasite development. The latter effect was also observed in vitro employing a model of infection of primary human hepatocytes by the human malaria parasite, P. falciparum [P. berghei and P.yoelii, and in vitro, using P. falciparum [Metformin, a biguanide antidiabetic drug that primarily targets the liver, was shown to inhibit hepatic infection in vivo in the lciparum . Despitelciparum ,115.P. yoelii has been evaluated. Complete prevention of hepatic infection was achieved with cyproheptadine and ketotifen at 5 mg/kg, as well as for terfenadine at a higher dose of 50 mg/kg of mouse body weight. Azatadine and loratadine also demonstrated partial activity against the liver stage of infection. It should be noted that the impact of these drugs on hepatic infection was not assessed directly on liver samples but was rather estimated from the ensuing appearance of blood stage parasitemia. Nonetheless, all compounds were shown to be inactive against erythrocytic parasite forms at the concentrations that were effective for prophylaxis, confirming that the impact observed in pre-patency results solely from their activity against hepatic parasites [P. falciparum in vitro [P. berghei in vivo [P. yoelii in vivo [P. yoelii infection [P. yoelii [P. berghei parasite screen, and were further shown to be active against the P. falciparum blood stage [Plasmodium infection is in agreement with a previous study where this compound impacted the erythrocytic infection of P. falciparum in vitro, as well as of chloroquine-sensitive P. vinckei and chloroquine-resistant P. yoelii in vivo [Plasmodium chaperonin TRiC/CCT was identified as the target of clemastine [The potential prophylactic activity of the antihistamine agents cyproheptadine, ketotifen, terfenadine, azatadine, and loratadine, employed in the management of allergies, against a chloroquine-resistant strain of arasites . In anotin vitro , P. berg in vivo , and P.  in vivo . Cyprohenfection . Cyprohe. yoelii . In huma. yoelii , whereas. yoelii . Two addod stage . Astemiz in vivo . Recentlemastine .P. berghei infection of a human hepatic cell line [P. berghei infection by 1.7 days [P. berghei, P. yoelii and/or P. chabaudi in vivo [P. falciparum [P. vivax [The immunosuppressive agent cyclosporin A, which is commonly used to inhibit the rejection of transplanted organs, displayed activity in the nanomolar range against ell line . When ad1.7 days . Besides in vivo ,126,127,lciparum ,125 and,P. vivax  in vitroP. vivax ,130.P. berghei, namely the proton pump inhibitors omeprazole and esomeprazole; the angiotensin receptor antagonist telmisartan; the ovulation inducer clomiphene; the anticancer drugs daunorubicin, doxorubicin, and idarubicin; the antirheumatic agent auranofin; the antihistamine agent clemastine fumarate; the immunosuppressive agent mycophenolic acid; the structurally-related estrogen receptor modulators tamoxifen citrate and toremiphene citrate employed in the treatment of breast cancer; and the prophylactic antianginal agent perhexiline maleate , but not for the antihistamine drug promethazine, nor for the antibiotics clindamycin, minocycline, doxycycline, azithromycin, and ciprofloxacin [P. cynomolgi model of infection, tinidazole was later proved to be ineffective in preventing relapse of P. vivax, in a pilot phase II clinical trial [Plasmodium liver infection, were also assessed for their activity against P. cynomolgi hypnozoites, and shown to be ineffective in preventing malaria relapse in rhesus monkeys [Tinidazole, an anti-infective drug that is used against protozoan infections, and triamterene, a diuretic drug that is employed in the management of hypertension, have been shown to moderately protect from relapse, in a model of rhesus macaque infection by floxacin . Despiteal trial . The pro monkeys .P. cynomolgi reporter parasite lines, and the establishment and optimization of drug screening procedures for this step of infection, will streamline drug repurposing towards the elimination of dormant parasite forms, an obstacle to the global effort of malaria eradication.The development of new tools for straightforward hypnozoite identification, such as the recently developed dual-fluorescent  and dualPlasmodium infection may identify bonus effects or suggest alternative treatments that can tackle both conditions simultaneously. Furthermore, these drugs may be readily available in malaria-endemic regions, facilitating their employment against this disease. In many cases, such drugs already have reported blood stage activity, but their impact on Plasmodium hepatic infection remains elusive. Further characterization of these medicines may create the possibility for their employment as malaria prophylaxis and/or multistage anti-plasmodial strategies.Malaria affects the poorest regions of the globe, where it poses huge health and economic challenges. These regions are also affected by many other infectious diseases, for which vaccination is not always available, and which may require recurrent treatment. Therefore, assessing the potential activity of the drugs employed in the management of such infectious diseases against Mycobacterium tuberculosis bacteria, is the leading cause of death from a single infectious agent [Plasmodium parasites could help control malaria in regions where both infections are co-endemic. In fact, most tuberculostatics have already been tested against the blood stage of Plasmodium infection. However, their effectiveness against the parasite’s liver stage bottleneck remains largely unknown. An exception to this rule is the panel of fluoroquinolones that belong to the second line of TB treatment, which have been tested in terms of their liver stage anti-plasmodial activity, as described above.TB, a disease caused by infection with us agent . Althougus agent , where iP. berghei infection has been assessed in vivo, revealing that only the combination of all three compounds, or of rifampicin and isoniazid, reduces blood parasitaemia and increases mouse survival [P. berghei parasites, an effect that was observed in vivo, but not in vitro, suggesting an indirect mechanism of action [P. berghei in vivo, increasing mouse survival [P. chabaudi in vivo, reducing parasitaemia, and against P. falciparum in vitro, reducing the viability of ring forms [P. vivax-infected patients [P. falciparum’s life cycle [The first line treatment against TB relies on the combination of ethambutol, isoniazid, rifampicin, and pyrazinamide . Except survival . The comsurvival ,191,192,survival . Isoniazf action . Rifampisurvival , againstng forms . Howeverpatients . Additiofe cycle ,198,199.P. falciparum’s erythrocytic development, an activity that was probably linked to the inhibition of a parasite’s acid phospholipase [P. falciparum blood stage cultures and did not impact this phase of the parasite’s life cycle [P. falciparum erythrocytic growth at an IC50 that is close to that of M. tuberculosis, by acting upon the essential sulfur mobilization pathway required for apicoplast maintenance, which is of bacterial origin [P. vivax blood stages by acting on the pathway that was demonstrated for P. falciparum. However, these observations still require experimental validation [P. yoelii [P. falciparum [P. falciparum blood stage [P. falciparum blood stage parasites [P. falciparum blood stages [Plasmodium infection may provide new insights into their possible application as multi-stage anti-plasmodial strategies.Besides fluoroquinolones, other drugs employed as second-line TB treatment have also been screened for their blood stage anti-plasmodial activity, but their efficacy against the parasite’s liver stages remains unaddressed. Such is the case of the injectable agents amikacin, kanamycin, and streptomycin. Amikacin was shown to delay holipase . Conversfe cycle ,201,202.l origin . In sililidation . The mac. yoelii , to disrlciparum ,207, andlciparum ,206. Anood stage ,209. Linarasites , whereasd stages . The antPlasmodium infections. To tackle this problem the WHO recommends periodical treatment with the inexpensive drugs albendazole and mebendazole [P. falciparum’s intraerythrocytic development in vitro, although at relatively high concentrations [P. berghei infection with contradicting results. Although albendazole was not effective in P. berghei-infected mice, it reduced parasitaemia in infected rats [Plasmodium co-infections [Plasmodium parasite. LF, commonly known as elephantiasis, may result in severe disability and, consequently, social stigma. Towards the long-term goal of elimination of this helminthic disease, WHO