Of 1,668 minipools tested, 1 was positive for DENV RNA, and 161 (9.7%) were positive for CHIKV RNA (Table 1). This finding indicates a minimum MP-NAAT–detectable infection rate of 0.6% (161 positive donations of 26,688 total donations), assuming only 1 of the 16 donations in each positive minipool was viremic. However, because the reactive minipool proportion peaked at 19.5% in September 2014 (Table 1), some pools would probably contain >1 viremic donation.

Individual donations comprising reactive minipools were not archived for further testing. Thus, we could not directly determine numbers of reactive IDS per reactive minipool. Therefore, we used a published algorithm (25) to estimate the proportion of donations that would contain CHIKV RNA at levels detectable by MP-NAAT (Table 1). This modification yielded an estimate for MP-NAAT detectable viremia of 0.65% for the overall season and an upper limit of 0.93%. The highest estimated proportion of MP-NAAT–detectable CHIKV RNA-positive donations was during September and October (1.34% and 1.31% of donations reactive for CHIKV RNA by MP-NAAT, respectively) (Table 1). This estimation represented a slightly delayed peak when compared with suspected and confirmed clinical cases reported in Puerto Rico (Figure 1).

Although not optimized to be quantitative, the TC-TMA assay provided approximate viral RNA copy numbers (Figure 2, panel A). Several minipools, particularly from early in the epidemic, had >107 copies/mL, although they were tested as a minipool, and thus effectively diluted 1:16. Of 161 reactive minipools, 125 had quantifiable viral loads. Remaining minipools had viral loads less than an estimated value of 0.5 log copies/mL (according to the calibration curve). The median viral load of 161 reactive minipools was 550 copies/mL (range <3.16 copies/mL–2.3 × 107 copies/mL). Donations from November and December had lower viral loads than donations from preceding months.

We also performed testing of archived IDS for CHIKV RNA for 3,007 donations collected in Puerto Rico during September–November 2014. We identified 56 confirmed positive donations, and ID-NAAT yields were 1.7%–2.1% for the 3 months tested (Table 2). When samples were diluted 1:16 to mimic minipools, proportions of RNA-positive samples detectable by MP-NAAT for September–November decreased to 0.4%–0.9%. Only 21 (37.5%) of 56 ID-NAAT–reactive specimens were reactive when tested for CHIKV RNA at a dilution of 1:16. Thus, 35 (62.5%) of 56 specimens would probably have been missed by routine MP-NAAT (Table 2). As expected, viral loads were low in donations reactive only by ID-NAAT. Only 8 of the ID-NAAT only–reactive samples had quantifiable viral loads (range 5.2–760 copies/mL) (Figure 2, panel B).

We performed assays to detect IgM and IgG in the 56 ID-NAAT–reactive specimens to characterize the relationship between development of IgG and IgM, viral load, and the ability of minipool testing to detect viremic donations (Table 2). Thirteen (23.2%) of 56 samples were seronegative; 2 were detectable only by ID-NAAT. These 2 samples are presumed to represent donors detected in the earliest stages of acute infection. The remaining 11 seronegative viremic donations had detectable viral loads (range 5 ×102–1.3 × 108 copies/mL) (Figure 2, panel B), including 8 (14.3%) of 56 with viral loads >104 copies/mL. These samples were probably from donors who were near the peak of viremia, but still collected before seroconversion occurred.

Most CHIKV RNA-reactive samples were IgM positive (75%) and IgG positive (64%); 1 sample was IgM negative and IgG positive. Development of IgG titers is an inverse correlate of CHIKV RNA detection (28); of the IgG-reactive samples, only 4 (11.1%) of 36 were detectable by the less sensitive MP-NAAT. Viral loads of samples sorted on the basis of NAAT results (ID only vs MP-NAAT detectable) and serologic data demonstrate a typical profile of acute viral infection (Figure 2, panel B). The 43 viremic IgM-positive or IgG-positive donations had significantly lower viral loads (median <3.16 copies/mL) than 13 viremic seronegative donations (60,000 copies/mL; p<0.0001 by 2-tailed Mann-Whitney test). Although similar proportions of ID-NAAT–positive samples were detected in November (1.7%) and September (1.8%), only 2 (11.8%) of 17 were seronegative in November compared with 6 (33.3%) of 18 in September, which suggested waning of the epidemic and a higher proportion of donations at the end of acute infection.

To estimate the incidence of CHIKV infection during the 2014 epidemic, we performed IgG serologic studies on blood donor specimens collected at the beginning of the epidemic (June 2014; preepidemic) and after the epidemic had subsided (March 2015; postepidemic). Collection was delayed until March to maximize detection of IgG seroconversion and to enable the maximum period for potential donors to recover from symptomatic infection, which would result in self-deferral, or deferral by the blood collection organization.