Review 2: "Malaria Parasite Resistance to Azithromycin is not Readily Transmitted by Mosquitoes"

RR:C19 Evidence Scale rating by reviewer:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review: 

Emerging drug resistance of front-line antimalarials continues to be an issue; the authors sought to investigate the utility of azithromycin as an additional possible partner or prophylactic drug by examining azithromycin-resistant human and mouse parasite strains throughout the parasite life cycle with particular interest in their transmission potential. They generated azithromycin strains, determined to harbor point mutations present in a conserved region of the 50S ribosomal protein L4 (Rpl4; G95D for P. berghei and G76V for P. falciparum), and subsequently compared their infectivity to mosquitos, parasite development within mosquitos, and onward transmission potential to and/or liver stage development within vertebrate hosts. They concluded G95D mutants disrupt parasite development during P. berghei mosquito stages as well as severely impair onward transmission to mice, correlating with apicoplast and nuclear defects noted during the mosquito and liver stages. They similarly concluded that, while the G76V mutant did not inhibit P. falciparum development in mosquitos, it did significantly reduce capacity to replicate in the liver (in a humanized mouse model), again correlating with apicoplast and nuclear defects. They discussed these results fitting with expected azithromycin drug pressure and the necessity of apicoplast function during certain parasite developmental stages and pointing towards liver stage development deficits for both mutant mouse and human parasite strains that would seemingly have significant fitness deficits regarding transmission.

The authors offer an interesting and unique addition to the malaria drug resistance research arena, which is much needed. Yet, I do have concerns about their conclusions and messaging. For instance, it is difficult to extrapolate findings from P. berghei mouse transmission models (which the paper relies on most) to real world human malaria transmission given the differences in parasites and models. In fact, the stark difference in P. berghei and P. falciparum azithromycin resistant clones generated here in terms of their mosquito development, highlight this. While the authors do attempt to infer P. falciparum transmission capabilities of the G76V mutant by injecting mice with humanized livers with sporozoites, and I certainly understand the experimental limitations, this of course does not equate to studying the complete transmission cycle. Without their mouse model supporting parasite ability to develop subsequent asexual blood stage infection, it cannot be said the degree to which the liver stage defects they observed would translate to a transmission impediment. Their reported 80% reduction in mutant P. falciparum liver parasite load, along with visual assessment of liver stage schizonts with atypical morphology and fewer nuclei does not definitively suggest P. falciparum mutants are incapable progressing through the liver stage, which is an important point considering that even some minor ability to complete the transmission cycle could be a big problem for propagating resistance, particularly in the face of drug pressure on a grand scale in the real world. Furthermore, the n they provide for the humanized mice experiments is notably very small (2 mice for WT and 3 mice for the mutant strain) to make any significant conclusions.

The authors’ general conclusions about azithromycin resistance mutations are also hard to extrapolate with only a few clones generated per species. They attribute azithromycin resistance and their experimental observations to the specific point mutations in the apicoplast Rpl4 gene, however even the two G95D mutant P. berghei strains had notably different characteristics in their assays, highlighting other potentially unknown influences on parasite development might be present in their mutant strains. Their drug pressure also generated another P. bergheiS89L Rpl4 mutant they were largely unable to work with due to more significantly impaired growth rates, but the fact that additional mutants could be generated is important to remember when contextualizing their data. Similarly, it is important to contextualize some of their conclusions with the recognition they were unable to genetically introduce specific Rpl4 apicoplast point mutations into WT strains to confer specificity of their observed parasite development defects (though again I understand the experimental limitations in being able to do so). Given the two G95D P. berghei mutant strains did have differential growth characteristics (one of them having less severe oocyst and sporozoite reduction in infected mosquitos and being able to initiate a liver stage mouse infection following inoculation), the fact that they only selected a single P. falciparum clone to work with for all their falciparum experiments is a shortcoming. It would have been beneficial in extrapolating the real world relevance of their findings to have characterized more falciparum clones. Taken together, while the selective pressure resulting in mutations in Rpl4 gene fit with their hypothesis about the apicoplast being differentially important in various aspects of the parasite life cycle, it must be kept in mind that the data they show here is certainly not an exhaustive repertoire of mutations nor azithromycin resistant parasite development characterization.

Overall, the manuscript and results within are experimentally sound, innovative, and very interesting, but I did feel the authors made more grandiose conclusions than their data alone specifically suggested – take the title alone, with reference to the above concerns. I also have some other data representation questions, such as ensuring all of their microscopy findings (which they rely on heavily for the results and conclusions) are not only described and depicted as representative, but actually quantified. As another example, there is a results section header stating “‘Azithromycin resistant P. berghei sporozoites lose their apicoplast,” yet in the results details they describe 12.6% of G95D_1 and 18.8% of G95D_2 retain intact apicoplast – thus I would prefer a more nuanced section title (especially given 20% of parasites potentially developing normally has big implications for the ability to still have some parasites develop through the life transmission life cycle). This reiterates the importance of quantifying their microscopy descriptions for other assays; for instance, with their P. falciparum IFA findings regarding liver stage schizonts, they only describe general observations without quantifying the prevalence of aberrant development. Finally, the n’s used in their experimental replicates also were often quite variable between strains – e.g. the number of mouse transmission attempts per strain of the P. berghei mutants was n=1 for G95D_1, n=4 for G95D_2, and n=6 for WT, and in multiple other scenarios their WT n measurements were much higher than the mutant strains. While I understand the mutant strains were likely more difficult to work with, this discrepancy is noted when interpreting their results and conclusions. 

The authors show lots of interesting correlative findings about azithromycin resistance, Rpl4 mutations, apicoplast dysfunction, and impact on parasite development in mosquitoes (P. berghei) and infectivity through the liver stage (P. berghei and P. falciparum), but as described above, it is hard to definitively make causative statements or to truly infer downstream or large scale transmission impact, and even a small amount of onward transmission could propagate resistance spread. In their discussion, the authors barely mention their own study limitations, nor do they discuss some other interesting findings related to potential impact of widespread azithromycin use in a background of non-resistant strains. (In particular, I refer to other recent studies suggesting potentially enhanced infectivity of P. falciparum parasites to mosquitos in the presence of azithromycin trialed in a chemoprophylactic context https://pubmed.ncbi.nlm.nih.gov/34315475/; albeit the azithromycin effect on gametocyte infectivity of non-resistant strains is seemingly nuanced, with others reporting reduced infectivity https://pubmed.ncbi.nlm.nih.gov/27419152/.)