recommends annual mass drug administration to at-risk populations. The recommended regimens vary with the co-endemicity with other filarial diseases, and include the already mentioned albendazole, as well as ivermectin [Plasmodium co-infection [Helminths, or parasitic worms, are responsible for a high burden of disease in developing countries . Among hendazole . While ttrations ,215. Albted rats , which wted rats . Althougfections ,221,222.ermectin , which iermectin . The antermectin , remainsermectin . Like alnfection ,221. Thunfection . OverallLeishmania parasites, which are transmitted by phlebotomine sandflies, cause leishmaniasis in humans, a disease that can present as visceral, cutaneous, and mucocutaneous leishmaniasis. The type of the disease, the Leishmania species involved, the geographical location, and the existence of concomitant pathologies in a given patient will determine the type of treatment to be administered. WHO-recommended treatment options include the injectable pentavalent antimonials meglumine antimoniate and sodium stibogluconate, amphotericin B deoxycholate, liposomal amphotericin B, paromomycin (aminosidine), and pentamidine isethionate, and the oral miltefosine [P. yoelii-infected mice increased the severity of infection [P. falciparum strains in vitro, being more effective against ring stage parasites than against the later trophozoite and schizont stages [P. falciparum phosphoethanolamine methyltransferase, as well as to impact this parasite’s blood stage development [Plasmodium infection has been described, their liver stage activity remains unknown. Further efforts towards this characterization may bring new insights to the possible employment of these drugs as multistage anti-plasmodial strategies.The protozoan tefosine . Adminisnfection . A heat-t stages . Furthert stages . Miltefoelopment . AlthougThe co-endemicity of malaria and other infectious diseases provides an opportunity for repurposing drugs from the latter to the former. Medicines currently employed in the management of TB, helminth infections and leishmaniasis have been mostly reported to possess blood stage activity, but their potential as prophylactic agents remain largely uncharacterized. Evaluation of these and other anti-infective medicines may reveal bonus effects, multistage activity, and ultimately contribute to the malaria prophylactic and therapeutic toolbox.Drug repurposing is an increasingly popular approach in the development of therapeutic tools to combat diseases or conditions for which novel pharmacological solutions are required. Drug repositioning is particularly attractive when dealing with malaria and other neglected diseases that exert their burden in some of the poorest regions of the globe. In fact, it has been argued that one of the key barriers to malaria treatment and chemoprevention lies in the reluctance of the pharmaceutical industry to invest in the development of drugs that would offer only limited marketing prospects . The cosPlasmodium parasite species, the liver stage of Plasmodium infection is an ideal but underexplored target for prophylactic interventions against malaria [Plasmodium infection increasingly amenable to investigation, including the assessment of drug activity against hepatic parasites. A wide array of parasite and host models, including in vitro and in vivo systems, is now available to researchers [Plasmodium infection especially appealing, as it permits screening the activity of large numbers of existing compounds against this phase of the parasite’s life cycle. Moreover, by enabling the evaluation of the hepatic stage activity of drugs known to inhibit other stages of the parasite’s life cycle, it significantly amplifies the possibility of identifying and developing much-sought-after compounds with multi-stage anti-plasmodial activity [In addition to constituting a reservoir for dormant forms of hypnozoite-forming  malaria . The scaearchers , and newearchers . This evactivity .Plasmodium parasite development in the liver. Such studies should, however, bear in consideration that drugs to tackle a disease such as malaria need to be not only widely available but also largely affordable, if they are to be used by those who need them most. The WHO’s EML includes a large number of drugs that address the minimum medicine needs for efficacious, safe, and cost–effective medicines for priority conditions [Plasmodium infection, possibly providing a bonus effect in malaria-endemic settings. Nevertheless, it should be noted that drugs targeting NCDs likely act on a host rather than a parasite target, raising concerns about undesired side-effects. On the other hand, as with all repurposed drugs, the safety and toxicity of these compounds will have been ascertained, limiting these risks. Moreover, it should also be noted that targeting host factors to combat malaria may also offer an opportunity to circumvent the development of resistance by the parasite [As outlined in the present review, the number of FDA-approved drugs with demonstrated activity against hepatic malaria parasites has been growing substantially. It is also safe to expect that this number will continue rising in the near future, as laboratories around the world invest their efforts into evaluating the ability of existing drugs to effectively inhibit nditions . This linditions . Althougparasite . Hence, Plasmodium life cycle [Plasmodium infection continue to be urgently needed. While this may entail the development of novel chemotypes for anti-plasmodial intervention, as recently reviewed in [Despite recent progress in the quest for an effective vaccine against malaria, with a particular emphasis on vaccine candidates that target the liver stage of the fe cycle ,242,243,iewed in ,245,246,


Malaria and HIV are common infections in Africa and cause substantial morbidity and mortality in pregnant women. We aimed to assess the association of malaria with anemia in pregnant women and to explore the joint effects of malaria and HIV infection on anemia in pregnant women.We used nationally representative, cross-sectional demographic and health surveys (DHS) that were conducted between 2012 and 2017 across 7 countries of sub-Saharan Africa . The outcome variables were anemia (defined as a hemoglobin concentration < 110 g/L), and hemoglobin concentration on a continuous scale, in pregnant women at the time of the interview. We used generalized linear mixed-effects models to account for the nested structure of the data. We adjusted models for individual covariates, with random effects of the primary sampling unit nested within a country.. Prevalence of malaria only, HIV only, and malaria- HIV coinfection in pregnant women was 31% , 1.3%  and 0.52%  respectively. Overall prevalence of anemia was 48.3% (95% CI: 45.1 to 51.5%). The anemia prevalence in pregnant women with malaria infection only was 56.0% (95% CI: 50.1 to 61.7%); HIV infection only, 62.5% (95% CI: 25.9 to 89.8%); malaria- HIV coinfection, 60.0 (95% CI: 17.0–92.7%) and without either infection, 44.6% (95% CI: 40.7 to 48.6%). In the fully adjusted models, malaria infection was associated with 27% higher prevalence of anemia , and 3.4 g/L lower hemoglobin concentration  compared to uninfected pregnant women. The prevalence of HIV infection and malaria-HIV coinfection was too low to allow meaningful analysis of their association with anemia or hemoglobin concentration.A total of 947 pregnant women, ages, 15–49 y, were analyzedMalaria was associated with an increased prevalence of anemia during pregnancy. Plasmodium falciparum malaria, are associated with the etiology of anemia in pregnancy in sub-Saharan Africa [Globally, 38% of pregnant women are anemic, and rates are higher in sub-Saharan Africa at 56%, compared with high income countries at 22% . Anemia n Africa . The burn Africa –9. Due tn Africa , 11. Suc+ T cell count was below 200 cells/μL. The pathological interaction between malaria and HIV in dually infected patients is synergistic and bidirectional [In a recent cross-sectional study aimed at characterizing the prevalence of malaria in people living with HIV, the prevalence of malaria was 7.3% . The preectional . Malariaectional , and an ectional , 16. Immectional . TherapeEpidemiological studies on the association of malaria- HIV coinfection with anemia in pregnancy are not population-based and suffer from low statistical precision , 19. Thehttps://dhsprogram.com/What-We-Do/Survey-Types/DHS-Methodology.cfm). In summary, data collection was based on a stratified two-stage probability sampling