Sensitive Immunoassay Detection of Plasmodium Lactate Dehydrogenase by Inductively Coupled Plasma Mass Spectrometry

Synchronous development of the erythrocytic stages of a human malaria parasite, Plasmodium falciparum, in culture was accomplished by suspending cultured parasites in 5% D-sorbitol and subsequent reintroduction into culture. Immediately after sorbitol treatment, cultures consisted mainly of single and multiple ring-form infections. At the same time, varying degrees of lysis of erythrocytes infected with the more mature stages of the parasite was evident. Approximately 95% of the parasites were in the ring stage of development at 48 and 96 hr after sorbitol treatment-likewise, a high percentage of trophozoite and schizont stages was observed at 24, 72, and 120 hr. D-Mannitol produced similar, selective, lytic effects.

The global burden of malaria has been substantially reduced over the past two decades. Future efforts to reduce malaria further will require moving beyond the treatment of clinical infections to targeting malaria transmission more broadly in the community. As such, the accurate identification of asymptomatic human infections, which can sustain a large proportion of transmission, is becoming a vital component of control and elimination programmes. We determined the relationship across common diagnostics used to measure malaria prevalence - polymerase chain reaction (PCR), rapid diagnostic test and microscopy - for the detection of Plasmodium falciparum infections in endemic populations based on a pooled analysis of cross-sectional data. We included data from more than 170,000 individuals comparing the detection by rapid diagnostic test and microscopy, and 30,000 for detection by rapid diagnostic test and PCR. The analysis showed that, on average, rapid diagnostic tests detected 41% (95% confidence interval = 26-66%) of PCR-positive infections. Data for the comparison of rapid diagnostic test to PCR detection at high transmission intensity and in adults were sparse. Prevalence measured by rapid diagnostic test and microscopy was comparable, although rapid diagnostic test detected slightly more infections than microscopy. On average, microscopy captured 87% (95% confidence interval = 74-102%) of rapid diagnostic test-positive infections. The extent to which higher rapid diagnostic test detection reflects increased sensitivity, lack of specificity or both, is unclear. Once the contribution of asymptomatic individuals to the infectious reservoir is better defined, future analyses should ideally establish optimal detection limits of new diagnostics for use in control and elimination strategies.

Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovale and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovale. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.