Transmission between the human host and the mosquito vector is a crucial step in the malaria parasite life cycle. It represents a bottleneck where parasite numbers shrink from millions in the human body to less than a hundred in the mosquito vector (Smith et al., 2014). Transmission through the mosquito is thus vulnerable to interruption and is a key focus of malaria research (Churcher et al., 2015; Sauerwein and Bousema, 2015), with research tools including membrane feeding assays designed to explore this transitioning phase of the parasite. DMFAs were initially developed by Rutledge and colleagues in 1964 in which malaria parasites were exposed to mosquitoes via a membrane feeding apparatus (Rutledge et al., 1964).

Direct Membrane Feeding Assays provide a means to investigate the still poorly understood process of human to mosquito transmission and the resulting mosquito infection. For example, DMFAs can be used to study the infectiousness of different human malaria reservoirs, and estimate their contribution towards transmission (Graves et al., 1988; Diallo et al., 2008). This can include symptomatic, patent infections as in the present study and asymptomatic, often low-density infections (Kiattibutr et al., 2017). In addition, DMFAs can be used to study the effect of drugs, vaccine candidates and immune factors on the development of the mosquito stages of the Plasmodium parasites (Bousema et al., 2012; Delves et al., 2012; Sattabongkot et al., 2015; Vallejo et al., 2016). Also, DMFAs provide an opportunity for circumventing some of the operational and ethical complicating factors associated with feeding mosquitoes directly on the skin of malaria infected individuals. Finally, there is evidence that there is no clustering of gametocytes in the skin as initially perceived thus making DMFAs a reliable tool for infection studies (Meibalan et al., 2019; Talman et al., 2020).

Despite these advantages, DMFAs are resource intensive, require an insectary and rely on stringent logistics for sample collection, handling, rapid transportation and processing as it has been shown that the time between blood collection and performance of the DMFA can impact assay outcome, most likely due to premature gametocyte activation (Churcher et al., 2012; Sattabongkot et al., 2015; Soumare et al., 2021). As a further complication, conducting DMFAs with P. vivax requires proximity to endemic areas in order to access infected samples as continuous culture of this parasite species remains elusive (Roobsoong et al., 2015). Papua New Guinea (PNG) is amongst the countries with the highest P. vivax burden in the world, thus P. vivax is a research priority for the country and infected blood samples can still easily be obtained (Cattani et al., 1986; Müller et al., 2003; Howes et al., 2016; World Health Organization, 2019; World Health Organization, 2020). Establishing DMFAs with P. vivax provides a tool to study P. vivax transmission that is of potentially global relevance.

DMFAs were performed in PNG previously in 1983 - 1985 in village-based malaria surveys, prior to diagnosis and on known gametocyte carriers in clinical outpatient populations in Madang and Goroka (Graves et al., 1988). In the present study, we investigated the infectivity of blood samples obtained from symptomatic, rapid diagnostic test (RDT)-positive individuals to Anopheles farauti colony mosquitoes.

Currently the limitation with doing research with P. vivax is it is still difficult to maintain a continuous culture of P. vivax, which necessitates access to naturally acquired infections in field settings, often associated with additional cost and operational constraints. As such, a reliable P. vivax DMFA setup in an endemic setting can be of great value.