design, with the samples drawn from the most recent census frame, which is the sampling frame. First, countries were divided into geographic regions. Within these regions, populations were stratified by urban and rural areas of residence. The DHS surveys never define the urban/rural, it always follows the national definition which was defined by the population census. Within these stratified areas, a random selection of enumeration areas (EAs) or primary sampling units (PSUs) were drawn. On average, surveys selected 300–500 EAs with probability proportional to the population [Data were from the latest Demographic and Health Surveys from 7 sub-Saharan Africa countries: Burundi, the Democratic Republic of the Congo, Ghana, The Gambia, Mali, Senegal, and Togo . Hemoglobin concentrations adjusted for altitude and cigarette smoking status using formulae provided by the US Centers for Disease Control and Prevention –23. HemoPlasmodium falciparum (Pf), Plasmodium vivax (Pv) and other malaria species in human whole blood. HIV testing was carried out using the standard testing algorithm of HIV antibody enzyme-linked immunosorbent assays (ELISAs) [The primary exposure variables of interest were malaria, HIV and malaria- HIV coinfection. Malaria testing was carried out using SD Pf/Pan Rapid diagnostic tests, Bioline Malaria Ag P.f/Pan  which is a qualitative test for the detection of histidine-rich protein II antigen of (ELISAs) .: First Possible confounders adjusted for were: age, iron supplementation, pregnancy trimesters and intermittent preventative therapy use.lme4 package in R version 3.5.1 [We assumed intra-country and intra-PSU correlation among the responses. Therefore, we fit generalized linear mixed-effects models (GLMM) using the on 3.5.1 . For theon 3.5.1 . We examon 3.5.1 . Also, won 3.5.1  and map . Data were collected between November 2012 and December 2017. Detailed in Additional File pyrimethamine (Table p = 0.01) and in those who were on intermittent preventive treatment with sulfadoxine/pyrimethamine . Western African countries had higher prevalence of anemia compared to Central and East African countries. No differences in household wealth quintile status, marital status, breast feeding status and number of births in the previous 5 years. Figure n = 293), 1.3%  and 0.52%  respectively. The prevalence of malaria in HIV positive pregnant women was 38% (95% CI: 15 to 68%). Prevalence of any anemia and moderate anemia was higher in pregnant women with malaria and HIV infections than in pregnant women without infections. Mild and severe anemia prevalence was not significantly different across the infection categories . Compared to the first trimester (n = 277), the mean hemoglobin concentration was lower in participants in the second (n = 369) and third trimester (n = 301) . In a sensitivity analysis examining the association of infection status and lower threshold of hemoglobin concentration of < 100 g/L, the results are consistent with the primary model and are reported in Additional File .In Table ster n = 77, the mWe showed that malaria infection had a significant association with anemia and lower hemoglobin concentration in pregnancy. The finding was robust to adjustment for various confounding factors. The absence of any association for HIV and malaria-HIV coinfection is possibly due to low statistical precision. The quantitatively small variation of 6% in hemoglobin concentrations and anemia explained by malaria and HIV infections shown in our analysis suggests that in sub-Saharan Africa, anemia in pregnancy is a result of a complex interplay of other factors in addition to malaria and HIV. Such factors include; nutritional (such as iron and folate deficiency) and genetics (such as inherited hemoglobin disorders) and inflammation , 30–32.The high prevalence of anemia in pregnant women was consistent with a previously reported prevalence of 56% in studies of the same region . The assPlasmodium falciparum erythrocytes membrane protein-1,VAR2CSA [The mechanisms by which malaria and HIV lead to anemia in pregnancy are complex. Malaria infection during pregnancy causes acute anemia mainly through direct destruction of erythrocytes, sequestration of infected and uninfected erythrocytes by the spleen, and decreased erythropoiesis. Furthermore, the placental sequestration of malaria-infected erythrocytes is typical in pregnant women , and occ,VAR2CSA . Hepcidi,VAR2CSA , 40. In ,VAR2CSA . On the ,VAR2CSA .pyrimethamine. Unexpectedly however, in our study, intermittent preventive treatment with sulfadoxine/pyrimethamine was associated with deleterious effects on maternal hemoglobin. Such a finding could be explained by increasing parasite resistance in various parts of sub-Saharan Africa resulting to the effect of IPTp transitioning from net benefit to neutral or net harm [Preventative strategies for anemia in pregnancy due to malaria and HIV are well documented. The current guidelines for the management of HIV in pregnant women include the use of trimethoprim-sulfamethoxazole (co-trimoxazole) in addition to antiretroviral therapy, which both lower odds of coinfection . In a prnet harm . There inet harm .Although we observed an association between malaria infection and anemia in pregnancy, the variance in hemoglobin concentration and anemia explained by this association was small (6%), suggesting that other factors other than malaria are attributable to the high prevalence of anemia in pregnant women. In sub-Saharan Africa, low hemoglobin concentration is also caused by nutritional deficiencies of iron , vitaminFactors contributing to the mean hemoglobin differences between the Western region and the rest of sub-Saharan Africa have been explored. Geospatial analysis of hemoglobin concentration in sub-Saharan Africa concluded that known environmental drivers of anemia-causing parasitic infections and nutritional iron deficiency, such as distance to a perennial water body, land surface temperature and normalized difference vegetation index show stronger effects for Western Africa than Eastern Africa . In addiOur study had some limitations. First, the cross-sectional nature of the study could not allow us to infer causality. Anemia is a chronic multifactorial syndrome and women are often anemic before becoming pregnant. Therefore, the temporal association between malaria during pregnancy and anemia occurrence could not be established in our study. Second, a onetime assessment of malaria and HIV in this cross-sectional study design could have led to an underestimation of the infection rate since infection testing was not repeated. Third, pregnancy is self-reported hence early pregnancy is likely to be under reported. The pregnancy reporting behavior from the DHS data shows that only 75% of women with a 10-week pregnancy report that they are pregnant. Fourth, variable selection for the infection-anemia models was mainly driven by information availability from the survey datasets. Consequently, other possible confounding variables such as gestational age, nutritional status  and helminths that were not adjusted in our analysis could have biased the observed association. Nevertheless, considering we adjusted for major possible confounders known to be associated with malaria and HIV and anemia, our study provides stable estimates of anemia in pregnancy in several countries in sub-Saharan Africa using a nationally representative sample. Such factors include pregnancy trimester, iron supplementation, and intermittent malaria preventive treatment use. Lastly, our study had low number of coinfected pregnant women resulting in excessively wide confidence intervals. The low prevalence of 1.3% in the study population is explained by the low prevalence of HIV in Western, Central and Eastern Africa, which represents our study sample. It is likely such a selection bias could have influenced the estimates towards the null. Consequently, our findings may not generalize to entire sub-Saharan Africa.Malaria was associated with an increased prevalence of anemia during pregnancy. The prevalence of HIV infection and malaria-HIV coinfection was too low to allow meaningful analysis of their association with anemia or hemoglobin concentration.Additional File 1. Country- specific sample size and population. Sample size summary for the analyzed DHS data indicating the country, year of survey, number of pregnant women, region and 2017 population size.Additional File 2. Sensitivity analysis. Association of infection status with anemia (Hb < 100 g/L) in pregnant women, 15–49 y old.


