Review 1: "Malaria Parasite Resistance to Azithromycin is not Readily Transmitted by Mosquitoes"

Philip Rosenthal

doi:doi:10.1162/2e3983f5.3819c927

Malaria parasite resistance to azithromycin is not readily transmitted by mosquitoes

by Hayley D. Buchanan, Robyn McConville, Lee M. Yeoh, Michael F. Duffy, Justin A. Boddey, Geoffrey I. McFadden, and Christopher D. Goodman

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Published on Jan 23, 2024
www.biorxiv.org

Description

Drug resistance in malaria parasites severely erodes our ability to control disease. Combination therapies—where multiple compounds are combined to reduce the probability of resistance emerging to two or more compounds simultaneously—are the favoured solution to stemming antimalarial resistance. Thus, identifying suitable partner compounds for combinations is a priority. We sought potential partner compounds with inherent refractoriness to the spread of resistance that might better protect the primary antimalarial. We focused on azithromycin, a cheap, safe antibacterial that kills malaria parasites by blocking protein synthesis in the apicoplast—a relict plastid in malaria parasites. Selection for azithromycin resistance in rodent (Plasmodium berghei) and human (P. falciparum) malaria parasites yielded various mutations in the apicoplast-encoded gene for the 50S large ribosomal subunit protein L4 (Rpl4). P. berghei Rpl4 mutant parasites developed poorly in mosquitoes, producing aberrant oocysts and low numbers of sporozoites. P. falciparum Rpl4 mutants developed normally in mosquitoes. The azithromycin resistant mutants of both P. berghei and P. falciparum parasites could establish liver stage infections, but both species developed aberrantly, and P. berghei failed to progress to blood stage infections without massive mechanical intervention. The mutations in the apicoplast-encoded ribosomal protein Rpl4 apparently confer azithromycin resistance in the vertebrate blood phase, but the mutations confer a fitness deficit—likely reduced efficiency of apicoplast protein synthesis—that becomes a severe problem for the parasites in the highly-replicative mosquito stage (for P. berghei) and liver stage (for both P. berghei and P. falciparum). The apicoplast is more active metabolically in mosquito and liver stages than blood stage, and we hypothesise that the mutations in apicoplast Rpl4 render it less able to deliver on these metabolic needs, thus retarding parasite development. Azithromycin resistance will therefore be less likely to spread geographically, making it an attractive option as a perennial partner compound to protect appropriate frontline antimalarials from resistance spread.

RR:C19 Evidence Scale rating by reviewer:

Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review:

This manuscript assesses the ability of P. berghei and P. falciparum parasites selected for resistance to the antibiotic azithromycin to be transmitted by mosquitoes. Resistance was selected and associated with mutations in Rp14, and mutant parasites had decreased transmission potential, albeit with different defects in the two studied plasmodial species. Thus, as the authors showed previously for a different drug, atovaquone, although resistance to azithromycin may be selected readily, there is evidence that it will not be readily transmitted, potentially limiting its public health importance. The relevance of these results is tempered somewhat by the appreciation that azithromycin is a slow-acting antimalarial with relatively modest potency, such that the drug is not currently recommended for the treatment or prevention of malaria, but as the authors suggest, azithromycin might become part of some future combination antimalarial regimen. Overall, this is a well-written report describing well conceived experiments to explore the transmissibility of parasites selected for azithromycin resistance. The results are complex, with important differences between results for the studied murine and human malaria parasites, but the important overall conclusion, that azithromycin resistance is accompanied by defects in transmission by mosquitoes, is very convincing. Some relatively minor concerns are as follows.

Line 108. The statement “Azithromycin is used as monotherapy to treat uncomplicated malaria” is misleading and arguably even dangerous. Monotherapies should not be used to treat uncomplicated falciparum malaria. Further, azithromycin is a rather feeble antimalarial, compared to artemisinins and other first-line agents, due both to relatively low potency and its slow mode of action. Azithromycin has been studied in combination with other agents in limited studies, but it is not part of any recommended regimen to treat or prevent falciparum malaria. For this report azithromycin can appropriately be considered a potential component of a combination regimen, but it should not be implied that it is, by itself, an effective antimalarial.
Line 274. “Apicoplast malaise” is an intriguing term, but what does it mean? The authors may wish to avoid a precise description here, but somewhat clearer explanation would be helpful.
Results. The authors present a well thought out and comprehensive set of experiments establishing the impacts of azithromycin resistance mutations on transmission in P. berghei and P. falciparum. It would have been helpful if more of the approaches utilized with P. berghei were also performed in P. falciparum, the relevant human malaria parasite, but the technological challenges (e.g. the difficulty of studying human hepatocytes and humanised mice) are appreciated, and P. berghei serves as an appropriate model to validate the more limited, yet convincing results with P. falciparum. Indeed, results in the two plasmodia differed importantly, yet both showed results expected to be accompanied by major defects in transmission from mosquitoes to vertebrate hosts.
Discussion. It is not surprising, but perhaps interesting that the phenotypes conferred by azithromycin resistance mutations were incomplete, i.e. some parasites progressed through various steps required for transmission despite the mutations. Considering this, might additional drug pressure in a subject newly infected by mosquito bite who happens to have azithromycin in the bloodstream enable some parasites to be further selected for successful transmission? Of course we cannot answer this question, but it might be raised in the discussion.
Line 521. In mention of azithromycin for prophylaxis the authors might acknowledge the qualification that the drug has not performed very well as a chemoprophylactic agent, underperforming comparator antimalarial regimens in some key studies.

Review 1: "Malaria Parasite Resistance to Azithromycin is not Readily Transmitted by Mosquitoes"

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