Molecular surveillance of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum isolates from Mozambique

Between 2010 and 2016, a total of 9124 blood samples were collected onto filter papers during cross-sectional studies conducted at the beginning (November) or end (May) of the malaria season in Southern Mozambique (Manhiça and Magude; Table 1). In Mozambique, a peak in transmission is usually seen during the rainy season, from November to April. Transmission intensity in southern Mozambique is generally low, although areas of high transmission may still be observed [9].

Malaria diagnosis was conducted using microscopy, HRP2-based RDT and qPCR; or only RDTs and qPCRs. The inclusion criteria for the deletion analysis were: 1) a negative HRP2-based RDT (SD BIOLINE Malaria Antigen P. f—05FK50) but positive by microscopy and qPCR (18S rRNA) or 2) a negative HRP2-based RDT but positive qPCR (18S rRNA) if microscopy was not performed. First, a nested PCR targeting single copy k13 gene (nPCR_k13) was performed to verify the presence of parasite DNA in the sample [10]. Second, pfhrp2 and pfhrp3 genes were amplified using standard primers as described elsewhere [5, 11]. Finally, pfhrp2 and pfhrp3 deletions were concluded if kelch13 gene PCR was positive, but PCRs for pfhrp2 and pfhrp3 failed to amplify the respective gene. The laboratory-adapted culture lines 3D7 as a positive control for both pfhrp2 and pfhrp3, and HB3 and DD2 as negative controls for pfhrp3 and pfhrp2, respectively, were amplified simultaneously. The National Mozambican Ethics Review Committee and the Hospital Clínic of Barcelona Ethics Review Committee approved the collection of samples and molecular analysis. Informed consent and permission (in the case of children under 18 years of age) were also obtained from each participant or a parent/legal guardian during the cross-sectional studies.

Thin and thick blood smears were air-dried, stained with Giemsa and examined using a light microscope fitted with a 100 × oil immersion lens and a 10 × eyepiece to quantify parasitaemia in the Centro de Investigação em Saúde de Manhiça (CISM) laboratory [9]. Slides were read twice by two different qualified microscopists, and if there was discordance in the results, a third reading was performed by an additional microscopist.

DNA was extracted from a half of the filter paper (Whatman, 903^TM), containing a 25 μL blood drop by using QIAamp DNA Mini kit (Qiagen). The ABI PRISM 7500 HT Real-Time System (Applied Biosystems) was used to amplify purified parasite DNA templates, using a previously described method [12, 13]. Parasitaemia in the clinical samples was quantified by extrapolation against the standard curve prepared from an in vitro culture of 3D7 strain.

Purified DNA templates were amplified using 2720 Thermal Cycler (Applied Biosystems) following a previously described method for the kelch13 gene [10].

Samples with intact parasite DNA confirmed by nPCR_k13 were used for further amplification of region covering exon 1 and 2, as well as exon 2 of pfhrp2 and pfhrp3 genes [5, 11], following previously described methods with minor changes. These changes include the use of 1× HOT FirePol Master Mix, annealing temperatures of 63 °C of 1 min for across regions of exon 1 and 2 of pfhrp2 gene and 60 °C of 1 min for exon 2 amplification for both pfhrp2 and pfhrp3 genes. PCR products were visualized using 2% agarose (Invitrogen) and a UV trans-illuminator.