Evidence suggests that Oxidative stress has been shown to plays an important role in gestational diabetes mellitus (GDM) etiology. On the other hand, women with GDM are at an increased risk for complications such as endothelial dysfunction and cardiovascular diseases.To investigate the effects of alpha-lipoic acid (ALA) on the maternal circulating values of lipid profile and lipid ratios in women with GDM.Sixty women with GDM were participated in the present study. The ALA group (n = 30) received ALA (100 mg/day) and the placebo group (n = 30) received cellulose acetate (100 mg/day) for eight wk. The maternal circulating values of hemoglobin A1C, triglyceride (TG), total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol, triglyceride-glucose (TyG) index, atherogenic index of plasma (AIP), non-HDL-C, and lipid ratios were assessed before and after the intervention. P-value The values of TyG index (p  Maternal circulating values of TyG index, TG, TG/HDL, AIP decreased after eight wk of ALA supplementation in women with GDM. Gestational diabetes mellitus (GDM) is a condition that is initiated or diagnosed for the first time in pregnancy, which is defined as glucose intolerance. This type of diabetes increases insulin resistance in body. Mothers suffering from GDM are at a high risk for type 2 diabetes in future. Clinical studies have shown that oxidative stress plays a role in the pathophysiology of numerous diseases (1). Normal pregnancy is regarded as a condition associated with increased oxidative stress. However, despite the increasing oxidative stress, there is a balance between antioxidants and oxidants in normal pregnancy. Bodies of evidence suggest that oxidative stress plays an important role in pregnancy complications such as GDM, preeclampsia, and hydatidiform mole . Alpha-lipoic acid (ALA)  is a sulfur-containing fatty acid  that plays a basic role as an antioxidant and is required for mitochondrial α-ketoacid dehydrogenase complex. ALA is produced by plants and animals. However, its synthesis in human is very low (4). Its antioxidant properties are attributed to several reasons including its capacity for direct scavenging of reactive oxygen species (ROS), regeneration of endogenous antioxidants such as glutathione, vitamins E and C, and its metal-chelating activity (5). Short-term treatment with ALA in patients with type 2 diabetes mellitus (T2DM) has been suggested to improve lipid profile by improving oxidative stress and inflammatory responses (6). A systematic review has shown that supplementation with ALA can improve lipid profile except HDL-C in patients with metabolic diseases (7).A review study has suggested that ALA not only has no side effects but also has protective effects against embryopathy, fetal mortality, and placental ultrastructural changes due to diabetes during pregnancy (8). Women with a history of GDM are at an increased risk for complications such as endothelial dysfunction and cardiovascular diseases (9). It has been shown that lipid profile controlling during GDM can prevent impairment of the feto-placental endothelial function (10). On the other hand, women who use insulin during GDM can face some cardiovascular complications compared to those who undergo nutritional therapy for controlling the disease (11). However, little data are available about the effects of ALA supplementation on the values of lipid profile and lipid ratios in women with GDM. Therefore, the current clinical trial study was designed to investigate into this matter.The current clinical trial study was conducted at the Taleghani Hospital and Diabetes Clinic of Arak University of Medical Sciences between February 2017 and January 2018. The study is a double-blind clinical trial in which the participants and the statistician were not aware of the type of drug to be administered and the group classification, respectively. GDM diagnosis was carried out according to the 75-gr oral glucose tolerance test when at least one or more of the following glucose values were exceeded: fasting Participants were divided into ALA and placebo groups using simple randomization method by the random number table. While 30 participants were included in the ALA group and received ALA  (1 capsule 100 mg/day), the remaining 30 were included in the placebo group and received manually prepared capsule containing cellulose acetate  (1 capsule 100 mg/day). The intervention lasted for eight weeks. Studies have shown that a single dosage administration (50-600 mg) of ALA is completely absorbed within 30-60 min (8). Since receiving 100 mg/day of ALA has been shown to have no significant side effects on mothers and newborns during pregnancy (8), this dosage was used in the current study. All subjects were under the same diet plan for controlling blood glucose levels. Participants were asked to follow the provided diet plan and not to change their ordinary daily physical activity during the trial. As a reminder, every two weeks, the patients were phone called for taking their capsules appropriately.Demographic data including participants' height, weight (light clothing without shoes weight), body mass index (BMI), blood pressure (right arm in sitting position) were measured at the beginning and end of the trial. BMI was determined as weight (kg)/height , high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), and total cholesterol (TC)  were measured using a chemical analyzer . Low-density lipoprotein cholesterol (LDL-C) levels were calculated using the Friedewald equation (13). The atherogenic index of plasma (AIP) (14), the triglyceride-glucose (TyG) index as Ln [ of the Taleghani Hospital and Diabetes Clinic of Arak University of Medical Sciences.t tests. Interactions between the corresponding biochemical variables and the ALA group were explored using the Analysis of Covariance (ANCOVA). Data with p-values Qualitative data were evaluated using the Chi-square test. The normal distribution of variables was explored by the Kolmogorov-Smirnov test. Comparisons of quantitative parameters were performed using paired and independent In this study, data of the participants in two groups (n = 30/each) were analyzed. No significant differences were observed in the demographic and biochemical data of the two groups at the baseline (Tables I and II). In addition, no significant changes were observed in the weight, systolic blood pressure, and BMI in each group following the intervention.Intragroup and intergroup comparisons of the variables are given in Tables III and IV, respectively. At the end of the intervention, values of FBS , TG  found that the combination therapy with ALA and myo-inositol significantly reduces BMI in women with polycystic ovary syndrome. Noori and colleagues (20) found that consumption of 800 mg of ALA in diabetic nephropathy significantly decreases systolic blood pressure in the supplement group compared with the placebo group.Orally consumed ALA is absorbed almost entirely from intestine (21). In the current study, values of ALA increased significantly in the treatment group compared with the placebo group after the intervention. Therefore, this finding can be accounted for ALA consumption by participants in the ALA group.In addition, in the current study, HbA1C levels decreased marginally in the ALA group after supplementation with ALA, albeit the absence of statistical significance according to the results of ANCOVA. Accordingly, marginally reduced values of HbA1C may not be attributed to the intervention effects. Bao and co-workers (22) found that the daily consumption of 600 mg of ALA in patients with T2DM does not change HbA1C levels significantly. Chang and co-workers (23) observed that using 600 mg of ALA for 12 weeks does not affect HbA1c levels in individuals with end-stage renal disease. Porasuphatana and co-workers (24) observed that taking different doses of 300, 600, 900, and 1200 mg/day of ALA for six months causes a significant reduction in the HbA1C levels in individuals with T2DM. Derosa and co-workers (5) found that taking 600 mg of ALA supplementation in individuals with T2DM reduces HbA1C levels significantly.An in vitro study conducted by Di Fulvio and co-workers (9) has shown that vascular cells obtained from umbilical cords of women with GDM maintain certain dysfunctions such as increased ROS and reduced nitric oxide (NO) bioavailability even after culturing for several times. Another in vitro study conducted by Di Tomo and