In the present study, we investigated the infectiousness of symptomatic, RDT-positive malaria cases obtained from local health facilities. In a resource constrained situation where diagnosis by microscopy is not readily available and where the primary diagnosis of malaria is performed by RDT, it is important to assess which RDT result will most likely lead to a mosquito infection. As commonly known, RDT results are not reliable in distinguishing between Plasmodium species in co-endemic settings, however, the present study shows that they can be used to prioritise samples selected for DMFAs to maximise the probability of a specific species being present in the sample and to increase infection success ( Table 2 ) (World Health Organization, 2006). We observed that in the group of samples positive for only pLDH with the CareStart RDT the proportion of successful DMFAs was highest (35.1%) compared to HRP2 (27.3%) or when positive for both antigens (11.1%). This difference in proportions was statistically significant between pLDH and both antigens (p<0.01) but not between pLDH and HRP2 (p=0.43). In PNG where both P. falciparum and P. vivax are present in roughly equal proportions, P. vivax infections were more likely when the RDT is positive for only the pLDH antigen (World Health Organization, 2017). We therefore decided that by selecting samples only positive for pLDH over HRP2 (or both antigens) the likelihood of the sample being P. vivax would be significantly increased and DMFA success can be increased up to 3-fold. There is a sound biological explanation as to why acute P. vivax infections result in mosquito infections more frequently. It has been shown that P. vivax gametocytes develop faster, and are present and infectious at the onset of an infection while P. falciparum gametocytes can take 10 days to mature (Bousema and Drakeley, 2011). Consequently, lower infectiousness in symptomatic P. falciparum patients as compared to P. vivax patients is expected since people are likely to seek treatment before P. falciparum gametocytes have matured (Kiattibutr et al., 2017). Furthermore, HRP2 based RDTs can remain positive for 35-42 days after treatment and clearance of parasitaemia, while for pLDH it takes only 2 days before the antigen is cleared from circulation giving a more reliable result (Grandesso et al., 2016).

We observed that the proportion of samples that infect mosquitoes was higher for P. vivax (44.2%) compared to P. falciparum (11.4%) according to microscopy. Interestingly, we observed a higher mosquito infection rate (58.3%) for samples with P. vivax gametocytes detectable by light microscopy while the mosquito infection rate with P. falciparum gametocytes was low (15%). Although our findings are in contrast to what was observed previously by Graves and colleagues in An. farauti where they showed a 37.5% (6/16) infectivity with P. vivax, 18.8% (3/16) infectivity in P. vivax with gametocytes and a 48.1% (13/27) infectivity in P. falciparum with gametocytes, this may be due to the difference in sample sizes used (Graves et al., 1988). Our results show that our DMFA with P. vivax is about 4 times more successful than with P. falciparum especially when considering samples with gametocytes. Although we are uncertain as to why we observed low P. falciparum infections a possible explanation that we did not evaluate in this study is that immunity-related factors are responsible. This could be further studied by comparing, in parallel, DMFAs conducted with autologous plasma (i.e., replacement of patient’s plasma with the individual’s own plasma), whole blood (directly added to the feeder) and malaria-naïve plasma (i.e., replacement of patient plasma with plasma from a P. falciparum naive donor). Furthermore, a possible explanation is that this strain of mosquitoes may have become refractory to wild-type P. falciparum infection. This was observed with cultured gametocytes of P. falciparum which were fed to this strain of An. farauti mosquitoes via standard membrane feeding which resulted in an extremely low mosquito infection rate (Smith et al., 2014). This indicated that the An. farauti strain was refractory to cultured gametocytes and that could also be the case with wild-type parasites as well. Interestingly, our observations that this does not apply to P. vivax may be the basis for further studies into species-specific mechanisms of mosquito infection.

