A Large Proportion of P. falciparum Isolates in the Amazon Region of Peru Lack pfhrp2 and pfhrp3: Implications for Malaria Rapid Diagnostic Tests

Abstract

BackgroundMalaria rapid diagnostic tests (RDTs) offer significant potential to improve the diagnosis of malaria, and are playing an increasing role in malaria case management, control and elimination. Peru, along with other South American countries, is moving to introduce malaria RDTs as components of malaria control programmes supported by the Global Fund for AIDS, TB and malaria. The selection of the most suitable malaria RDTs is critical to the success of the programmes.MethodsEight of nine microscopy positive P. falciparum samples collected in Iquitos, Peru tested negative or weak positive using HRP2-detecting RDTs. These samples were tested for the presence of pfhrp2 and pfhrp3 and their flanking genes by PCR, as well as the presence of HRP proteins by ELISA. To investigate for geographic extent of HRP-deleted parasites and their temporal occurrence a retrospective study was undertaken on 148 microscopy positive P. falciparum samples collected in different areas of the Amazon region of Peru.FindingsEight of the nine isolates lacked the pfhrp2 and/or pfhrp3 genes and one or both flanking genes, and the absence of HRP was confirmed by ELISA. The retrospective study showed that 61 (41%) and 103 (70%) of the 148 samples lacked the pfhrp2 or pfhrp3 genes respectively, with 32 (21.6%) samples lacking both hrp genes.ConclusionsThis is the first documentation of P. falciparum field isolates lacking pfhrp2 and/or pfhrp3. The high frequency and wide distribution of different parasites lacking pfhrp2 and/or pfhrp3 in widely dispersed areas in the Peruvian Amazon implies that malaria RDTs targeting HRP2 will fail to detect a high proportion of P. falciparum in malaria-endemic areas of Peru and should not be used. RDTs detecting parasite LDH or aldolase and quality microscopy should be use for malaria diagnosis in this region. There is an urgent need for investigation of the abundance and geographic distribution of these parasites in Peru and neighbouring countries.

False-Negative Rapid Diagnostic Tests for Malaria and Deletion of the Histidine-Rich Repeat Region of the hrp2 Gene

“…Although the negative PCR results could have resulted from sequence variability in the upstream and downstream regions used for the primers, that finding alone would not produce a negative RDT for HRP2. For that reason, because we are not aware of sequence variation in those regions that interfere with this PCR and because previous reports detail hrp2 deletions from Papua New Guinea 16 and Peru, 17 these results suggest that spontaneous deletions of hrp2 occur in many (perhaps most) areas with P. falciparum transmission.…”

“…These results confirm that hrp2 deletions occur under field conditions as well as in the laboratory [14][15][16] and show that P. falciparum parasites without the histidine-rich repeat region of the hrp2 gene or its HRP2 gene product produce bloodstream infection in sub-Saharan Africa. In addition, recent reports indicate that hrp2 deletions are present in both Papua New Guinea and the Amazon region of Peru 16,17 and thus, suggest that false-negative results for RDTs based on HRP2 may also occur in those regions. However, it is not clear whether parasites without hrp2 are at a selective disadvantage or whether they are more frequent at times of intense transmission, such as the rainy season, when these studies were performed in Mali.…”

Evaluación de campo de la precisión diagnóstica de la prueba de diagnóstico rápido SD Bioline Malaria Antigen Pf/Pv® en Colombia

“…Es así que en Colombia se han evaluado en los últimos años pruebas que detectan estos antígenos, como Now ICT Malaria Pf/ Pv ®, Diamed OptiMAL-IT ® Malaria y Paracheck-Pf ®, con el fin de conocer su capacidad diagnóstica, generalmente con buenos resultados (25)(26)(27)(28) Sin embargo, es importante seguir profundizando en la investigación de otros antígenos blanco con el fin de lograr niveles más altos en la sensibilidad y especificidad de las pruebas de diagnóstico rápido, y para evitar diagnósticos con resultados falsos negativos como ocurre en el caso de las cepas de P. falciparum que no expresan la HRP II, por deleción del gen que codifica para este antígeno, situación que se ha presentado principalmente en aislamientos de parásitos de la región amazónica (14,29,30).…”

“…El almacenamiento de las muestras se llevó a cabo en bolsas de cierre hermético individuales con una bolsa de sílica gel y se mantuvieron en un lugar fresco y a temperatura ambiente hasta su llegada al Instituto Nacional de Salud para su procesamiento. Las muestras fueron diagnosticadas en ciego (13)(14)(15).…”

Malaria Diagnostics in Clinical Trials

“…However, a small proportion of P. falciparum parasites appear to lack intact HRP2 or have altered forms, which can lead to false-negative results. 42,43 In addition, HRP2 antigen can persist in the blood for up to two weeks after resolution of blood-stage parasite infection, 44 partly because of carriage by gametocytes. 45 Thus, HRP2-based RDTs should not be used for patient follow-up for at least one month after treatment.…”