colleagues (25) has shown that ALA significantly improves detrimental effects of hyperglycemia on umbilical vein endothelial cells obtained from mothers with GDM, concluding that this may point to new strategies for prevention of diabetes-related vascular complications.A systematic review study has shown that supplementation with ALA can improve lipid profile except HDL-C in individuals with metabolic diseases (7). However, little data are available in this matter in GDM. In the current study, the mean TC/HDL and LDL/HDL level changes in the ALA and placebo groups did not show a statistically significant difference. However, the mean TyG index, TG, TG/TC, TG/HDL, and AIP levels in the ALA group significantly decreased after the intervention. It has been reported that the increased values of TyG index, TG, and TG/HDL-C in early pregnancy are associated with high incidence of GDM development (15). The TyG index is the best indicator of insulin resistance compared with other lipid parameters as a noninvasive, effective, and simple tool. On the other hand, TyG index is a better predictor of atherosclerosis than homeostatic model assessment of insulin resistance (HOMA-IR) . AIP value has been shown to be a good biomarker for the risk of atherosclerosis and cardiovascular diseases. In addition, the calculation of AIP, especially when lipid profile is in normal range, has been proposed, which can show more useful data than traditional lipid analysis about patient's situation . Results from the ANCOVA did not point to a statistically significant difference for TG/TC. Therefore, reduced TG/TC values may not be attributed to the intervention effects. An increase in the TG/HDL-C ratio was reported in GDM compared with healthy pregnancy (28). The TC/HDL-C, LDL-C/HDL-C, and TG/HDL ratios showed significant positive correlations with HOMA-IR. Therefore, these ratios could be used as simple alternative markers for assessing insulin resistance during pregnancy and predicting women at a high risk for GDM (29). It has been shown that ALA can increase adenosine monophosphate-activated protein kinase (AMPK) activity which causes inhibition of fatty acid synthesis, while simultaneously fatty acids oxidation is activated. On the other hand, expression of enzymes acetyl-CoA carboxylase and fatty acid synthase, two rate-limiting enzymes in fatty acid synthesis, are depressed in subjects supplemented with ALA (7).A major limitation of the present study was the lack of a healthy control group to allow the comparison of the results obtained from the participants with them.The results of the current study in women with GDM showed a significant decrease in the maternal circulating values of TyG index, TG, TG/HDL, AIP, and TBARS in the ALA group compared to the placebo group at the end of the intervention. More studies are required to evaluate the potential effects of ALA on the reduction of birth defects in women suffering from GDM.The authors declare that they have no conflict of interest.


Plasmodium sporozoites are the infective stage of the malaria parasite. Though this is a bottleneck for the parasite, the quantitative dynamics of transmission, from mosquito inoculation of sporozoites to patent blood-stage infection in the mammalian host, are poorly understood. Here we utilize a rodent model to determine the probability of malaria infection after infectious mosquito bite, and consider the impact of mosquito parasite load, blood-meal acquisition, probe-time, and probe location, on infection probability. We found that infection likelihood correlates with mosquito sporozoite load and, to a lesser degree, the duration of probing, and is not dependent upon the mosquito’s ability to find blood. The relationship between sporozoite load and infection probability is non-linear and can be described by a set of models that include a threshold, with mosquitoes harboring over 10,000 salivary gland sporozoites being significantly more likely to initiate a malaria infection. Overall, our data suggest that the small subset of highly infected mosquitoes may contribute disproportionally to malaria transmission in the field and that quantifying mosquito sporozoite loads could aid in predicting the force of infection in different transmission settings. Anopheles mosquitoes inject sporozoites as they look for blood. Though transmission is a bottleneck for the parasite and thus a good point for intervention, many aspects of transmission remain poorly understood. In this study, using a rodent model of malaria, we found that the majority of infective bites do not result in malaria infection. Furthermore, we found that the bites of mosquitoes with heavy parasite burdens are significantly more likely to result in blood stage infection. These data have important implications for designing interventions targeting transmission stages of the malaria parasite as they suggest that reducing parasite loads, even without completely eliminating them, could be effective against disease spread. We also found that mosquitoes that probe but do not succeed in finding blood are equally likely to initiate infection, an important finding for human vaccine trials. Overall this work contributes to our understanding of the epidemiology of malaria and should aid in the development of malaria elimination strategies.Malaria is a leading cause of death in many parts of the world. Infection is initiated when infected  Plasmodium are the causative agents of the disease and are transmitted by Anopheline mosquitoes. Sporozoites, the infective stage of the parasite, reside in mosquito salivary glands and are injected into the host’s skin as the mosquito searches for blood  to ] to Eqs  weights . Akaike  weights  and to cLogistic regression models with probability of malaria infection as the dependent variable were used to assess the associations with blood meal status, location of the bite, probe time, and sporozoite load. All models included robust variance estimates to adjust for potential within-experiment correlations of malaria infections . CategorFor the probe time experiments, the relationship between probe time and malaria infection was analyzed using the Fisher’s exact test. Following this, pairwise comparisons were performed using logistic regression analysis where malaria infection was the dependent variable and probe time was the covariat, represented by 2 indicator variables (1 min and 5 min probe time groups) with the 10 sec probe time group being the reference. The p-value for the slope of each group represents the comparison of that group to the reference. Comparison of the 2 slopes gives the p-value for the 1 min versus the 5 min groups. The model included a robust variance estimate to account for potential correlation between malaria risk within the same experiment.P) and sporozoite load (S), logistic regression model was fit to the data. The results of the exploratory analyses indicated a non-linear relationship between sporozoite load and log-odds of malaria infection. This non-linear relationship is represented with linear spline of sporozoite load with a knot at 20,000 copies (i.e. piece-wise linear slope model). The model is formulated as follows:1 < 20,000 copies and S2 ≥ 20,000 copies, β0 is the log-odds of malaria infection with no sporozoite load (i.e. the intercept), β1 is the change in log-odds of malaria infection for every 500 copies increase in sporozoite load up to 20,000 copies , and β2 is the change in log-odds of malaria infection for every 500 copies increase in sporozoite load between 20,000 and 647,714 copies . After obtaining maximum likelihood estimates If you have any questions or concerns while you make these revisions, please let us know.Sincerely,Tim J.C. AndersonGuest EditorPLOS PathogensKirk DeitschSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XGrant McFaddenEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-2556-3526***********************Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: The manuscript by Aleshnick and colleagues is a very timely, important and robust paper. The data are unique and provide important insights in developmental bottlenecks for malaria parasites. Whilst a non-human malaria model is used, it is plausible that the findings also have implications for Plasmodium species that are relevant for humans. I have only minor comments.Reviewer #2: This manuscript investigated the impact of Plasmodium sporozoite load, blood-meal acquisition, probe-time, and