Vestigators were un-blinded to the qPCR result. If this was positive the volunteer was treated for malaria. In the case of participants with positive thick film microscopy, but no symptoms consistent with P. falciparum malaria infection, clinical investigators were un-blinded to the qPCR results and the volunteer only treated if any preceding samples had .500 parasites per mL. This was to reduce the likelihood of falsepositive diagnosis by microscopy. Time to diagnosis was measured in hours and converted to days for analysis.SafetyVolunteers were reviewed in clinic the day after CHMI. All volunteers were called daily on days 2? post CHMI (C+2?) to enquire as to adverse events and use of medications. As in previous mosquito bite CHMI studies at our centre, volunteers were reviewed in clinic on dC+6 in the evening and then twice a day, morning and evening between dC+7 and dC+14. Undiagnosed volunteers were reviewed once a day in the morning between dC+15 and C+21. At each visit, blood was sampled for microscopy and qPCR, physical observations performed and AEs solicited. AE were graded according to the criteria in Tables S1 S2. On diagnosis, volunteers were treated with a 3 day curative course of oral Malarone (atovaquone/proguanil 250 mg/100 mg) where each dose was directly observed in clinic. Volunteers were reviewed 24 and 48 hours post diagnosis where blood was sampled for microscopy. Provided these two blood-films after treatment were negative for parasites volunteers were not reviewed again in clinic until dC+35. If one of these blood films was positive, volunteers continued to be reviewed in clinic at 24-hour intervals until two consecutive blood films were negative. Volunteers were then reviewed at dC+35 and dC+90 where safety assessments were performed. Full blood count with differential, platelet count and serum biochemistry (including electrolytes, urea, creatinine, bilirubin, alanine aminotransferase, alkaline phosphatase and albumin) were measured at screening, the day before CHMI, atParasite Growth ModellingQuantitative real-time PCR (qPCR) was conducted as previously described. [29] Briefly, DNA was extracted from 0.5 mL blood, filtered to reduce WBC content, using POR8 Qiagen Blood Mini Kit. 10 of each extraction was run in triplicate qPCR ?equivalent to 150 uL blood directly assessed. Parasites per mL equivalent mean values were generated by a standard Taqman absolute quantitation, against a defined plasmid standard curveOptimising CHMI Using Needle Syringewith an ABi StepOne Plus machine and v2.1 software using default Universal qPCR and QC settings, apart from the use of 45 cycles and 25 uL reaction volume. Based upon results obtained using a dilution series of microscopically-counted cultured parasites this method has a lower limit of quantification (LLQ, defined as CV,20 ) of around 20 parasites/mL blood (p/mL). [30] The number of infected erythrocytes in the first generation after parasite release from the liver (liver-to-blood CASIN inoculum, LBI) and the parasite multiplication rate in the blood (PMR) were estimated (full modelling details are provided in Materials Methods S1). Time in hours between challenge and collection of each blood-sample was calculated using data specific to each volunteer for each of these events and then converted to days. Data from 1676428 non-immunised infectivity control subjects from three previous mosquito-bite CHMI trials were used as comparators (Ewer et al. unpublished). [29].All participants.Vestigators were un-blinded to the qPCR result. If this was positive the volunteer was treated for malaria. In the case of participants with positive thick film microscopy, but no symptoms consistent with P. falciparum malaria infection, clinical investigators were un-blinded to the qPCR results and the volunteer only treated if any preceding samples had .500 parasites per mL. This was to reduce the likelihood of falsepositive diagnosis by microscopy. Time to diagnosis was measured in hours and converted to days for analysis.SafetyVolunteers were reviewed in clinic the day after CHMI. All volunteers were called daily on days 2? post CHMI (C+2?) to enquire as to adverse events and use of medications. As in previous mosquito bite CHMI studies at our centre, volunteers were reviewed in clinic on dC+6 in the evening and then twice a day, morning and evening between dC+7 and dC+14. Undiagnosed volunteers were reviewed once a day in the morning between dC+15 and C+21. At each visit, blood was sampled for microscopy and qPCR, physical observations performed and AEs solicited. AE were graded according to the criteria in Tables S1 S2. On diagnosis, volunteers were treated with a 3 day curative course of oral Malarone (atovaquone/proguanil 250 mg/100 mg) where each dose was directly observed in clinic. Volunteers were reviewed 24 and 48 hours post diagnosis where blood was sampled for microscopy. Provided these two blood-films after treatment were negative for parasites volunteers were not reviewed again in clinic until dC+35. If one of these blood films was positive, volunteers continued to be reviewed in clinic at 24-hour intervals until two consecutive blood films were negative. Volunteers were then reviewed at dC+35 and dC+90 where safety assessments were performed. Full blood count with differential, platelet count and serum biochemistry (including electrolytes, urea, creatinine, bilirubin, alanine aminotransferase, alkaline phosphatase and albumin) were measured at screening, the day before CHMI, atParasite Growth ModellingQuantitative real-time PCR (qPCR) was conducted as previously described. [29] Briefly, DNA was extracted from 0.5 mL blood, filtered to reduce WBC content, using Qiagen Blood Mini Kit. 10 of each extraction was run in triplicate qPCR ?equivalent to 150 uL blood directly assessed. Parasites per mL equivalent mean values were generated by a standard Taqman absolute quantitation, against a defined plasmid standard curveOptimising CHMI Using Needle Syringewith an ABi StepOne Plus machine and v2.1 software using default Universal qPCR and QC settings, apart from the use of 45 cycles and 25 uL reaction volume. Based upon results obtained using a dilution series of microscopically-counted cultured parasites this method has a lower limit of quantification (LLQ, defined as CV,20 ) of around 20 parasites/mL blood (p/mL). [30] The number of infected erythrocytes in the first generation after parasite release from the liver (liver-to-blood inoculum, LBI) and the parasite multiplication rate in the blood (PMR) were estimated (full modelling details are provided in Materials Methods S1). Time in hours between challenge and collection of each blood-sample was calculated using data specific to each volunteer for each of these events and then converted to days. Data from 1676428 non-immunised infectivity control subjects from three previous mosquito-bite CHMI trials were used as comparators (Ewer et al. unpublished). [29].All participants.