vol21no7_pdf-version

Transdermal Diagnostics of Malaria(a proxy for malaria parasite–infected blood [22]). Thesample cuvette modeled the blood vessel by using askin-colored film, a channel with blood 1 mm deep, andan acoustically dampening bottom. The bulk optical absorptionof the laser pulse at 532 nm by normal wholeblood (mainly by hemoglobin) produced the backgroundtrace (Figure 2, panel A, black line) associated with thethermo-elastic effect (30) (a heat-driven transient pressurerise). However, no vapor nanobubbles were generated becausethe laser fluence applied was well below the vapornanobubble generation threshold for any normal bloodcomponents (22). The absence of vapor nanobubbles wasconfirmed experimentally by monitoring the laser pulseexposedsample volume with an optical scattering method(22,27) by using a low-power probe laser beam at 633 nmand monitoring its time-response to the excitation laserpulse (Figure 2, panel A, black line in inset). In normalblood, the optical scattering time-response to a single laserpulse indicated incremental transient bulk heating withoutgenerating a vapor nanobubble. Previously we haveshown that such a bulk photothermal effect does not causeany detectable detrimental effects at the molecular andcellular levels (22). Adding hemozoin nanocrystals to theblood at a concentration of 23 μg/mL, (which correspondsto ≈0.8% of parasitemia [9]), resulted in a completely differentacoustic trace under the same excitation and detectionconditions (Figure 2, panel A, red line). This tracewas attributed to H-VNBs, which were directly detectedin the same sample by optical scattering with optical timeresponsesof 50–100 ns duration and the H-VNB-specificshape, which revealed the vapor bubble expansion andcollapse without any recoil (Figure 2, panel A, red linein inset). Unlike the background acoustic traces obtainedfrom the normal blood, the H-VNB acoustic traces yielded5-fold higher peak-to-peak amplitudes and thus were easilydifferentiated from the blood background traces in thetrace amplitude histograms (Figure 2, panel B).To obtain proof of the device feasibility, the prototypewas further tested in human volunteers who did not havemalaria and on a malaria patient with a similar skin tone(dark). We applied the probe to wrists and earlobes andpositioned it over subcutaneous vessels. Thus, laser pulseswere delivered to blood through the skin. No blood sampleswere taken, and no reagents were applied. Acoustic tracesin response to each of 400 laser pulses (of the same fluenceas described earlier) were collected simultaneouslywith laser irradiation (within 20 seconds total) and analyzedstatistically in real time. In the malaria patient, theparasitemia (percentage of infected erythrocytes) was determinedby microscopy (thin blood film) and varied from2% (corresponding to ≈69,000 parasites/μL) 4 hours beforethe device test to 0.3% (corresponding to ≈8,600 parasites/μL)9 hours after the device test. As malaria-negativecontrols, we used healthy volunteers with similar skintone and under the conditions and procedure applied tothe malaria patient. Acoustic traces from the wrist of themalaria patient (Figure 2, panel C, red line) showed the H-VNB–specific pattern similar to that of the hemozoin-positivesamples in vitro (Figure 2, panel A, red line) and hadmuch higher amplitudes than those obtained from a healthyvolunteer with a similar skin tone (Figure 2, panel C, blackline). The amplitudes for the traces from the malaria patientwere significantly higher than those from the control,and the 2 histograms barely overlapped (Figure 2, panel D).Therefore, these acoustic traces indicated malarial infectionin a clinically ill patient. Similar traces were obtainedwhen the device was applied to the earlobes of the malariapatient and volunteers (3 volunteers with dark skin tonewere studied) (Figure 2, panel E). The similarity betweenthe wrist and ear lobe results further validates the successfuldetection of malarial infection by the H-VNB method.A quantitative analysis used the volunteers’ histograms todetermine the malaria threshold amplitude and revealed HIvalues as 42.4 mV and 1.3 mV in the malaria patient for thewrist and earlobe, respectively.The safety of H-VNB generation in humans is ensuredby the safe level of the laser fluence applied, 36 mJ/cm 2 ,which is considered to be skin-safe according to federalregulations (31). In addition, no short-term (10 min) orlong-term (3–4 d) signs of skin damage or irritation wereobserved in the study participants. These observations arealso in line with our previous observation of no damageto the laser-exposed malaria parasite–negative blood cells(22). Therefore, the device and procedure developed appearto be safe, and coupled with their blood- and reagentfreenature, deliver a completely noninvasive diagnosis ofmalaria in humans.We further studied the influence of skin tone on thebackground trace for 5 healthy volunteers with differentskin tones (Figure 2, panel F). For 532-nm wavelengthlight, we observed a predictable increase in the backgroundtrace amplitude resulting from the increase in the concentrationof melanin.In the third model, we evaluated the device for the rapidnoninvasive transcuticle analysis of individual malariainfected(oocyst-positive) Anopheles mosquitoes. Tenacoustic traces were obtained for each mosquito (7 in eachgroup) by scanning the body across the probe under thesame laser pulse fluence as described for the human studies.The negative control group (fed with uninfected humanblood, no oocysts) returned acoustic traces similar tothose for hemozoin- and malaria parasite–negative humanblood (Figure 2, panel G, black line). In the malaria-infectedmosquitoes, the trace shape and amplitude (Figure 2,panel G, red line) were similar to those obtained for hemozoin-and malaria parasite–positive traces. The histogramEmerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 7, July 2015 1125