probe location, on malaria infection probability. The results showed that mosquitoes with higher salivary gland sporozoites are more likely to initiate malaria infection, which provides reference data of a new useful phenotype for field studies. They also proved that infection probability was not impacted by whether a blood meal had been acquired by the mosquito. Overall, this paper will be interesting for readership working on malaria control and vaccine development.However, some methods used in this study are not very appropriate or not well described. For example, the qPCR primers indicated in this paper are able to amplify three locations of the Plasmodium yoelii yoelii 17XNL genome, which was not mentioned anywhere in the manuscript.Reviewer #3: This is animportant manuscript which will have a strong impact on the field. In a tour-de-force experiments, the authors quantify the dynamics of Plasmodium transmission from mosquito to a mammalian host and reveal that only one fith of the infectious bites will result in malaria infections. The authors develop a model that explains non-linear relationships between the sporozoite loads in the mosquito salivary glands and transmission success, defining that malaria infections are transmitted by a small number of highly infected mosquitoes. These conclusions have important consequences for malaria epidemiology and for design of anti-sporozoite vaccine trials.Although the number of sporozoites that are sufficient to initiate infection was not addressed in this manuscript, the authors provide strong evidence that only bites of mosquitoes that have more than 10,000 sporozoites will initiate the disease. Such quantitative approaches can be directly applied to the field studies instead of the currently used metrics of entomological innoculation rates that only consider the number of bites per person as a poxy for infectious bites.Based on the novelty and significance of the presented data I strongly recommend this manuscipt for publication in PLoS Pathogens.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: None.Reviewer #2: 1. qPCRhttps://plasmodb.org/common/downloads/release-46/Pyoeliiyoelii17XNL/). The templates are contig AABL01000525, AABL01001425 and AABL01002193. Normally, genes with multiple copies in the genome are not good candidate reference genes for qPCR analysis, due to the instability of such kind of genes. This should be mentioned in the manuscript.There will be three amplification products using the qPCR primers indicated in this paper, according to the Plasmodium yoelii yoelii 17XNL genome . It’s better to include the DNA recovery rates for different numbers of sporozoites in the manuscript or in the Supplementary files. Method for measuring 18s rRNA plasmid copy numbers should also be included.Also, a supplementary file/figure to show the primer amplification efficient and the standard curve would be useful for other people to re-use the data.2. Controlled/uncontrolled probe time experimentsThere were two mosquito feeding methods used in this study: controlled/uncontrolled probe time experiments. 1) In the controlled probe time experiments (page 29), some of the mosquitoes were forced to resume probing, which increased the probing by human manipulation. 2) Mosquitoes in the controlled probe time experiment groups also had more sporozoite load than in the controlled probe time experiment groups . 3) The explore time of mosquito to mice were different in these two experiment groups: 30min for uncontrolled groups, while variety for the controlled groups. It seems there were multiple variances in these two experiments, should it be better to separate the analysis of these two experiments ?Reviewer #3: I do not think that additional experiments are required.**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: Abstract: ‘being 7.5 times more likely to initiate a malaria infection’ suggests a precision that is not supported by the data. I would suggest to rephrase this and make it more descriptive  literature and current-day relevance of the question addressed.The first section of the results explains why P. yoelii was chosen. Part of the argument is missing. I believe that the authors want to say that because yoelii has such a high likelihood of resulting in detectable infections, it provides a sensitive system to detect/quantify potentially infectious bites. It would be good to mention this specifically.The direct observation of effective contact of mosquitoes is a considerable strength of the paperFigure 1 and 5b should come with error barsPage 8, ‘over 400’: give exact number.Figure 2c. the step appears completely driven by one data point that lies well above the fitted line. It would be good to discuss this in the discussion. The data overall are very convincing but the strong claim on this step is a weaker part of the  paper. I am unconvinced the step would be observed again if the study would be repeated.Fig3b is confusing. Too much attention is drawn to the first bar with a very low number of observations. It would be more appropriate to combine the two lower binsMethods are great and very complete; the discussion is appropriate and well written. It is great to see the Ross McDonald model being criticized in a positive and data-supported manner.Reviewer #2: Page2, Abstract: “sporozoites to patent blood-stage infection”, patient?Page 6, Supplemental Table. An extra column will be much helpful to clarify to the experimental methods, other than by probe time values of . Maybe also include Probe time (s) for mosquitoes without infection .Page 8 parag 2 & page 23 parag 2: what’s the range of sporozoite load in field samples?Page 8 second last lane: add reference.Page 9, page 12: The abbreviation for p value should be unified.Page 12 & Figure 2: the infection probability seems to reach a plateau after 20,000 spz load, any possible explanations?There were multiple logistic regressions mentioned in this manuscript, a summary of the parameters in these regressions should make the results much clearer.Figure 3: The authors mentioned that spz load may explain the different patterns in panel A and B. An extra scatter/box diagram of the spz load in different bin would help to clarify. panel A, how many samples in the 5min group have been forced to resume probing? What’s the significant level between 10 sec and 1min group? panel B, there was no mouse infected by mosquito (more than 100 samples) with >5min probing, any reason? The spz loads between “>5-10 min” and “>1-5 min” were not significantly different while the infection rates were quite different (page 15), why?Page 15: does “11,766 +/- 16,786” mean “range from 11,766 to 16,786”?Figure 4: legend for pie chart.Figure 5: it’s better to mention that it’s based on 30min of mosquito-mouse exposure experiment. Any differences in probe time and spz load, between mosquitoes that able to get a blood-meal and not able to get blood-meal in 30min?Page 21 lane 4: what’s the proportion in this study and in the field?Page 24: what’s the infection probabilities estimated from field studies?Reviewer #3: To increase the clarity of the manuscript I would like the authors to restructure the information split between the Introduction and Discussion. Ross-MacDonald formula should be described in the Introduction to setup the stage for the current study. otherwise, the information appears in both places but is very imprecise in the Introduction which is cryptic for a wide audience of readers. Eliminating repetitions between the Results and Discussion will also make the stronger the Discussion part.I was also confused by the discussion of the results presented in Figure 3A: "As shown in Figure 3A, mosquitoes that were allowed to probe longer were more likely to initiate a malaria infection  with pairwise comparisons showing a statistically significant difference in infection probability between the 10 sec and 5 min probe times (p=0.021) and a less robust but significant difference between the 1 min and 5 min probe times (p=0.025). The difference between the 1 min and 5 min groups was not significant by non-parametric tests such as the Mann-Whitney (p=0.15)."Could the authors use the appropriate statistical tools? If the data is normally distributed, the use of parametric tests is justified. If the data is not normally distributed, only non-parametric tests should be used. The authors have to make clear whether the observed differences are statistically significant or not and show variability and p-values on the graph.The Figure 3B is also confusing. If the authors identify problems in the dataset, it should not be presented in the main figure as such. Instead, the authors could only present the data for the groups that can be compared .