In the present study, we observed an 85% concordance between the microscopy diagnosis and qPCR diagnosis. We observed a 94% sensitivity and a 49% specificity when comparing qPCR with light microscopy as the reference method. This is a result of the lower limit of detection of the qPCR method, which is able to detect many more infections as compared to light microscopy. As qPCR is able to detect these sub-microscopic infections, the proportion of false positive is overestimated when compared to light microscopy leading to an apparently low specificity. The possibility of an infection (or no infection) by microscopy being confirmed by qPCR is expressed by a moderate PPV and NPV (88% and 67%). We note that the lack of having microscopy diagnosis being done prior to bleeding was a limitation in this study and light microscopy results were only obtained retrospectively by highly trained microscopists. We found that species and parasite stage determination by light microscopy was a very good predictor of infection success, as P. vivax with gametocyte infections resulted in approximately 4-fold increased infection success in the mosquitoes as compared to P. falciparum. Based on our results we estimate that light microscopy diagnosis before bleeding would enable a further increase of DMFA success rate with P. vivax to around 60% if suitable P. vivax samples (those with gametocytes by light microscopy) were selected. Similar infectivity rates (45-60%) were measured in Anopheles aquasalis, Anopheles albitarsis in Brazilian Amazon, Anopheles albamanus in Colombia, An. dirus in Thailand and Anopheles arabiensis in Ethiopia (Sattabongkot et al., 2003; Solarte et al., 2011; Rios-Velásquez et al., 2013; Vallejo et al., 2016; Tadesse et al., 2018). We did also observe a significant but moderate correlation between P. vivax gametocytes and mosquito infection ( Figure 2B ). Other studies observed similar but often stronger positive associations between P. vivax gametocyte densities and the proportion of infected mosquitoes in An. dirus in Thailand and An. arabiensis in Ethiopia (Kiattibutr et al., 2017; Tadesse et al., 2018). However, there are other studies which describe the relationship between P. vivax gametocytaemia and mosquito infection as weak with An. dirus in Thailand (Sattabongkot et al., 1991; Sattabongkot et al., 2003).

The observed correlation between mosquito infection prevalence and oocyst density was moderate but significant for both P. falciparum and P. vivax ( Figures 3A–D ). That is, the more mosquitoes are infected during a DMFA, the higher the average number of oocysts in the infected mosquitoes. Our findings is in contrast with a previous study where a strong correlation was observed between the mosquito infection rate and the oocyst rates for An. dirus with P. vivax (Kiattibutr et al., 2017).

We noted that only 2/45 (4.4%) of samples resulted in mosquito infections from DMFA using blood from the Madang Town Clinic while 36/137 (26.2%) of samples from Yagaum clinic infected mosquitoes. The low infection rate from Madang Town Clinic was mainly because most of the samples were without gametocytes especially P. vivax gametocytes. Of the 45 samples 3 samples had only P. falciparum gametocytes while 2 had only P. vivax gametocytes and one with both P. falciparum and P.vivax gametocytes. Of the 2 samples that led to successful infections, one had only P. vivax gametocytes while the other had both P.falciparum and P. vivax gametocytes. Another factor which could have influenced the infectivity of the mosquitoes but was not investigated here is impact of temperature fluctuations of the thermal flask while transporting it from Madang to the laboratory, and the longer duration between collection of the sample and the DMFA. It has been shown elsewhere that temperature of thermal flask does influence the infectivity of the mosquitoes (Soumare et al., 2021).

This study provides important insights into the infectivity of symptomatic malaria cases to An. farauti in PNG. Overall, we show that symptomatic P. vivax infections are more likely to be infectious to mosquitoes as compared to symptomatic P. falciparum infections. This may be a result of the differences in gametocyte dynamics that exist between P. falciparum and P. vivax. We have re-established a DMFA set up in PNG, where frequent access to P. vivax infections is provided. This could serve as a platform to test potential transmission blocking vaccines and antimalarials, which act on gametocytes or the mosquito developmental stages of P. vivax.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Papua New Guinea Medical Research Advisory Council. Written informed consent to participate in this study was provided by the participants or their legal guardian/next of kin.

Designed the study: LR, SK, ML. Conducted the laboratory work: LT, RV, MK, TK, EN. Secured funding: IM, IF, SK. Drafting and preparation of the manuscript: LT, SK. Critically revising the manuscript: SK, RV, MK, TB, LS, IF, LR, CC. All authors contributed to the article and approved the submitted version.

This work was supported in part by the Bill and Melinda Gates Foundation (OPP1034577), National Institute of Allergy and Infectious Diseases (NIAID) (5U19AI089686-03), Swiss National Science Foundation (310030_134889), and National Health and Medical Research Council (NHMRC) of Australia (GNT1127356). LT is supported by a PhD scholarship from James Cook University. IM is supported by a Research Fellowship from NHMRC. LR and SK are supported by Career Development Fellowships from NHMRC of Australia.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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