Field evaluation of a real time loop-mediated isothermal amplification assay (RealAmp) for malaria diagnosis in Cruzeiro do Sul, Acre, Brazil.

Conventional molecular methods, such as nested polymerase chain reaction (PCR), are very sensitive for detection of malaria parasites, but require advanced laboratory equipment and trained personnel. Real-time loop-mediated isothermal amplification (RealAmp), a loop-mediated isothermal amplification-based molecular tool (LAMP), facilitates rapid target amplification at a single temperature setting, reducing the need for sophisticated equipment. We evaluated the performance of a field-adapted RealAmp assay for malaria diagnosis in Cruzeiro do Sul, Acre State, Brazil, a remote area in Brazil with limited laboratory capabilities. We enrolled 1,000 patients with fever (axillary temperature ≥ 37.5 C) or history of fever in last 24 h presenting for malaria diagnosis from February through June 2015. DNA was extracted from dried blood spots using a boil and spin method (heat treatment) at the sample processing site, and also using commercial kits at a Brazilian national reference laboratory. RealAmp was performed for Plasmodium genus, P. falciparum, and P. vivax identification. In addition, Giemsa-stained blood smears were prepared and examined by two independent well-trained study microscopists. A combination of Real-time PCR and nested PCR was used as reference test. The sensitivity and specificity of RealAmp in the field site laboratory were 94.1% (95% confidence interval [CI]: 90.1-96.8) and 83.9% (95% CI: 81.1-86.4), respectively. The sensitivity and specificity of local microscopy were 87.7% (95% CI: 82.6-91.7) and 98.9% (95% CI: 97.8-99.4), respectively, while study microscopy showed sensitivity of 96.4% (95% CI: 93.0-98.4) and specificity of 98.2% (95% CI: 97.0-99.0). None of the three tests detected 20 P. falciparum and P. vivax mixed infections identified by the reference test. Our findings highlight that it is possible to implement simple molecular tests in facilities with limited resources such as Cruzeiro do Sul in Brazil. RealAmp sensitivity was similar to that of microscopy performed by skilled professionals; both RealAmp and study microscopy performed poorly in detection of mixed infection. Attempts to develop and evaluate simpler molecular tools should continue, especially for the detection of malaria infection in remote areas.

Histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia.

More than 80% of available malaria rapid diagnostic tests (RDTs) are based on the detection of histidine-rich protein-2 (PfHRP2) for diagnosis of Plasmodium falciparum malaria. Recent studies have shown the genes that code for this protein and its paralog, histidine-rich protein-3 (PfHRP3), are absent in parasites from the Peruvian Amazon Basin. Lack of PfHRP2 protein through deletion of the pfhrp2 gene leads to false-negative RDT results for P. falciparum. We have evaluated the extent of pfhrp2 and pfhrp3 gene deletions in a convenience sample of 198 isolates from six sites in three states across the Brazilian Amazon Basin (Acre, Rondonia and Para) and 25 isolates from two sites in Bolivia collected at different times between 2010 and 2012. Pfhrp2 and pfhrp3 gene and their flanking genes on chromosomes 7 and 13, respectively, were amplified from 198 blood specimens collected in Brazil. In Brazil, the isolates collected in Acre state, located in the western part of the Brazilian Amazon, had the highest percentage of deletions for pfhrp2 25 (31.2%) of 79, while among those collected in Rondonia, the prevalence of pfhrp2 gene deletion was only 3.3% (2 out of 60 patients). In isolates from Para state, all parasites were pfhrp2-positive. In contrast, we detected high proportions of isolates from all 3 states that were pfhrp3-negative ranging from 18.3% (11 out of 60 samples) to 50.9% (30 out of 59 samples). In Bolivia, only one of 25 samples (4%) tested had deleted pfhrp2 gene, while 68% (17 out of 25 samples) were pfhrp3-negative. Among the isolates tested, P. falciparum pfhrp2 gene deletions were present mainly in those from Acre State in the Brazilian Amazon. These results indicate it is important to reconsider the use of PfHRP2-based RDTs in the western region of the Brazilian Amazon and to implement appropriate surveillance systems to monitor pfhrp2 gene deletions in this and other parts of the Amazon region.