**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article : 18 May 2020Dear Dr. Sinnis,We are delighted to inform you that your manuscript, "Experimental Determination of the Force of Malaria Infection Reveals a Non-Linear Relationship to Mosquito Sporozoite Loads," has been formally accepted for publication in PLOS Pathogens.We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles  are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. 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Plasmodium falciparum parasites are known to exhibit extensive genetic diversity in areas of high transmission intensity and infected individuals in such communities often harbour several complex mixtures of parasite clones with different genetic characteristics. However, in the micro-environment, the extent of genetic diversity of P. falciparum parasites remain largely unknown. In this study therefore, the complexity of P. falciparum infections in households was investigated among symptomatic siblings, living under the same roof in north-central Nigeria.P. falciparum isolates was done by allele-specific nested PCR of the highly polymorphic merozoite surface protein-2 (msp-2) gene.Children were enrolled into the study if they were at least two from a household and presented with symptoms of uncomplicated malaria. Clinical malaria was confirmed by light microscopy of Giemsa-stained thick and thin blood films. Genomic DNA was isolated from blood spots on filter paper. Molecular characterization of msp-2 alleles were identified from 215 fragments (range: 180–480 bp). Majority of the isolates [65.6% (n = 61)] were polyclonal infections consisting of 2–6 clones and were significantly more common with the FC27 allelic family (p = 0.036). The multiplicity of infection (MOI) per household ranged from 1.0 to 4.5 while the overall MOI in the study population was 2.31. The pattern of distribution of msp-2 allele types among the households fell into two categories: households where both msp-2 allele types (FC27 and 3D7) were present; households where only one msp-2 allele type (FC27 or 3D7) was present. Majority of the households [88.4% (n = 38)], had both msp-2 allele types but they were disproportionately distributed among the children while in a few households [11.6% (n = 5)], all the children were infected with only one type of msp-2 allele.Ninety-three children from 43 households were enrolled into this study. A total of 26 different P. falciparum isolates exhibit remarkable degree of genetic diversity in the micro-environment and are composed mainly of multiclonal infections, which is an indication of a high ongoing parasite transmission. This suggests that the micro-environment is an important area of focus for malaria control interventions and for evaluating intervention programmes.These findings showed that  Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi [P. falciparum occurs predominantly in Africa and is responsible for most of the severe and life-threatening forms of the disease [P. falciparum to develop resistance to almost all available anti-malarials, the emergence of insecticide resistant mosquitoes, and the non-availability of a malaria vaccine have been major obstacles to the effective control and eradication of malaria. Considerable progress was made in the control of malaria between 2000 and 2015 when global malaria mortality and incidence rates fell by 62 and 41%, respectively [P. falciparum parasites to undermine control measures such as chemotherapy, insecticide-treated nets, and the development of an effective vaccine is its characteristic phenotypic and genetic diversity.Malaria is a major parasitic disease of public health concern in tropical and sub-tropical regions of the world. Human malaria is caused by knowlesi , 2. Of t disease , 4. The ectively . About 2ectively . NigeriaPlasmodium falciparum exhibits enormous genetic diversity in natural populations, and this is evident in the number of antigenically diverse parasite populations found among infected individuals in malaria-endemic regions. Genetic diversity in the parasite population is characteristically a reflection of the transmission intensity in an area and is required for the acquisition of protective immunity against malaria [ malaria –12. Indi malaria –17. On t malaria –20.msp-2 locus alone or together with the msp-1 locus to assess genetic diversity and complexity of P. falciparum infections in different communities with varying transmission intensities and among infected individuals with different clinical presentations of malaria [P. falciparum infections at the level of the micro-environment, among members of the same household. The aim of this study, therefore, was to determine the genetic diversity of P. falciparum isolates in children of the same household, living under the same roof, in Lafia, north-central Nigeria, using the highly polymorphic msp-2 gene.Genetic diversity in natural populations of the malaria parasites has been characterized in several studies using polymorphic, unlinked genetic markers in the parasite genome. Notable amongst these markers are the genes encoding surface antigens found at the developmental stages of the parasite life cycle, particularly the blood-stage antigens merozoite surface protein 1 (MSP-1) and (MSP-2), which are known to be highly polymorphic. Several studies have utilized PCR typing of the  malaria –29. HoweAnopheles gambiae sensu stricto (s.s.), Anopheles arabiensis, Anopheles funestus, Anopheles moucheti, Anopheles melas, and Anopheles nili [This study was conducted in Lafia, a city located within the middle belt region in north-central Nigeria. Lafia lies within Nigeria’s Guinea savannah ecological zone and in this area malaria is known to be endemic and perennial as described previously , 31. Theles nili , 32P. falciparum-infected children" after satisfying the inclusion criteria as follows:(i) at least 2 children of the same household living under the same roof; and, (ii) they must have been residing in the same house for at least 6 months. Uncomplicated or mild malaria was defined as symptomatic malaria with presentations that included chills, pyrexia at presentation (axillary temperature > 37.5 °C) or history of fever within the preceding 48 h, presence of asexual forms of P. falciparum in peripheral blood smears and absence of any indication of severe illness or vital organ dysfunction.Children aged 9 months to 12 years who presented with symptoms compatible with uncomplicated or mild malaria at the Dalhatu Araf Specialist Hospital Lafia, between 2006 and 2011, were enrolled into the study on "determinants of disease outcome in Participation in the study was voluntary. Before being included in the study, the study protocol was explained to the parent/guardian of the children from each family, and then informed consent was obtained. Ethical approval was obtained from the Ethics Committees of the Nasarawa State Ministry of Health and the Dalhatu Araf Specialist Hospital, Lafia, Nasarawa State, Nigeria, with reference numbers S/MH/519/VOL.1/84 and DASH/ADM/MR/VOL.1/0001, respectively. There were no selection criteria in the enrolment process of children in the households as children were registered at random. The first child presented by a parent during the enrolment process, irrespective of age or sex, was documented as child 1 for that particular household; the second child presented was child 2 and so on. All malaria-confirmed cases were appropriately treated by the Hospital clinicians.Approximately 0.5 ml of venous blood samples were collected from each child for parasitological and haematological analyses. All samples were de-identified at the point of collection for the confidentiality of participants and only labelled with alphanumeric codes. Three drops of blood collected were blotted on labelled 3MM Whatman filter paper, air dried, individually sealed in plastic bags, and stored at room temperature until they were used for DNA extraction. Thick and thin blood smears were also prepared for microscopic examination. The slides were labelled and allowed to dry, after which the thin smears were fixed with methanol and subsequently allowed to dry. Slides were stained with freshly prepared 5% Giemsa stain for 20 min at room temperature  and examDNA was isolated from the dried blood spots on filter paper using the QIAamp® DNA Mini Kit  following the manufacturer’s instructions; 150 μl of distilled water was used to elute DNA, which was then stored at − 20 °C until use.P. falciparum msp-2 gene by nested PCR according to previously described amplification procedures [msp-2 gene using the MSP2-1 and MSA2-4 primer pairs. Two sets of nested reactions were subsequently employed to amplify the central polymorphic region of the gene using the allele-specific primers FC 27-1/FC 27-2 and 3D7-1/3D7-2 for the FC27 and 3D7 allele types, respectively , 2.0 µl of each primer (10 µM) and 5 µl of genomic DNA. The reaction was performed using the following cycling condition: initial denaturation step at 94 °C for 5 min followed by 35 cycles of 10 s at 94 °C, 30 s at 57 °C and 40 s at 72 °C and a final extension step of 72 °C for 3 min. All nested reactions were performed in a final volume of 25 µl containing 2.0 µl of PCR product from the primary reaction, 12.5 µl of Go Taq® Green Master Mix  and 2.0 µl of each primer (10 µM). The PCR cycling condition was: initial denaturation step at 94 °C for 5 s followed by 30 cycles of 10 s at 94 °C, 30 s at 57 °C and 40 s at 72 °C, and a final extension step of 72 °C for 3 min. Primer sequences were synthesized by Invitrogen Life Technologies, UK. All PCR assays were performed using a BIOMETRA TB1 ThermalCycler .All samples were genotyped for ocedures . BrieflyPCR products were subjected to electrophoresis on 2% agarose and visualized under ultraviolet trans-illumination after staining with SYBR® Green. Fragment sizes were visually calculated relative to a standard size (100 bp) molecular weight DNA marker . The DNA fragments were grouped into bins if their fragment sizes were within 20 bp intervals.t-test was used to compare different normally distributed continuous variables. Numerical data not conforming to normal distribution were log-transformed. The multiplicity of infection (MOI) was defined as the minimum number of parasite genotypes per infected individual and was calculated as the mean number of PCR fragments or parasite genotypes per infected child. Clonality of infection was defined as the number of distinct PCR fragments per infected child. An infection was defined as polyclonal if more than one distinct PCR fragments or parasite genotypes were present in an isolate. Statistical significance was defined as p < 0.05.Data were analysed using XLSTAT Version 2019.1.2 . The StuP. falciparum infection, although the majority  were those with 2 infected children. The clinical and demographic characteristics of the study participants are shown in Table p = 0.041).A total of 93 children from 43 unrelated households/families were enrolled in this study. Each of the households had either 2 or 3 children with microscopically confirmed MSP-2 locus with 100% efficiency in the amplification reaction. The distribution of MSP-2 alleles among the 93 children from the 43 households studied showed high genetic diversity of the P. falciparum isolates as reflected in the inter-allelic as well as intra-allelic diversity. A total of 215 MSP-2 fragments were detected. The FC27 allele type was observed to be predominantly higher  than the 3D7 allele type . A total of 26 different MSP-2 alleles were identified from the 215 fragments after the fragments were grouped into bins within 20 bp intervals. The FC27 alleles included 14 different band sizes (range: 200–480 bp) of which the 310 bp was the most frequent (17.42%); while the 3D7 alleles included 12 different band sizes (range: 180–430 bp) and the most frequent (17.44%) was the 230 bp .Isolates from all the children enrolled in the study were genotyped for allelic polymorphisms at the p = 0.036). The MOI was 2.31 .The clonality of infection (number of distinct parasite clones) ranged from 1–6. The majority of the isolates  were polyclonal infections consisting of 2–6 clones; and were significantly more common with the FC27 allelic family  were present; households where only one msp-2 allele type (FC27 or 3D7) was present.The distribution of MSP-2 allele types (FC27 and 3D7) were present, it was observed that in the majority of the households , both msp-2 allele were present but disproportionately distributed among the children. In some of these households, all the infected children had both the FC27 and the 3D7 allele types  was present, few households  have all the children infected with only one type of MSP-2 allelic family, which may either be the FC27 allele type or the 3D7 allele type  were present, while the second were households where only one MSP-2 allele type (FC27 or 3D7) was present. The majority of the households (88.4%) investigated belonged to the first category where both msp-2 alleles were present, showing that parasite clones carrying FC27 and 3D7 alleles are widely distributed in the study region. This observation was in agreement with a previous study in Tanzania where most of the households investigated had parasites of mixed genotypes [msp-2 allelic families were prevalent was that the FC27 and 3D7 alleles were disproportionately distributed among the infected children. Thus, in some households, all the infected children had mixed allelic infections with parasites carrying both FC27 and 3D7 alleles. In contrast, in other households, one of the children had parasite isolates carrying a particular type of msp-2 allele and the other child had parasite isolates carrying the other type of msp-2 allele. Nevertheless, in some other households, one or two of the children may be infected with multiple parasite clones or genotypes which may belong to either of the msp-2 alleles or both. Although about 65% of the households have at least one child with isolates carrying both the FC27 and the 3D7 allele types, only a few households (30.2%) were observed to have all the children carrying isolates belonging to both the FC27 and the 3D7 allelic families. In a previous study in Gabon, it was observed that about 80% of the members of the household investigated had parasite isolates carrying both FC27 and 3D7 alleles [msp-2 multiclonal infection in this study could be an indication of a high ongoing parasite transmission, suggestive of effective genetic recombination of the parasite population within the female Anopheles [The results showed that enotypes . An impo alleles  althoughnopheles , 42. Altnopheles , althougnopheles .msp-2 allele type (FC27 or 3D7). This was also consistent with the findings from Tanzania, where they observed a few instances in which different people in the same household had parasites of similar genotypes [It was also interesting to note that a few households (11.6%) belonged to the second category, where all the children had parasites carrying only one enotypes . Apart fAnopheles [Anopheles can lead to independent chromosomal re-assortment of genes and is the principal mechanism for generating novel combination of genes, and consequently, new parasite strains with novel genotypes [Majority of the participants in this study were infected with a mixture of more than one parasite clone. On the whole, it was found that about 65% of the study participants had polyclonal infections consisting of 2–6 clones with an overall MOI of 2.31 clones per infected child. This observation is consistent with previous reports from Nigeria , 31, 42 nopheles , 54. Recenotypes –56. Howeenotypes . Neverthenotypes , 58, 59.P. falciparum in the micro-environment. Future studies will need to take cognisance of the above limitations, use more robust techniques, and consider other regions with different malaria transmission intensity.The present study has some limitations, including: i) the use of a single genetic marker; and, ii) the fact that PCR may not be able to resolve between alleles of similar size but different sequences or those with a size difference of about 10 bp. All of these may potentially underestimate the complexity of infections in this study. However, these data have provided more insight into the genetic complexity of P. falciparum populations in the micro-environment. This is an indication of intense ongoing parasite transmission in the study region and shows that the micro-environment should be a priority target for appropriate malaria control interventions.This study showed high genetic complexity of